CN117731544B

CN117731544B - Almond acid-containing spot-lightening and seal-lightening whitening synergistic composition with targeting transdermal delivery function, and preparation method and application thereof

Info

Publication number: CN117731544B
Application number: CN202311755351.9A
Authority: CN
Inventors: 鲁旺旺; 刘倩; 承静; 汪冰洁; 麦丽芬; 刘卫; 王曦
Original assignee: Guangzhou Jiyan Cosmetics Technology Co ltd
Current assignee: Shanghai Jiyan Cosmetics Technology Co.,Ltd.
Priority date: 2023-12-19
Filing date: 2023-12-19
Publication date: 2024-11-15
Anticipated expiration: 2043-12-19
Also published as: CN117731544A

Abstract

The invention provides a spot-lightening, seal-lightening and whitening synergistic composition containing mandelic acid and having a targeting transdermal delivery function. Compared with the prior art, the outermost layer of the spot-lightening and seal-lightening whitening synergistic composition provided by the invention is a stripping active substance containing high-concentration mandelic acid, and the stripping active substance is released firstly, so that pigmentation formed by epidermis can be quickly lightened; the inner layer contains high-concentration phenethyl resorcinol and other whitening active ingredients, can permeate to a target part of melanocytes of a basal layer, and has the effects of blocking blacking and inhibiting source blacking; the skin care concept of highly fitting and accurately releasing is realized through the inner water phase, the oil phase and the outer water phase; in addition, the dual antiallergic peptide is added into the oil phase, so that the inflammation of the vaccinia skin residue can be reduced, the pigmentation induced by the inflammation can be prevented, the irritation caused by high-concentration mandelic acid and phenethyl resorcinol can be reduced, the secondary irritation to acne skin crowds can be reduced, and further the acne skin can be better reduced.

Description

Almond acid-containing spot-lightening and seal-lightening whitening synergistic composition with targeting transdermal delivery function, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to a spot-lightening, seal-lightening, whitening and synergy composition containing mandelic acid and having a targeting transdermal delivery function, a preparation method and application thereof.

Background

Acne has become the eighth chronic disease worldwide, and studies have found that more than 80% of people have varying degrees of acne in their lifetime. Acne sequelae are typically post-acne scars (moderately severe acne), post-acne erythema (PIE) (red acne marks), and post-acne Pigmentation (PIH) (black brown acne marks). Among them, erythema and pigmentation after acne have a high incidence of erythema up to 90% and pigmentation up to 70% (Li Qiaoling, xue Li, li Li. Investigation of factors related to skin lesions of acne sequelae. Chinese cosmesis 2018,27 (10): 24-26.). Post-acne pigmentation is a pigmentation spot or plaque that generally leaves a color ranging from tan to black after the erythema at the vaccinia site subsides, and its causes include intrinsic factors of inflammatory stress such as inflammatory cascade reaction that erupts at the vaccinia site, extrinsic factors such as ultraviolet irradiation stimulus. Wherein the extent and depth of the inflammatory stress response, the stability of the melanocytes, together lead to epidermal and dermal melanin deposition.

There have been many studies showing that many inflammatory factors can overactivate melanocytes and produce more melanin, such as 1L-1 alpha, 1L-33, PGE2, etc. (Md Razib Hossain, int J Mol Sci.2021Apr;22 (8): 3970). Pigmentation is one of the most headache sequelae in acne people, as most of the long-standing after the acne patients develop black spots, it has been shown based on epidemiological investigation that more than 65% of acne patients have pigmentation for more than 1 year (Abad-CASINTAHAN ET al.j dermotol.2016:43:826-828), and that even the light and medium acne people can develop pigmentation. Thus, for acne patients, timely intervention and importance of the intervention in early stages of melanogenesis can avoid the formation of intractable and difficult-to-remove pigmentation.

As pigmentation involves a number of influencing factors, in order to more effectively address pigmentation, it is generally desirable to cover multiple targets of pigmentation, and common therapeutic strategies include sun protection, antioxidant, stress signal disruption, melanin production inhibition, and melanin metabolism acceleration. Currently, in 2019 edition of chinese acne treatment guidelines, treatment recommendations for acne sequelae are newly added, wherein post-acne pigmentation recommended medication mainly covers two target pathways: pathway 1 is a drug that inhibits melanogenesis by using some melanogenesis-limiting enzyme inhibitors, such as arbutin; pathway 2 is achieved by using some agents that have exfoliative effects, i.e. lighten pigmentation that has formed on the epidermis, such as retinoids, fruit acids, etc.

