A whitening nano-composition targeting fibroblasts and melanocytes, its preparation method and application

Abstract

The present invention belongs to the technical field of cosmetics, and particularly relates to a whitening nano-composition targeting fibroblasts and melanocytes, a preparation method thereof and an application. The present invention provides a whitening nano-composition targeting fibroblasts and melanocytes, which comprises diglucosyl gallic acid, Glycyrrhiza glabra root extract, Olea europaea leaf extract, Thalassiothrix elongata extract, oil, emulsifier, co-emulsifier and the balance of water. The whitening nano-composition provided by the present invention can simultaneously target melanocytes and fibroblasts, inhibit the melanin generation pathway (MITF), regulate the SDF-1 pathway, reduce the activities of melanin generation-related enzymes, prevent melanin transfer, activate proteasome activity, reduce oxidative inflammatory damage and UV stimulation; moreover, the nano-composition provided by the present invention can promote the efficient penetration of the above four active ingredients through the skin barrier, can be highly concentrated and retained in the target tissue for a long time, and has sustained release and controlled release, so that the whitening effect is remarkable.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and particularly relates to a whitening nano-composition targeting fibroblasts and melanocytes, a preparation method thereof, and an application thereof. Background Art

[0002] Melanocytes are mainly distributed in the basal layer of the skin epidermis, embedded between epidermal basal cells, and are neural crest-derived cells that produce melanin. The synthesis, secretion, transfer, and shedding of melanin play a particularly important role in determining skin color. When melanocytes are stimulated by ultraviolet rays, they become activated, promoting the synthesis and transport of melanin, resulting in the darkening of the skin color. Research has found that the key enzymes involved in melanin formation mainly include tyrosinase (TYR), DHICA oxidase (TRP-1), and dopachrome isomerase (TRP-2). There are mainly three signal pathways involved in regulation, namely the cyclic adenosine monophosphate-dependent signal pathway (cAMP signal pathway), the signal pathway mediated by protein Wnt (Wnt signal pathway), and the mitogen-activated protein kinase signal pathway (MAPK signal pathway). Among them, microphthalmia-associated transcription factor (MITF) is an important target of each pathway.

[0003] Fibroblasts located in the dermis are the main cell components of loose connective tissue and are differentiated from mesenchymal cells. They not only synthesize and secrete collagen and elastin to generate collagen fibers, reticular fibers, and elastic fibers, but also synthesize and secrete matrix components such as glycosaminoglycans and glycoproteins. Fibroblasts and melanocytes are closely related in the process of skin pigmentation. Stromal cell-derived factor-1 (SDF-1) is a key protein for communication between fibroblasts and melanocytes, keeping tyrosinase under control. With the increase of age, the SDF-1 in senescent fibroblasts decreases, leading to excessive secretion of melanin. On the other hand, the activity of the proteasome decreases with age. The proteasome is the main degradation pathway for proteolysis. The decrease in its activity will cause the formation of undegradable lipofuscin from protein and lipid residues, making the skin dull and showing age spots. By regulating the key enzyme activities, signal pathways, acting factors, etc. of related substances such as melanin and lipofuscin, it is expected to become a new direction for skin whitening. At the same time, as is well known, external factors such as ultraviolet rays and pollution, as well as internal factors such as hormones and inflammation levels, will also act on the skin together, affecting the function of melanocytes through different mechanisms and pathways, thereby changing the skin color.

[0004] However, from the ingredients of current market whitening products, it can be seen that at present, most products mainly achieve corresponding effects by inhibiting, blocking, or affecting melanin production and transfer, and some products achieve whitening effects by promoting melanin metabolism and detachment from the stratum corneum. Generally speaking, the current whitening products have limited action targets and single action mechanisms, ultimately resulting in unsatisfactory whitening effects. Summary of the Invention

[0005] The object of the present invention is to provide a whitening nano - composition targeting fibroblasts and melanocytes, and its preparation method and application. The whitening nano - composition provided by the present invention can target fibroblasts and melanocytes simultaneously, and has significant whitening effects by inhibiting the melanin generation pathway (MITF), regulating the SDF - 1 pathway, reducing the activities of melanin - generating related enzymes, preventing melanin transfer, activating proteasome activity, reducing oxidative inflammatory damage, and UV stimulation.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a whitening nano - composition targeting fibroblasts and melanocytes, which, by mass percentage, comprises: 1 - 10% diglucosyl gallic acid, 1 - 10% Glycyrrhiza glabra root extract, 1 - 10% Olea europaea leaf extract, 1 - 10% Thalassiothrix elongata extract, 1 - 10% oil, 1 - 20% emulsifier, 1 - 30% co - emulsifier, and the balance of water;

[0008] The oil includes one or more of triglyceride caprylate / caprate, triglyceride palmitate, isopropyl palmitate, tri(ethylhexanoic acid) glyceride, isopropyl myristate, ethylhexyl cocoate, and ethylhexyl palmitate;

[0009] The emulsifier includes one or more of glucosides, polyglycerols, polysorbates, polyoxyethylene hydrogenated castor oils, and phospholipids;

[0010] The co - emulsifier includes one or more of diethanol distearate, ethoxydiglycol, dipropylene glycol, glycerol, butanediol, sorbitol, isopropanol, 1,2 - hexanediol, 1,2 - pentanediol, and 1,3 - propanediol.

[0011] Preferably, the glucosides include one or more of octyl glucoside, coco - glucoside, and arachidyl glucoside;

[0012] The polyglycerols include one or more of polyglycerol - 10 oleate, polyglycerol - 10 myristate, polyglycerol - 10 diisostearate, polyglycerol - 4 oleate, and polyglycerol - 6 polyricinoleate;

[0013] The polysorbates include one or more of polysorbate - 20, polysorbate - 40, polysorbate - 60, and polysorbate - 80;

[0014] The polyoxyethylene hydrogenated castor oils include one or more of PEG - 40 hydrogenated castor oil, PEG - 60 hydrogenated castor oil, and PEG - 80 hydrogenated castor oil;

[0015] The phospholipids include one or more of hydrogenated lecithin, lecithin and soy lecithin.

[0016] Preferably, the oil includes one or more of triglyceride caprylate / caprate, tri(ethylhexanoate) glyceride and ethylhexyl cocoate.

[0017] Preferably, the emulsifier includes one or more of octyl glucoside, polyglyceryl-10 oleate, polyglyceryl-10 myristate and lecithin.

[0018] Preferably, the co-emulsifier includes one or more of glycerol, 1,2-hexanediol, 1,2-pentanediol and 1,3-propanediol.

[0019] Preferably, the particle size of the whitening nano-composition targeting fibroblasts and melanocytes is 10 - 500 nm.

[0020] The present invention provides a method for preparing the whitening nano-composition targeting fibroblasts and melanocytes according to the above technical solution, comprising the following steps:

[0021] Mix the extract of Glycyrrhiza glabra root, oil and emulsifier to obtain an oil phase;

[0022] Mix diglucosyl gallic acid, extract of Olea europaea leaf, extract of Thalassiothrix elongata, co-emulsifier and water to obtain an aqueous phase;

[0023] Mix and emulsify the oil phase and the aqueous phase, and then perform micronization treatment to obtain a micron-sized dispersion;

[0024] Perform nanosization treatment on the micron-sized dispersion to obtain the whitening nano-composition targeting fibroblasts and melanocytes.

[0025] Preferably, the micronization treatment is shear mixing, the rotation speed of the shear mixing is 4000 - 30000 rpm, and the time is 1 - 20 min.

[0026] Preferably, the nanosization treatment is high-pressure homogenization treatment or high-pressure microfluidization treatment;

[0027] The pressure of the high-pressure homogenization treatment is 300 - 1600 bar, the temperature is 20 - 70 °C, and the number of cycles is 1 - 10 times;

[0028] The pressure of the high-pressure microfluidization treatment is 3000 - 16000 psi, the temperature is 20 - 70 °C, and the number of cycles is 1 - 10 times.

[0029] The present invention provides the application of the whitening nano-composition targeting fibroblasts and melanocytes as described in the above technical solution, or the whitening nano-composition targeting fibroblasts and melanocytes prepared by the preparation method as described in the above technical solution, in the preparation of whitening cosmetics.

[0030] The present invention provides a whitening nano-composition targeting fibroblasts and melanocytes, which comprises, by mass percentage: 1-10% of diglucosyl gallic acid, 1-10% of Glycyrrhiza glabra root extract, 1-10% of Olea europaea leaf extract, 1-10% of Thalassiosira elongata extract, 1-10% of oil, 1-20% of emulsifier, 1-30% of co-emulsifier and the balance of water; the oil comprises one or more of triglyceride caprylate / caprate, triglyceride palmitate, isopropyl palmitate, tri(ethylhexanoic acid) glyceride, isopropyl myristate, ethylhexyl cocoate, ethylhexyl palmitate; the emulsifier comprises one or more of glucosides, polyglycerols, polysorbates, polyoxyethylene hydrogenated castor oils and phospholipids; the co-emulsifier comprises one or more of diethanol distearate, ethoxydiglycol, dipropylene glycol, glycerol, butanediol, sorbitol, isopropanol, 1,2-hexanediol, 1,2-pentanediol and 1,3-propanediol. The present invention comprehensively considers various whitening mechanisms, and uses the combination of diglucosyl gallic acid, Glycyrrhiza glabra root extract, Olea europaea leaf extract and Thalassiosira elongata extract to achieve the synergistic effect of multi-effect and multi-target active ingredients. Specifically: diglucosyl gallic acid can prevent the generation of lipofuscin in the skin by inhibiting the production of reactive oxygen species (ROS); act on melanocytes, effectively prevent melanin synthesis by inhibiting the activity of tyrosinase; inhibit the expression of MITF, a key gene for melanin production, through the Wnt signaling pathway; at the same time, it can prevent the oxidation of sebum in pores and repair the phenomenon of enlarged pores; inhibit DNA damage caused by UV and repair the phenomenon of dull skin; inhibit the production of inflammatory factor IL-1 and repair skin inflammation; Glycyrrhiza glabra root extract not only has an obvious inhibitory effect on the activity of tyrosinase, but also can act on dopachrome tautomerase (TRP-2), DHICA oxidase and α-MSH melanocyte-stimulating hormone; in addition, Glycyrrhiza glabra root extract also has the effects of inhibiting inflammation and the generation of reactive oxygen species; the seaweed polyphenols produced by Thalassiosira elongata can activate the expression of SDF-1, a key target for pigmentation disorders, and achieve the regulation of melanocytes by fibroblasts; Olea europaea leaf extract has an anti-inflammatory effect, improves telangiectasia and post-inflammatory hyperpigmentation caused by inflammation; helps to enhance the skin's own ability to scavenge oxygen free radicals and protect skin cells from ultraviolet damage. The present invention provides the combination of the above four whitening active ingredients, which can effectively target melanocytes and fibroblasts, inhibit the melanin production pathway (MITF), regulate the SDF-1 pathway, reduce the activity of melanin production-related enzymes, prevent melanin transfer, activate proteasome activity, reduce oxidative inflammatory damage and UV stimulation, and form a multi-effect whitening nano-composition. On the other hand: the present invention uses the above oil, emulsifier and co-emulsifier to nano-encapsulate and deliver the four active ingredients.Based on the nanostructure and characteristics formed by the above-mentioned oils, emulsifiers, and co-emulsifiers, the composition obtained by nano-encapsulation in the present invention has good stability and water dispersibility, increases the solubility of multi-component whitening active ingredients, and also improves the irritation of the active ingredients, enabling the active ingredients to reach a sufficient concentration in the product to exert corresponding functional effects. While effectively improving the solubility of each active ingredient, it provides a stable storage space for them, avoiding unnecessary degradation or inactivation of the active ingredients before storage and use, which is beneficial to increasing the concentration of active substances in whitening products. More importantly, the present invention makes full use of the advantages of the percutaneous penetration characteristics, biocompatibility, controlled release and sustained release characteristics of the nanocarriers. The nano-composition provided by the present invention can promote the efficient penetration of the above four active ingredients through the skin barrier, and can quickly reach the basal layer where melanocytes are distributed and the dermis deep in the skin. The whitening nano-composition has good skin permeability and retention performance, can be highly concentrated and retained for a long time in the target tissue, is slowly released and controlled released, is effectively taken up by target cells, improves the bioavailability of the active ingredients, enhances the whitening effect, and prolongs the action time. Description of the Drawings

[0031] Figure 1 Results of cell safety evaluation;

[0032] Figure 2 Results of chicken embryo stimulation evaluation;

[0033] Figure 3 Results of skin cumulative permeation amount and skin retention amount;

[0034] Figure 4 Results of skin penetration observed by laser confocal microscope;

[0035] Figure 5 Results of laser confocal observation of B16F10 cell uptake behavior;

[0036] Figure 6 Results of cell uptake detected by flow cytometry;

[0037] Figure 7 Results of determination of melanocyte tyrosinase activity;

[0038] Figure 8 Results of determination of melanin content in melanocytes;

[0039] Figure 9 Results of 3D skin model related determination;

[0040] Figure 10 Results of comparison of changes before and after using the whitening nano-composition essence;

[0041] Figure 11 Results of changes before and after using the whitening nano-composition essence. Detailed implementation mode

[0042] The present invention provides a whitening nano - composition targeting fibroblasts and melanocytes, which, by mass percentage, comprises: 1 - 10% diglucosyl gallic acid, 1 - 10% Glycyrrhiza glabra root extract, 1 - 10% Olea europaea leaf extract, 1 - 10% Thalassiothrix elongata extract, 1 - 10% oil, 1 - 20% emulsifier, 1 - 30% co - emulsifier and the balance of water;

[0043] The oil includes one or more of triglyceride caprylate / caprate, triglyceride palmitate, isopropyl palmitate, tri(ethylhexanoic acid) glyceride, isopropyl myristate, ethylhexyl cocoate, ethylhexyl palmitate;

[0044] The emulsifier includes one or more of glucosides, polyglycerols, polysorbates, polyoxyethylene hydrogenated castor oils and phospholipids;

[0045] The co - emulsifier includes one or more of diethanol distearate, ethoxydiglycol, dipropylene glycol, glycerol, butanediol, sorbitol, isopropanol, 1,2 - hexanediol, 1,2 - pentanediol and 1,3 - propanediol.

[0046] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well - known to those skilled in the art.

[0047] By mass percentage, the whitening nano - composition targeting fibroblasts and melanocytes provided by the present invention includes 1 - 10% diglucosyl gallic acid, preferably 1 - 5%.

[0048] In the present invention, diglucosyl gallic acid can prevent the generation of lipofuscin in the skin by inhibiting the production of reactive oxygen species (ROS); act on melanocytes, effectively prevent melanin synthesis by inhibiting the activity of tyrosinase; inhibit the expression of MITF, a key gene for melanin production, through the Wnt signaling pathway. At the same time, it can prevent the oxidation of sebum in pores, repair the phenomenon of enlarged pores; inhibit DNA damage caused by UV, repair the phenomenon of dull skin; inhibit the production of inflammatory factor IL - 1, repair skin inflammation. Diglucosyl gallic acid can also inhibit the transport of melanosomes from the dendrites of melanocytes to the stratum corneum, preventing skin darkening.