Chinese patent publication No. CN108743430B discloses a co-delivery nano composition containing phenethyl resorcinol, which contains phenethyl resorcinol and other whitening active ingredients, and the whitening active ingredients with multiple target points and multiple action mechanisms are proved to have whitening effects through examples. However, in this patent, for phenethyl resorcinol and other whitening active ingredients, the phase separation preparation is simply performed based on the dissolution characteristics of the raw materials in the process, for example, the water-soluble raw materials are in the same phase, the oil-soluble raw materials are in the same phase, and the phenethyl resorcinol and exfoliating active ingredients are in the same phase, so that the precise release cannot be realized, and the related active ingredients cannot play the key roles more efficiently. The accurate release is critical in both the medical field and the cosmetic field, on one hand, the active ingredients can be released to the target position to the greatest extent, the bioavailability of the active ingredients is fully improved, and the high-efficiency effect on the target position is further exerted; on the other hand, the attack on other non-target parts can be reduced, and side effects are reduced.

Disclosure of Invention

In view of the above, the technical problem to be solved by the invention is to provide a spot-lightening, seal-lightening and whitening synergistic composition containing mandelic acid and having a targeting transdermal delivery function, and a preparation method and application thereof.

The invention provides a spot-lightening and seal-lightening and whitening synergistic composition containing mandelic acid and having a targeting transdermal delivery function, which is formed by an inner water phase, an oil phase and an outer water phase;

The internal aqueous phase comprises an alpha-MSH antagonist;

The oil phase comprises phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, an emulsifier, neutral phospholipid and grease;

the outer aqueous phase comprises a exfoliating agent and a polyglycerol ester emulsifier;

The exfoliating agent includes mandelic acid.

Preferably, the mass of the alpha-MSH antagonist is 0.01% -1% of the mass of the spot-lightening and seal-lightening synergistic composition;

the mass of the phenethyl resorcinol is 1-10% of the mass of the spot-lightening and seal-lightening synergistic composition;

the mass of the palmitoyl tetrapeptide-7 is 0.01% -1% of the mass of the spot-lightening and seal-lightening synergistic composition;

The mass of the acetyl dipeptide-1 cetyl ester is 0.01% -1% of the mass of the spot-lightening and seal-lightening synergistic composition;

the mass of the cutin stripping agent is 1-10% of the mass of the spot-lightening and seal-lightening synergistic composition.

Preferably, the mass ratio of the phenethyl resorcinol, the palmitoyl tetrapeptide-7 and the acetyl dipeptide-1 cetyl ester is (1-10): (0.01-0.5): (0.01-0.5).

Preferably, the α -MSH antagonist is selected from one or more of the group consisting of nonapeptide-1, undecylenoyl phenylalanine and hexapeptide-2;

The exfoliating agent further includes salicylic acid and/or octanoylsalicylic acid.

Preferably, the internal aqueous phase further comprises a first polyol; the outer aqueous phase further comprises a second polyol;

the first polyol and the second polyol are each independently selected from one or more of glycerol, propylene glycol, butylene glycol, 1, 2-pentanediol, 1, 2-hexanediol, 1, 3-propanediol, dipropylene glycol, PEG-10 propanediol, and polyethylene glycol-400;

The mass of the first polyol is 1-5% of the mass of the spot-lightening and seal-lightening synergistic composition;

the mass of the second polyol is 10-20% of the mass of the spot-lightening and seal-lightening synergistic composition.