[0049] By mass percentage, the whitening nano - composition targeting fibroblasts and melanocytes provided by the present invention includes 1 - 10% Glycyrrhiza glabra root extract, preferably 1 - 5%.

[0050] In the present invention, the extract of Glycyrrhiza glabra root is a flavonoid substance extracted from Glycyrrhiza glabra, and its main active ingredient is glabridin. It not only has a significant inhibitory effect on tyrosinase activity, but also acts on dopachrome tautomerase (TRP-2), DHICA oxidase and α-MSH melanocyte-stimulating hormone. In addition, the extract of Glycyrrhiza glabra root also has the effects of inhibiting inflammation and the generation of reactive oxygen species. Experimental data show that the whitening effect of the extract of Glycyrrhiza glabra root is 232 times higher than that of vitamin C.

[0051] By mass percentage, the whitening nano-composition targeting fibroblasts and melanocytes provided by the present invention comprises 1-10% of olea europaea leaf extract, preferably 1-5%.

[0052] In the present invention, the olea europaea leaf extract is rich in active molecules such as biological polyphenols and flavonoids. The main active ingredients are oleuropein and hydroxytyrosol, which can stimulate macrophage activity, reduce the expression of pro-inflammatory factors, have an anti-inflammatory effect, and improve telangiectasia and post-inflammatory hyperpigmentation caused by inflammation. Oleuropein can help enhance the ability of the skin to scavenge oxygen free radicals by itself and protect skin cells from ultraviolet damage. Hydroxytyrosol is one of the powerful antioxidants, and its antioxidant ability is higher than that of coenzyme Q10. On the other hand, the olea europaea leaf extract can also activate the activity of proteasome, thereby reducing the area of pigmentation, reducing lipofuscin and age spots.

[0053] By mass percentage, the whitening nano-composition targeting fibroblasts and melanocytes provided by the present invention comprises 1-10% of thalassiosira elongata extract, preferably 1-5%.

[0054] In the present invention, the seaweed polyphenols produced by thalassiosira elongata can activate the expression of SDF-1, a key target for pigmentation disorders, and realize the regulation of melanocytes by fibroblasts. Tests show that it can significantly reduce the melanin content in the dark spots of Caucasian volunteers (a 12.6% decrease within one month, and the effect is twice that of the placebo), significantly reduce the number of visible spots on Asian skin (a 156% decrease within one month), and significantly reduce the melanin content of pigmented spots on African skin (a 327% decrease within two months).

[0055] Although scientifically selecting and combining different whitening mechanisms and taking effect from several target pathways simultaneously can effectively improve the overall whitening effect. However, the multi-target effect also means that it is necessary to adopt a combination of multiple different efficacy components or different components with multiple functions. In this process, it is necessary to avoid the interference or reaction of the components in order to increase the overall whitening effect. The present invention creatively adopts four whitening efficacy components, namely diglucosyl gallic acid, extract of Glycyrrhiza glabra root, olea europaea leaf extract and thalassiosira elongata extract, for synergistic compatibility, realizing the synergistic effect among the components, being able to achieve multi-efficacy whitening, and thus enhancing the whitening effect.

[0056] The present invention uses four whitening functional components, namely diglucosyl gallic acid, Glycyrrhiza glabra root extract, Olea europaea leaf extract, and Thalassiosira elongata extract, in synergistic combination. The mass percentage content of the four whitening functional components should neither be too high nor too low. When the content of the functional components in the whitening nano-composition is too low, the whitening effect is not significant; as the content of the functional components increases, the whitening effect increases, but when the content of the functional components exceeds a certain value, it will cause a significant increase in the particle size of the whitening nano-composition, which is not conducive to the long-term stability of the product.

[0057] Based on mass percentage, the whitening nano-composition targeting fibroblasts and melanocytes provided by the present invention comprises 1-10% of oil, preferably 1-8%.

[0058] In the present invention, the oil includes one or more of triglyceride caprylate / caprate, triglyceride palmitate, isopropyl palmitate, tri(ethylhexanoic acid) glyceride, isopropyl myristate, ethylhexyl cocoate, and ethylhexyl palmitate; more preferably, it includes one or more of triglyceride caprylate / caprate, tri(ethylhexanoic acid) glyceride, and ethylhexyl cocoate.

[0059] Based on mass percentage, the whitening nano-composition targeting fibroblasts and melanocytes provided by the present invention comprises 1-20% of emulsifier, preferably 5-20%.

[0060] In the present invention, the emulsifier includes one or more of glucosides, polyglycerols, polysorbates, polyoxyethylene hydrogenated castor oils, and phospholipids.

[0061] In the present invention, the glucosides preferably include one or more of octyl glucoside, coco glucoside, and arachidyl glucoside.

[0062] In the present invention, the polyglycerols preferably include one or more of polyglycerol-10 oleate, polyglycerol-10 myristate, polyglycerol-10 diisostearate, polyglycerol-4 oleate, and polyglycerol-6 polyricinoleate.

[0063] In the present invention, the polysorbates preferably include one or more of polysorbate-20, polysorbate-40, polysorbate-60, and polysorbate-80.

[0064] In the present invention, the polyoxyethylene hydrogenated castor oils preferably include one or more of PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, and PEG-80 hydrogenated castor oil.

[0065] In the present invention, the phospholipids preferably include one or more of hydrogenated lecithin, lecithin, and soy lecithin.

[0066] In the present invention, the emulsifier preferably includes one or more of octyl glucoside, polyglyceryl-10 oleate, polyglyceryl-10 myristate, and lecithin.

[0067] By mass percentage, the whitening nano-composition targeting fibroblasts and melanocytes provided by the present invention includes 1-30% of a co-emulsifier, preferably 5-20%.

[0068] In the present invention, the co-emulsifier includes one or more of diethanol distearate, ethoxydiglycol, dipropylene glycol, glycerol, butanediol, sorbitol, isopropanol, 1,2-hexanediol, 1,2-pentanediol, and 1,3-propanediol; more preferably, it includes one or more of glycerol, 1,2-hexanediol, 1,2-pentanediol, and 1,3-propanediol.

[0069] The present invention combines the types and mass percentages of the oils, emulsifiers, and co-emulsifiers defined above with the four whitening efficacy components in the present invention, and can form a nano-carrier encapsulation structure. The prepared whitening nano-composition has a smaller particle size, uniform size, and high stability.

[0070] By mass percentage, the whitening nano-composition targeting fibroblasts and melanocytes provided by the present invention includes the balance of water.

[0071] In the present invention, the water is preferably distilled water or purified water.

[0072] In the present invention, the particle size of the whitening nano-composition targeting fibroblasts and melanocytes is preferably 10-500 nm, more preferably 10-200 nm. When the particle size of the whitening nano-composition provided by the present invention is preferably in the range of 10-200 nm, the whitening nano-composition has higher stability.