Preferably, the mass of the emulsifier is 3-7% of the mass of the spot-lightening and seal-lightening synergistic composition;

The emulsifier is selected from one or more of octyl glucoside, arachidyl glucoside, polyglycerol-10 oleate, polyglycerol-10 myristate, polyglycerol-4 oleate, polyoxyethylene hydrogenated castor oil and polysorbate-80;

the mass of the neutral phospholipid is 1-5% of the mass of the spot-lightening and seal-lightening synergistic composition;

The neutral phospholipid is one or more selected from lecithin, soybean phospholipid, hydrogenated soybean phospholipid, dimyristoyl phosphatidylcholine, dioleoyl phosphatidylcholine, phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, cephalin, and sphingomyelin;

the mass of the grease is 1% -5% of the mass of the spot-lightening and seal-lightening whitening synergistic composition;

The oil is selected from one or more of caprylic/capric triglyceride, palmitic triglyceride, isopropyl palmitate, glycerol tri (ethylhexanoate), isopropyl myristate, ethylhexyl cocoate, ethylhexyl palmitate, isopentyl laurate, squalane, olive oil, soybean oil and jojoba seed oil;

the mass of the polyglycerol ester emulsifier is 1-5% of the mass of the spot-lightening and seal-lightening synergistic composition;

The polyglycerol ester emulsifier is selected from one or more of polyglycerol-10 myristate, polyglycerol-10 oleate, polyglycerol-10 dipalmitate and polyglycerol-10 diisostearate.

The invention also provides a preparation method of the mandelic acid-containing spot-lightening and seal-lightening whitening synergistic composition with the targeting transdermal delivery function, which comprises the following steps:

s1) mixing an alpha-MSH antagonist with water to obtain an inner water phase;

mixing phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, emulsifier, neutral phospholipid and oil to obtain oil phase;

Mixing a keratolytic agent and a polyglycerol ester emulsifier with water to obtain an external water phase;

S2) dripping the inner water phase into the oil phase for mixing to obtain a water-in-oil system;

s3) dropwise adding the water-in-oil system into an external water phase, and mixing to obtain a water-in-oil-in-water emulsion;

s4) carrying out micronization treatment on the water-in-oil-in-water emulsion to obtain a micron-sized fraction;

S5) carrying out nanocrystallization treatment on the micrometer-sized particles to obtain the spot-lightening, seal-lightening and whitening synergistic composition containing mandelic acid and having the targeted transdermal delivery function.

Preferably, the dropping speed in the step S2) and the step S3) is 1-5 drops/S independently; the rotational speeds of the mixing in the step S2) and the step S3) are respectively and independently 10-60 rpm;

the step S4) is characterized in that the micron-sized treatment is high-speed shearing; the rotating speed of the high-speed shearing is 4000-10000 rpm; the high-speed shearing time is 1-10 min;

The nanocrystallization treatment in the step S5) is high-pressure homogenization treatment or high-speed micro-jet treatment; the pressure of the high-pressure homogenization treatment is 300-1600 bar; the cycle times of the high-pressure homogenizing treatment are 1 to 10 times; the pressure of the high-speed micro-jet treatment is 5000-16000 psi; the cycle number of the high-pressure micro-jet treatment is 1-10.

The invention also provides a cosmetic, which comprises the spot-lightening, seal-lightening and whitening synergistic composition containing mandelic acid and having a targeted transdermal delivery function.

Preferably, the mass of the spot-lightening, seal-lightening and whitening synergistic composition containing mandelic acid and having the targeted transdermal delivery function is 0.01-10% of the mass of the cosmetic.

The invention provides a spot-lightening and seal-lightening and whitening synergistic composition containing mandelic acid and having a targeting transdermal delivery function, which is formed by an inner water phase, an oil phase and an outer water phase; the internal aqueous phase comprises an alpha-MSH antagonist; the oil phase comprises phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, an emulsifier, neutral phospholipid and grease; the external aqueous phase includes a exfoliating agent and a polyglycerol ester emulsifier. Compared with the prior art, the outermost layer of the spot-lightening and seal-lightening whitening synergistic composition provided by the invention is a stripping active substance containing high-concentration mandelic acid, and the stripping active substance is released firstly, so that pigmentation formed on the epidermis can be quickly lightened, and the effect of accelerating light black is exerted; the inner layer contains high-concentration phenethyl resorcinol and other whitening active ingredients, can permeate to a target part of melanocytes of a basal layer, and has the effects of blocking blacking and inhibiting source blacking; the skin care concept of highly fitting and accurately releasing is realized through the inner water phase, the oil phase and the outer water phase, so that the skin care composition can help to overcome pigmentation of acne people more efficiently and pointedly; in addition, the dual antiallergic peptide is added into the oil phase, so that the inflammation of the vaccinia skin residue can be reduced, the pigmentation induced by the inflammation can be prevented, the irritation caused by high-concentration mandelic acid and phenethyl resorcinol can be reduced, the secondary irritation to acne skin crowds can be reduced, and further the acne skin can be better reduced.