[0073] The present invention adopts a combination of multiple skin-whitening active ingredients. Due to differences in the molecular weight, hydrophilicity-hydrophobicity, solubility, etc. of various active ingredients, it is difficult to maintain a fully dissolved and dispersed state in the same dispersion system for a long time at the target concentration; this will lead to unstable phenomena such as precipitation, layering, and sedimentation of multiple active ingredients in the system, which will directly affect the use of the product. In addition, for some active ingredients, their chemical structures are prone to dissociation to show acidity or alkalinity, or undergo denaturation and inactivation. When applied, these substances may cause skin irritation or even allergic reactions. The present invention uses four skin-whitening active components, namely diglucosyl gallic acid, Glycyrrhiza glabra root extract, Olea europaea leaf extract, and Thalassiothrix elongata extract, in synergistic combination. At the same time, the types and mass percentages of the above-defined oils, emulsifiers, and co-emulsifiers are combined with the four skin-whitening active components. The obtained skin-whitening composition has good stability and water dispersibility, increases the solubility of the active ingredients, and also improves the irritation of the active ingredients, so that the active ingredients can reach a sufficient concentration in the product to exert the corresponding functional effects. While effectively improving the solubility of each active ingredient, it provides a stable storage space for them, avoiding unnecessary degradation or inactivation of the active ingredients before storage and use.

[0074] At the same time, the present invention fully considers that due to the existence of the skin barrier and the differences in the physicochemical properties of multi-functional active ingredients such as molecular weight, solubility, and oil-water partition coefficient, there will be significant differences in the effect of the ingredients penetrating through the skin barrier into the target site, and even some ingredients cannot effectively penetrate the barrier. The present invention preferably sets the particle size of the skin-whitening nano-composition to be 10 - 500 nm. Through the types and mass percentages of the above-defined oils, emulsifiers, and co-emulsifiers, a nano-carrier for the skin-whitening active components is formed. By making full use of the advantages of the percutaneous penetration characteristics, biocompatibility, controlled release and sustained release characteristics, etc. of the nano-carrier, the formed nano-composition can promote the efficient penetration of the above four active ingredients through the skin barrier, and can quickly reach the basal layer where melanocytes are distributed and the dermis layer deep in the skin. The skin-whitening nano-composition has good skin permeability and retention performance, can be highly concentrated and retained in the target tissue for a long time, with controlled release and sustained release, can be effectively taken up by target cells, improve the bioavailability of the active ingredients, enhance the skin-whitening effect, and extend the action time.

[0075] The present invention provides a preparation method of the skin-whitening nano-composition targeting fibroblasts and melanocytes as described in the above technical solution, including the following steps:

[0076] Mix the Glycyrrhiza glabra root extract, oil, and emulsifier to obtain an oil phase;

[0077] Mix diglucosyl gallic acid, Olea europaea leaf extract, Thalassiothrix elongata extract, co-emulsifier, and water to obtain an aqueous phase;

[0078] Mix the oil phase and the water phase, emulsify the mixture, and then perform micronization to obtain a micron-sized dispersion;

[0079] Perform nanosizing on the micron-sized dispersion to obtain the whitening nano-composition targeting fibroblasts and melanocytes.

[0080] In the present invention, the extract of Glycyrrhiza glabra root, oil, and emulsifier are mixed (hereinafter referred to as the first mixing) to obtain an oil phase. In the present invention, the temperature of the first mixing is preferably 40-50°C, more preferably 50°C. In the present invention, the first mixing is preferably carried out under water bath heating conditions.

[0081] In the present invention, diglucosyl gallic acid, Olea europaea leaf extract, Thalassiothrix elongata extract, co-emulsifier, and water are mixed (hereinafter referred to as the second mixing) to obtain a water phase. In the present invention, the temperature of the second mixing is preferably 40-50°C, more preferably 50°C. In the present invention, the second mixing is preferably carried out under water bath heating conditions.

[0082] After obtaining the water phase and the oil phase, in the present invention, the oil phase and the water phase are mixed and emulsified, and then micronization is performed to obtain a micron-sized dispersion. In the present invention, the mixing of the water phase and the oil phase is preferably: dropping the water phase into the oil phase, and the dropping speed is preferably 8 drops / second. In the present invention, the mixing and emulsification are carried out under stirring conditions, the rotation speed of the mixing and emulsification is preferably 500-600 rpm, and the temperature of the mixing and emulsification is preferably 40-50°C.

[0083] In the present invention, the micronization is preferably shear mixing, the rotation speed of the shear mixing is preferably 4000-30000 rpm, more preferably 10000-30000 rpm; the time is preferably 1-20 min, more preferably 3-15 min. The temperature of the micronization is preferably 40-50°C.

[0084] After obtaining the micron-sized dispersion, in the present invention, the micron-sized dispersion is subjected to nanosizing to obtain the whitening nano-composition targeting fibroblasts and melanocytes.

[0085] In the present invention, the nanosizing treatment is preferably high-pressure homogenization treatment or high-pressure microfluidization treatment. In the present invention, the pressure of the high-pressure homogenization treatment is preferably 300 to 1600 bar, more preferably 800 to 1400 bar; the temperature is preferably 20 to 70 °C, more preferably 30 to 50 °C; the number of cycles is 1 to 10 times, more preferably 3 to 8 times. In the present invention, the pressure of the high-pressure microfluidization treatment is preferably 3000 to 16000 psi, more preferably 8000 to 14000 psi; the temperature is preferably 20 to 70 °C, more preferably 30 to 50 °C; the number of cycles is preferably 1 to 10 times, more preferably 2 to 8 times.

[0086] Through the above preparation method, the present invention co-packages the above four active ingredients in a nanocarrier. After encapsulation, the integrity and biological activity of the ingredient structure are still maintained. At the same time, the light and heat stability of the active ingredients is improved, the solubility and aqueous dispersibility of poorly soluble active ingredients are improved, the irritation of some active ingredients is reduced, and it can effectively promote the efficient penetration of high-content active ingredients through the skin barrier, quickly reach the basal layer where melanocytes are distributed and the dermis layer deep in the skin, stay for a long time, release and control release, be effectively taken up by target cells, improve the bioavailability of active ingredients, enhance the whitening effect, and extend the action time.

[0087] The present invention provides the application of the whitening nanocomposition targeting fibroblasts and melanocytes described in the above technical solution or the whitening nanocomposition targeting fibroblasts and melanocytes prepared by the preparation method described in the above technical solution in the preparation of whitening cosmetics.

[0088] In the present invention, the form of the whitening cosmetics includes but is not limited to toner, cream, lotion, essence and gel. The whitening nanocomposition of the present invention is easily soluble in water and is convenient to use. The mass percentage content of the whitening nanocomposition added in the whitening cosmetics is preferably 0.1 to 30%, more preferably 1 to 30%.

[0089] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they cannot be understood as limiting the protection scope of the present invention.

[0090] Example 1

[0091] By mass percentage, 2% Glycyrrhiza glabra root extract, 3% caprylic / capric triglyceride, 3% triethylhexanoin, 5% octyl glucoside, and 5% polyglyceryl-10 oleate are mixed and dissolved by heating in a water bath at 50 °C to obtain an oil phase;

[0092] Add 2% diglucosyl gallic acid, 2% olive leaf extract, 2% Thalassiothrix elongata extract, 10% 1,2-pentanediol, and 10% 1,2-hexanediol to the balance purified water, and dissolve by heating in a water bath at 50 °C to obtain an aqueous phase;

[0093] Drop the aqueous phase into the oil phase at a rate of 8 drops per second and continuously stir at a rotation speed of 50 °C and 600 rpm. After mixing evenly, perform high-speed shearing treatment at a rotation speed of 10,000 rpm for 3 min to obtain a micron-sized dispersion;

[0094] Perform high-pressure homogenization treatment on the micron-sized dispersion under the conditions of a temperature of 50 °C and a pressure of 800 bar for 4 cycles, and cool to room temperature to obtain a whitening nano-composition.