Drawings

FIG. 1 is a microscopic topography of the spot-lightening and seal-whitening synergistic composition prepared in example 5 of the present invention;

FIG. 2 is a graph showing cumulative percent release of phenethylresorcinol (377) and mandelic acid from the spot-lightening and whitening synergistic compositions prepared in example 5, comparative example 1, comparative example 9 and comparative example 10 and the free whitening composition prepared in comparative example 3 according to the present invention;

FIG. 3 is a graph showing the cumulative percent release of phenylethene resorcinol (377) and mandelic acid for the spot-lightening and seal-whitening synergistic composition prepared in example 5 of the present invention;

FIG. 4 is a graph showing the cell safety results of the spot-lightening, seal-lightening, whitening synergistic composition prepared in example 5, the spot-lightening, seal-lightening, whitening synergistic compositions prepared in comparative examples 1 and 2, and the free whitening composition prepared in comparative example 3 according to the present invention;

FIG. 5 is a graph showing the in vitro release behavior of the spot-lightening and seal-whitening synergistic composition prepared in example 5 of the present invention;

FIG. 6 is a graph showing the results of measuring the cumulative penetration amounts and skin retention amounts of the spot-lightening and whitening synergistic composition prepared in example 5 and the free whitening composition prepared in comparative example 3 according to the present invention;

FIG. 7 is a confocal microscopic section of the spot-lightening and seal-whitening synergistic composition prepared in example 5 of the present invention and the free whitening composition prepared in comparative example 3;

FIG. 8 is a graph showing the results of cell uptake test by laser confocal microscopy of the spot-lightening and seal-whitening synergistic composition prepared in example 5 of the present invention and the free whitening composition prepared in comparative example 3;

FIG. 9 is a graph showing the results of cell uptake test by flow cytometry of the spot-lightening and whitening synergistic composition prepared in example 5 and the free whitening composition prepared in comparative example 3 of the present invention;

FIG. 10 is a graph showing the effect of the spot-lightening and seal-whitening synergistic composition prepared in example 5 of the present invention on B16F10 cell and HaCaT cell uptake;

FIG. 11 is a graph showing the results of intracellular tyrosinase activity measurement of the spot-lightening and whitening synergistic compositions prepared in examples 1, 2 and 5, the spot-lightening and whitening synergistic compositions prepared in comparative examples 1 and 2, and the free whitening composition prepared in comparative example 3 according to the present invention;

FIG. 12 is a graph showing the effect of the spot-lightening and whitening synergistic compositions prepared in examples 1,2 and 5, the spot-lightening and whitening synergistic compositions prepared in comparative examples 1 and 2, and the free whitening composition prepared in comparative example 3 on melanogenesis in cells according to the present invention;

FIG. 13 is a graph showing the results of the whitening efficacy test of the spot-lightening and seal-lightening synergistic composition prepared in example 5 of the present invention;

FIG. 14 is a graph showing the results of whitening efficacy test of the 3D skin melanin model of the spot-lightening and whitening synergistic composition prepared in example 5, the spot-lightening and whitening synergistic compositions prepared in comparative examples 1 and 2, and the free whitening composition prepared in comparative example 3 according to the present invention;

Fig. 15 is a graph showing the peel test results of the spot-lightening and whitening synergistic composition prepared in example 5 of the present invention and the free whitening composition prepared in comparative example 3.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The invention provides a spot-lightening and seal-lightening whitening synergistic composition containing mandelic acid and having a targeting transdermal delivery function, which is formed by an inner water phase, an oil phase and an outer water phase, namely the oil phase wraps the inner water phase, and the outer water phase wraps the oil phase; the internal aqueous phase comprises an alpha-MSH antagonist; the oil phase comprises phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, an emulsifier, neutral phospholipid and grease; the outer aqueous phase comprises a exfoliating agent and a polyglycerol ester emulsifier; the exfoliating agent includes mandelic acid.