[0095] Examples 2 to 12

[0096] The formulations of Examples 2 to 12 are shown in Table 1, with the balance being purified water. The preparation method is the same as that of Example 1, except that the types or contents of the substances in the formulation are changed.

[0097] Example 13

[0098] The formulation is the same as that of Example 1. The difference is that after obtaining the micron-sized dispersion according to the steps of Example 1, perform high-pressure microfluidization treatment under the conditions of a pressure of 8000 psi and a temperature of 50 °C for 2 cycles, and cool to room temperature to obtain a whitening nano-composition.

[0099] Example 14

[0100] The formulation is the same as that of Example 1. The difference is that after obtaining the micron-sized dispersion according to the steps of Example 1, perform high-pressure homogenization treatment under the conditions of a pressure of 500 bar and a temperature of 50 °C for 2 cycles, and cool to room temperature to obtain a whitening nano-composition.

[0101] Example 15

[0102] The formulation is the same as that of Example 1. The difference is that after obtaining the micron-sized dispersion according to the steps of Example 1, perform high-pressure homogenization treatment under the conditions of a pressure of 800 bar and a temperature of 50 °C for 2 cycles, and cool to room temperature to obtain a whitening nano-composition.

[0103] Table 1 Compositions of Examples 1 to 15

[0104]

[0105]

[0106]

[0107] Comparative Example 1

[0108] The formulation is shown in Table 2, with the balance being purified water. The preparation method is the same as that of Example 1, except that the types of oil agents are cyclomethicone and isononyl isononanoate.

[0109] Comparative Example 2

[0110] The formulation is shown in Table 2, with the balance being purified water. The preparation method is the same as that of Example 1, except that the types of emulsifiers are Tween 80 and Span 80.

[0111] Comparative Example 3

[0112] The formulation is shown in Table 2, with the balance being purified water. The preparation method is the same as that of Example 1, except that the type of co-emulsifier is n-butanol.

[0113] Comparative Examples 4 to 7

[0114] The formulation is shown in Table 2, with the balance being purified water. The preparation method is the same as that of Example 1, except that the types and contents of the active ingredients in the formulation are changed.

[0115] Comparative Example 8

[0116] The formulation is shown in Table 2, with the balance being purified water. All the active ingredients, oils, and co-emulsifiers are added to the purified water, and ultrasonic dissolution is carried out at 50 °C to obtain a free whitening composition.

[0117] Comparative Example 9

[0118] The preparation method is the same as that of Example 1, except that the active ingredient in the formulation is only 2% olea europaea leaf extract, and a single-packaged nanocarrier containing 2% olea europaea leaf extract is obtained.

[0119] Comparative Example 10

[0120] The preparation method is the same as that of Example 1, except that the active ingredients in the formulation are 2% glycyrrhiza glabra root extract, 2% diglucosyl gallic acid, and 2% thalassiosira elongata extract raw materials, and a nano-composition containing 2% glycyrrhiza glabra root extract, 2% diglucosyl gallic acid, and 2% thalassiosira elongata extract is obtained.

[0121] Table 2 Compositions of Comparative Examples 1 to 10

[0122]

[0123]

[0124] Application Example 1

[0125] Preparation of human efficacy test samples

[0126] Dissolve 2.0% glycerol, 0.3% carbomer, 0.1% xanthan gum, 0.5% phenoxyethanol, and 97.2% purified water by stirring at room temperature to obtain a blank essence. And prepare each group of test samples according to the following table.

[0127] Table 3 Preparation methods of test essences

[0128] Control Group 1 Blank Essence Control Group 2 Blank Essence + 5% of the sample obtained in Comparative Example 4 Control Group 3 Blank Essence + 5% of the sample obtained in Comparative Example 5 Control Group 4 Blank Essence + 5% of the sample obtained in Comparative Example 6 Control Group 5 Blank Essence + 5% of the sample obtained in Comparative Example 7 Control Group 6 Blank Essence + 5% of the sample obtained in Comparative Example 8 Sample Group Blank Essence + 5% of the sample obtained in Example 1

[0129] Test Example 1

[0130] Storage stability

[0131] Place the whitening nano-compositions prepared in Examples 1 - 15 and Comparative Examples 1 - 3 in airtight containers, and place them at room temperature, 4°C, and 45°C for 3 months respectively, and conduct freeze-thaw experiments. Check the appearance of the samples under each condition before and after storage, test the particle size of the nano-compositions under each condition before and after storage, and comprehensively evaluate the stability of the nano-compositions.

[0132] Freeze-thaw experiment: Place the samples in airtight containers, place them at -20°C for 48 hours and then at 45°C for 48 hours, and cycle twice to observe the changes in the samples.

[0133] Both the particle size and Zeta potential of the nano-compositions are detected using a Zetasizer Nano-ZS90 nano particle size and potential analyzer. Take an appropriate amount of the nano-composition, dilute it 50 times with ultrapure water to make the average light intensity of the sample solution 200 - 300, the particle size measurement angle of the laser particle size analyzer is 90°, and the test temperature is 25°C.

[0134] The test results are shown in Table 4.

[0135] Table 4 Stability results

[0136]

[0137]

[0138]

[0139] As can be seen from the data in Table 4, the different whitening nano-compositions prepared under the conditions of the types and contents of the active ingredients, oils, emulsifiers, and co-emulsifiers according to the present invention have a uniform appearance, and there are no unstable phenomena such as delamination and precipitation after storage at room temperature, 4 °C, and 45 °C for 3 months. The particle size distribution range is 20-500 nm, and the Zeta potential is -5 to -50 mV. The whitening nano-compositions prepared according to the more preferred conditions (Examples 1, 3, 4, and 8) have smaller particle sizes, all less than 100 nm, and have more advantages in application. Their Zeta potential is in the range of -15 to -35 mV, which is beneficial to the long-term stability of the nano-composition. Example 13 is a nano-composition obtained by high-pressure microfluidization treatment, and its particle size stability has no obvious difference compared with the nano-composition treated by high-pressure homogenization with the same formula. Adjusting the high-pressure homogenization pressure and the number of homogenization times will affect the properties of the nano-composition (Examples 1, 14, and 15). The nano-composition obtained after treatment 4 times under the condition of 800 bar has the optimal particle size (Example 1). Replacing the types of oils, emulsifiers, and co-emulsifiers described in the present invention will lead to a decrease in the stability of the nano-composition. In Comparative Example 1, obvious oil floating occurred after storage at 45 °C for 3 months. In Comparative Example 2, a stable nano-composition could not be formed. In Comparative Example 3, the low-temperature stability was extremely poor, and precipitation was likely to occur.

[0140] Test Example 2

[0141] Centrifugal stability

[0142] There is no industry standard for the centrifugal stability of nano-carrier products for cosmetics. Therefore, based on the regulations of the centrifugal stability experiment of raw materials in the cosmetics industry, a nano-carrier centrifugal acceleration experiment was carried out on Examples 1, 3, 4, 8, and 13 at the standards of 3000, 5000, 8000, and 10000 r / min for 30 min. The experimental results are shown in Table 5.

[0143] Table 5 Centrifugal stability of whitening nano-composition

[0144]

[0145] As can be seen from the results in Table 5, after centrifugation at 3000, 5000, 8000, and 10000 r / min for 30 min, the above-mentioned whitening nano-compositions in the examples still have a uniform, clear, and transparent appearance, without turbidity, delamination, and precipitation phenomena, indicating that the whitening nano-composition has good centrifugal stability.

[0146] Test Example 3

[0147] Determination of encapsulation efficiency and drug loading

[0148] The diluted whitening nano-composition of Example 1 was centrifuged to determine the content W of free liquiritigenin F , and at the same time, after the sample was demulsified and centrifuged, the content W of liquiritigenin in the supernatant was determinedT The drug loading efficiency (DLE) of the whitening nano - composition was calculated according to Formula 1, and the encapsulation efficiency (EE) of the whitening nano - composition was calculated according to Formula 2.

[0149]

[0150]

[0151] W T : The total mass of glabridin, W F : The mass of free glabridin, W L represents the mass of the nano - carrier.

[0152] Detected by HPLC, the encapsulation efficiency of glabridin in the whitening nano - composition was 88.7 ± 0.6%, and the drug loading was 1.77 ± 0.12%.

[0153] Test Example 4

[0154] Cell safety evaluation

[0155] Using the survival rate of HaCaT cells (human keratinocytes) as an index, the cell safety of the whitening nano - composition described in Example 1 at different concentrations and the free whitening composition described in Comparative Example 8 was tested. The control group was complete medium.

[0156] The results of the cell safety evaluation of the whitening nano - composition and the free whitening composition at different concentrations on HaCaT cells are shown in Figure 1 (Note: Compared with the free composition at the same concentration, **P < 0.01). Figure 1 The experimental results showed that when the concentration of the whitening nano - composition was 400, 800, 1600 μg / mL, the cell viability of the free component group was significantly higher than that of the free component group. When the concentration of the carrier was in the range of 100 - 800 μg / mL, the whitening nano - composition had no toxicity to HaCaT cells. The results indicated that the nano - composition could reduce the toxicity of the whitening active ingredient.

[0157] Test Example 5

[0158] Chicken embryo chorioallantoic membrane irritation evaluation

[0159] The whitening nano - compositions described in Examples 1 - 15 were diluted and dropped on the surface of the chorioallantoic membrane. The vascular changes were observed and data were recorded to calculate the irritation score IS.

[0160] The final results of Examples 1 - 15 of the present invention were similar. The irritation evaluation results of the whitening nano - composition in Example 2 are shown in Figure 2After the whitening nano - composition diluted 10 - fold was in contact with the chorioallantoic membrane of chicken embryos for 300 s, there was no bleeding, vascular lysis, or coagulation in the capillaries, and the reaction score was 0.7, indicating that the whitening nano - composition diluted 10 - fold had good safety and no irritation.

[0161] Test Example 6

[0162] Patch test

[0163] Thirty subjects were selected. The 10% whitening nano - compositions described in Examples 1 - 15 and the blank control were both applied to the flexor side of the forearms of the subjects for 24 h. After the indentation disappeared, the skin reactions were observed.

[0164] The results showed that none of the 30 subjects had faint erythema, erythema, edematous erythema, significant redness and swelling, infiltration, or papules and papule - or blister - accompanied lesions, indicating that the whitening nano - composition in this test had no irritation to human skin.

[0165] Test Example 7

[0166] Determination of cumulative permeation amount and skin retention amount

[0167] The Franz diffusion cell method was used to conduct a transdermal experiment on ex vivo porcine skin, and the cumulative permeation amount per unit area and skin retention amount per unit area of the active ingredients in the 10% whitening nano - composition essence of the sample group in Application Example 1 and the 10% free whitening composition essence of the control group 6 were analyzed and calculated. The skin cumulative permeation amount of the active ingredient at different sampling times was calculated according to Formula 3.

[0168]

[0169] Among them, Q n is the cumulative drug permeation amount, C n is the drug concentration measured at the nth time, C i is the drug concentration measured at the i - th point, V0 is the volume of the diffusion cell, that is, the amount of release medium added, and V i is the sampling amount each time. The cumulative permeation amount per unit area Q = Q n / S, where S is the area of the diffusion cell, 2.27 cm 2 .

[0170] The cumulative skin permeation amount per unit area and skin retention amount of the main active ingredient glabridin in the licorice root extract in the sample were calculated. The results are shown in Figure 3 (Note: Compared with the free composition, **P < 0.01). The 24 - h cumulative skin permeation amount per unit area of glabridin in the free composition essence and the nano - composition essence were 1.88 μg / cm 2 and 8.42 μg / cm 2 , and the skin retention amounts were 13.29 μg / cm2 and 55.47 μg / cm 2 。Compared with the free composition essence, the skin cumulative permeation amount per unit area of glabridin in the nano-composition essence increased by 347.87%, and the skin retention amount increased by 317.38%. This indicates that after the active ingredient is encapsulated by the nano-carrier, it can effectively promote the transdermal absorption of the active ingredient and its retention in the skin, and improve its skin bioavailability.

[0171] Test Example 8

[0172] Observation of skin penetration behavior by laser confocal microscopy

[0173] The test experimental device is the same as the above transdermal experimental device. The samples of the 10% whitening nano-composition essence in Sample Group 1 of Application Example 1 and the 10% free whitening composition essence in Control Group 6 labeled with Rhodamine B (RhoB) were cryosectioned after the test, and observed by laser confocal microscopy.

[0174] The skin tissue penetration behaviors of free RhoB and RhoB nano-carriers at different time points observed by laser confocal microscopy are shown in Figure 4 。It can be seen that as time prolongs, the fluorescence penetration depth of the skin increases. The results show that within the same time, the fluorescence intensity of the RhoB-labeled whitening nano-composition essence in the skin is significantly stronger than that of the free whitening composition essence, and the whitening nano-composition essence can penetrate into the deep skin tissue. The nano-carrier can promote the rapid penetration of the encapsulated component into the skin.

[0175] Test Example 9

[0176] (1) Observation of cell uptake behavior by laser confocal microscopy

[0177] Replace the whitening active ingredient with fluorescein isothiocyanate (FITC) with an addition amount of 0.1%, and prepare FITC-labeled nano-carriers and free FITC according to the formulations of Example 1 and Comparative Example 8 in Tables 1 and 2. After treating B16F10 cells and staining respectively, observe using a laser confocal microscope.

[0178] The uptake behavior of B16F10 cells on the nano-carrier observed by laser confocal microscopy, the results are shown in Figure 5 , Figure 5 The free FITC and FITC carrier in correspond to Comparative Example 8 and Example 1 respectively. At 4 h of incubation, the fluorescence intensity of the free FITC group in B16F10 cells was weak, while the fluorescence of the FITC nano-carrier had entered the cells, and the fluorescence intensity was significantly stronger than that of free FITC. The experimental results show that compared with free FITC, the FITC nano-carrier can be taken up by B16F10 cells more, and effectively deliver the encapsulated active substance into the skin target cells to play a role.

[0179] (2) Detection of cell uptake behavior by flow cytometry

[0180] After treatment with free FITC and FITC nanocarriers, the intracellular fluorescence intensity of B16F10 cells was detected by flow cytometry.

[0181] The results of the detection of the uptake behavior of nanocarriers by B16F10 cells by flow cytometry are shown in Figure 6 (Note: compared with free, **P<0.01). It can be seen from Figure 6 that after incubation of B16F10 cells for 2 h and 4 h, compared with free FITC, the average fluorescence intensity of the nanocarrier samples increased by 175.40% and 111.63%, respectively. The experimental results show that the nanocarrier can significantly improve the cellular uptake and intracellular accumulation of its encapsulated substances by B16F10 cells.

[0182] Test Example 10

[0183] Determination of intracellular tyrosinase activity

[0184] The L-Dopa oxidation method was used to determine the activity of intracellular tyrosinase. B16F10 cells in the logarithmic growth phase were inoculated into 24-well plates at a density of 5.0×10 4 cells / well. After culturing for 24 h, the cells were divided into a control group, a model group, and an experimental group, with 3 replicates in each group. The control group was only added with DMEM complete medium, the model group was only added with DMEM complete medium containing 100 nmol / L α-MSH, and the experimental group was added with DMEM complete medium containing 100 nmol / L α-MSH and the compositions prepared in Comparative Example 8, Comparative Example 9, Comparative Example 10, and Example 1 diluted 2000 times. After continuing to culture for 48 h, 200 μL of cell lysate containing 1% (V%) Triton X-100 was added to each well, and the cells were lysed by freezing at -80 °C for 30 min. The cell lysate was collected and centrifuged, 100 μL of the supernatant was taken and placed in a 96-well plate, and 100 μL of 0.1% (W / V) L-Dopa solution was added. After incubation at 37 °C for 2 h, the absorbance (A) of each well was measured at a wavelength of 495 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Figure 7 The results of the determination of melanocyte tyrosinase activity. Note: compared with the model group, ** P<0.01; compared with Comparative Example 8, ## P<0.01; compared with Comparative Example 10, && P<0.01.

[0185] It can be seen from Figure 7It can be seen that, compared with the model group, Comparative Example 8, Comparative Example 9, Comparative Example 10 and Example 1 can all significantly reduce tyrosinase activity. Compared with Comparative Example 8, Example 1 has a more significant effect on reducing the tyrosinase activity of B16F10 cells (P<0.01). Compared with Comparative Example 9, Example 1 has a more significant effect on reducing the tyrosinase activity of B16F10 cells (P<0.01). Compared with Comparative Example 10, Example 1 has a more significant effect on reducing the tyrosinase activity of B16F10 cells (P<0.01). The results show that the inhibition rate of tyrosinase activity of the single-pack nanocarrier with only olive leaf extract (Comparative Example 9) is 17%, the inhibition rate of tyrosinase activity of the nano-composition with three raw materials of glabra root extract, diglucosyl gallic acid and Thalassiothrix elongata extract (Comparative Example 10) is 28%, and the inhibition rate of tyrosinase activity of the nano-composition with four raw materials of glabra root extract, diglucosyl gallic acid, Thalassiothrix elongata extract and olive leaf extract (Example 1) is 66%. The tyrosinase inhibition rate of Example 1 is higher than the sum of the tyrosinase inhibition rates of Comparative Example 9 and Comparative Example 10. Compared with Comparative Example 9, the tyrosinase activity inhibition rate of Example 1 increased by 288%. Compared with Comparative Example 10, the tyrosinase activity inhibition rate of Example 1 increased by 136%. The inhibition rate of tyrosinase activity of the free composition with four raw materials (Comparative Example 8) is 22%. Compared with Comparative Example 8, the tyrosinase activity inhibition rate of Example 1 increased by 187%. It is proved that the compounding of olive leaf extract with the raw materials of glabra root extract, diglucosyl gallic acid and Thalassiothrix elongata extract can promote the improvement of the effect of whitening active ingredients, and the whitening effect of the nano-composition after encapsulation is greatly enhanced compared with the free ingredients.

[0186] Test Example 11

[0187] Determination of Cellular Melanin Content and Observation of Generation

[0188] The content of intracellular melanin was determined by the NaOH lysis method. B16F10 cells in the logarithmic growth phase were taken, at 5.0×10 4Inoculate the cells at a density of [[number]] / well in a 24-well plate. After culturing for 24 h, divide the cells into a control group, a model group, and an experimental group, with 3 replicate wells in each group. The control group was added with only DMEM complete medium, the model group was added with only DMEM complete medium containing 100 nmol / L α-MSH, and the experimental group was added with DMEM complete medium containing 100 nmol / L α-MSH and the compositions prepared in Comparative Example 8, Comparative Example 9, Comparative Example 10, and Example 1, all diluted 2000 times. After continuing to culture for 48 h, collect the cells in each well into a centrifuge tube, add 200 μL of 1 mol / L NaOH solution containing 10% DMSO to each centrifuge tube, then place the EP tube in an 80 °C constant temperature water bath for 1 h to lyse the cells, dissolve the melanin, centrifuge, take the supernatant and transfer it to a 96-well plate, and measure the absorbance (A) of each well at a wavelength of 405 nm using an enzyme-linked immunosorbent assay reader. Figure 8 Determination results of melanin content in melanocytes. Note: Compared with the model group, ** P < 0.01; compared with Comparative Example 8, ## P < 0.01; compared with Comparative Example 10, && P < 0.01.

[0189] It can be seen from Figure 8 that compared with the model group, Comparative Example 8, Comparative Example 9, Comparative Example 10, and Example 1 can all significantly reduce the intracellular melanin content. Compared with Comparative Example 8, Example 1 has a more significant effect on reducing the intracellular melanin content in B16F10 cells (P < 0.01). Compared with Comparative Example 9, Example 1 has a more significant effect on reducing the intracellular melanin content in B16F10 cells (P < 0.01). Compared with Comparative Example 10, Example 1 has a more significant effect on reducing the intracellular melanin content in B16F10 cells (P < 0.01). Figure 8The results showed that the inhibition rate of intracellular melanin content of the single-pack nanocarrier with only olive leaf extract (Comparative Example 9) was 13%, and the inhibition rate of intracellular melanin content of the nano-composition (Comparative Example 10) with 3 raw materials including licorice root extract, diglucosyl gallic acid and Thalassiosira elongata extract was 22%. The inhibition rate of intracellular melanin content of Example 1 with 4 raw materials including licorice root extract, diglucosyl gallic acid, Thalassiosira elongata extract and olive leaf extract was 59%. The inhibition rate of intracellular melanin content of Example 1 was higher than the sum of the inhibition rates of intracellular melanin content of Comparative Example 9 and Comparative Example 10. Compared with Comparative Example 9, the inhibition rate of intracellular melanin content of Example 1 increased by 353%. Compared with Comparative Example 10, the inhibition rate of intracellular melanin content of Example 1 increased by 168%. The inhibition rate of intracellular melanin content of the free composition (Comparative Example 8) with 4 raw materials was 19%. Compared with Comparative Example 8, the inhibition rate of intracellular melanin content of Example 1 increased by 211%. It was proved that the compounding of olive leaf extract with licorice root extract, diglucosyl gallic acid and Thalassiosira elongata extract raw materials could promote the improvement of the action effect of whitening active ingredients, and the whitening effect of the nano-composition after encapsulation was greatly enhanced compared with the free components.

[0190] Test Example 12

[0191] Evaluation of the Whitening Effect of 3D Skin Melanin Model

[0192] The apparent chromaticity, apparent brightness (L* value) and melanin content of the 3D skin melanin model were measured for the negative control (NC), positive control (PC, kojic acid 500 μg / mL) and sample group (the whitening nano-composition described in Example 1, with a content of 100 μg / mL). The results are shown in Figure 9 .

[0193] Figure 9 . (A) in shows that compared with the blank group, the apparent chromaticity of each group became significantly darker after UVB irradiation treatment. Compared with the negative control group, after treatment with the whitening nano-composition, the apparent chromaticity of the skin model became significantly lighter, and the whitening effect was better than that of high-dose kojic acid (PC 500 μg / mL).

[0194] Figure 9 . (B) in shows that compared with the blank group, the apparent brightness of each group decreased significantly after UVB irradiation treatment (P<0.01). Compared with the negative control group, after treatment with the whitening nano-composition, the apparent brightness of the skin model increased significantly (P<0.01), and the effect was better than that of high-dose kojic acid (PC 500 μg / mL). Figure 9 . (C) in shows that compared with the blank group, the melanin content of each group increased significantly after UVB irradiation treatment (P<0.01). Compared with the negative control group, after treatment with the whitening nano-composition, the melanin content of the skin model decreased significantly (P<0.01).

[0195] Test Example 13

[0196] Human Efficacy Evaluation

[0197] Recruit 35 experimental volunteers aged 20 - 55 years old, with good health, no skin diseases, not pregnant or lactating, and not allergic to the product ingredients. After informing the evaluation process, possible effects, risks, precautions and signing the informed consent form for efficacy evaluation, conduct the efficacy evaluation test of different samples in Application Example 1. Regularly use the samples on the face twice a day, and do not use other products or drugs that may affect the skin condition during the test. Truthfully and accurately record and feedback the skin condition and feelings after use. Observe and compare the efficacy indicators of the volunteers four weeks after applying the samples. Use the Shanghai Fuhuan Intelligent Skin Analysis System and German CK Skin Melanin Tester MX18 to measure the ITA angle of facial skin; skin glossiness (L value); total area of black and pigmented spots; MI value of black and pigmented spots, calculate the changes of each index parameter after using the sample and take the average value.

[0198] Table 6 Changes in ITA° and L* values of facial skin of volunteers before and after using the sample

[0199]

[0200]

[0201] Table 7 Changes in skin melanin, MI and total area of pigmented spots of volunteers before and after using the sample

[0202]

[0203] Principle for measuring skin color and uniformity index: By detecting the reflected light on the skin surface structure through the Vplus device, the change of skin color can be comprehensively evaluated by the L*a*b colorimetric system and ITA°. The larger the ITA° value, the lighter the skin color. Among them, the larger the L* value, the whiter the skin; the larger the a* value, the redder the skin; the larger the b* value, the yellower the skin. The ITA° is calculated according to Formula 4:

[0204]

[0205] Principle for measuring melanin and pigmented spot indicators: Based on the principle of spectral absorption (RGB), the reflectance of light with a specific wavelength on the skin is measured through the Vplus device, and the absorbance of melanin in the skin is measured. The melanin index (MI) is calculated from this value. The smaller this value, the lower the melanin content in the skin, and vice versa, the higher the melanin content in the skin.

[0206] The facial conditions of some volunteers before and after using the 5% Whitening Nano - composition Essence for 28 days were collected by the Vplus device, as shown inFigure 10 , Figure 11 。 Figure 10 In (A), it is the comparison of facial pigments and age spots; Figure 10 In (B), it is the comparison of facial skin color; Figure 11 In (A), it is the comparison of facial pigments and age spots; Figure 11 In (B), it is the comparison of facial skin color. As shown in Tables 6 and 7, after the volunteers used the blank essence of Control Group 1 for four weeks, the ITA° value increased by 0.27%, the skin L* value increased by 0.19%; the MI value decreased by 1.65%, and the area of melasma decreased by 0.99%. There were no significant changes in the above indicators, indicating that the blank essence had no improvement effect on skin color, uniformity, melanin, and age spots. In comparison, Control Groups 2-6 and the sample group containing whitening agents showed significant improvement in skin ITA° and L* values after four weeks of use; the MI value and the total area of melasma decreased significantly. Among them, Control Groups 2-5 contained a single type of whitening agent respectively. When the four agents were used in combination, the effect of Control Group 6 with the same concentration of the agents was significantly improved. After using Control Group 6, the ITA° value increased by 7.08%, the L* value increased by 5.27%, the MI value decreased by 15.85%, and the total area of melasma decreased by 13.26%. The above results illustrate the scientificity of the combination of the four active ingredients in the whitening composition. The combination of different whitening mechanisms works simultaneously to achieve excellent whitening effects. Finally, after using the whitening nano-composition essence of the sample group obtained based on the nano-carrier delivery technology, the ITA° value increased by 10.47%, the L* value increased by 7.47%, the MI value decreased by 17.60%, and the total area of melasma decreased by 20.04%. Its effect on improving skin color, uniformity, melanin, and age spots was further enhanced.

[0207] As can be seen from the above, the whitening nano-composition provided by the present invention has excellent stability. Under the condition of high content of active ingredients, after the nano-composition is stored at room temperature, 4°C, 45°C, etc. for 3 months, the particle size does not change significantly, and there is no precipitation, stratification, etc. in the appearance. The particle size is between 10 and 200 nm, meeting the actual application requirements. The whitening nano-composition has good safety and no skin irritation. After being encapsulated by the nano-carrier, it can effectively promote the transdermal absorption and retention amount of the four whitening ingredients in the skin, penetrate into the deep tissue of the skin to take effect, and the uptake amount of the whitening nano-composition by melanocytes is significantly increased. The whitening nano-composition can significantly reduce the tyrosinase activity of B16F10 cells and inhibit melanin production, and the effect is better than that of the free ingredients with the same dose. It has a significant effect on scavenging ROS free radicals. The results of human whitening tests show that after using 5% whitening nano-composition essence for 28 days, after using the sample, the number and average area of melanin and age spots decrease, and the skin color uniformity and gloss are significantly improved, showing excellent whitening effects.

[0208] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments can also be obtained based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A whitening nano-composition targeting fibroblasts and melanocytes, characterized in that, By mass percentage, it includes: 1-10% of diglucosyl gallic acid, 1-10% of Glycyrrhiza glabra root extract, 1-10% of Olea europaea leaf extract, 1-10% of Thalassiothrix elongata extract, 1-10% of oil, 1-20% of emulsifier, 1-30% of co-emulsifier and the balance of water; The oil is triglyceride caprylate / caprate and triglyceride (ethylhexanoate), or tripalmitin; The emulsifier is polyglyceryl-10 oleate and octyl glucoside, or polyglyceryl-10 diisostearate; The co-emulsifier is 1,2-pentanediol and 1,2-hexanediol, or 1,3-propanediol.

2. The whitening nano-composition targeting fibroblasts and melanocytes according to claim 1, characterized in that, The particle size of the whitening nano-composition targeting fibroblasts and melanocytes is 10-500 nm.

3. The preparation method of the whitening nano-composition targeting fibroblasts and melanocytes according to claim 1 or 2, characterized in that, It includes the following steps: Mix the Glycyrrhiza glabra root extract, oil and emulsifier to obtain an oil phase; Mix diglucosyl gallic acid, Olea europaea leaf extract, Thalassiothrix elongata extract, co-emulsifier and water to obtain an aqueous phase; Mix and emulsify the oil phase and the aqueous phase, and then perform micronization treatment to obtain a micron-sized dispersion; Perform nanosization treatment on the micron-sized dispersion to obtain the whitening nano-composition targeting fibroblasts and melanocytes.

4. The preparation method according to claim 3, wherein The micronization treatment is shear mixing, the rotation speed of the shear mixing is 4000-30000 rpm, and the time is 1-20 min.

5. The preparation method according to claim 3, characterized in that, The nanosization treatment is high-pressure homogenization treatment or high-pressure microfluidization treatment; The pressure of the high-pressure homogenization treatment is 300-1600 bar, the temperature is 20-70 °C, and the number of cycles is 1-10 times; The pressure of the high-pressure microfluidization treatment is 3000-16000 psi, the temperature is 20-70 °C, and the number of cycles is 1-10 times.

6. Use of the whitening nano-composition targeting fibroblasts and melanocytes according to claim 1 or 2, or the whitening nano-composition targeting fibroblasts and melanocytes prepared by the preparation method according to any one of claims 3-5 in the preparation of whitening cosmetics.

Images