In the present invention, the internal aqueous phase comprises an alpha-MSH antagonist; human keratinocytes release a large amount of alpha-MSH upon receiving ultraviolet rays, and melanocyte stimulating hormone (alpha-MSH) is a major factor affecting melanogenesis, and can bind to melanocortin-1 receptor (MCIR) on the surface of melanocyte membrane, so that tyrosinase activity in cells is increased through signal transduction, and thus synthesis of melanin by melanocytes is increased. MC1R is highly expressed in melanocytes, and then a ligand which can be specifically combined with MC1R, namely an alpha-MSH antagonist, is selected to play a key role in inhibiting melanogenesis dimension. In the present invention, the α -MSH antagonist is an α -MSH antagonist known to those skilled in the art, and is not particularly limited, and one or more of nonapeptide-1, undecylenoyl phenylalanine and hexapeptide-2 are preferred in the present invention; the nonapeptide-1 is also called whitening peptide, is used as a bionic peptide, has very good matching property with MC1 receptors on melanocytes, can be combined with MC1R receptors on the surfaces of melanocytes in a competitive manner to inhibit MSH (melanocyte stimulating hormone) to play a role in blocking transmission of melanin signals, namely, can play a role in blocking blacking effectively, reduces generation of melanin, can be combined with other small molecular peptides, forms protein through peptide bond combination and folding of peptide chains, is absorbed by skin, enables skin color to be bright, reduces color spots and pigment deposition in the skin, and achieves the effects of whitening and lightening spots, fading acne marks and brightening the skin color; the mass of the alpha-MSH antagonist is preferably 0.01% -1% of the mass of the spot-lightening and seal-lightening synergistic composition; in some embodiments provided herein, the α -MSH antagonist is present in an amount of about 0.02%, 1%, 0.01%, 0.03%, or 0.015% by mass of the spot-lightening, seal-whitening synergistic composition.

In the present invention, the inner aqueous phase preferably further comprises a first polyol; the first polyalcohol can be used as an auxiliary emulsifier to change the rigidity of the nanoemulsion, improve the emulsion stability of an oil phase, reduce the interfacial tension so as to lead the nanoemulsion to spontaneously form and enlarge the formation area of the nanoemulsion, and the polyalcohol has the transdermal promoting effect, can promote the penetration of an active substance through the skin cuticle and can also improve the solubility and the water dispersibility of insoluble active substances; the first polyol is preferably one or more of glycerol, propylene glycol, butylene glycol, 1, 2-pentanediol, 1, 2-hexanediol, 1, 3-propanediol, dipropylene glycol, PEG-10 propanediol, and polyethylene glycol-400; the mass of the first polyol is preferably 1-5% of the mass of the spot-lightening and seal-lightening synergistic composition; in some embodiments provided herein, the first polyol is present in an amount of 3%, 5%, 1% or 4% by mass of the spot-lightening whitening synergistic composition.

According to the invention, the oil phase comprises phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, an emulsifier, neutral phospholipids and oils and fats; the mass of the phenethyl resorcinol is preferably 1-10% of the mass of the spot-lightening and seal-lightening synergistic composition; in some embodiments provided by the invention, the mass of the phenethyl resorcinol is specifically 5%, 10%, 1%, 6% or 4% of the mass of the spot-lightening and whitening synergistic composition; the phenethyl resorcinol (377) is derived from the natural whitening component-pinosylvin in Pinus sylvestris, and is a novel and efficient whitening active substance screened out after chemical transformation. The phenethyl resorcinol can effectively inhibit the activity of tyrosinase, tyrosine is a rate-limiting enzyme for melanin generation, and the inhibition effect is 22 times of that of the gold mark whitening component kojic acid, so that the regeneration of melanin can be inhibited from source, the regression of melanin is facilitated, and the whitening effect is achieved; the mass of the palmitoyl tetrapeptide-7 is preferably 0.01% -1% of the mass of the spot-lightening and seal-lightening synergistic composition; in some embodiments provided by the invention, the mass of the palmitoyl tetrapeptide-7 is specifically 0.02%, 1%, 0.01%, 0.03% or 0.015% of the mass of the spot-lightening and seal-whitening synergistic composition; palmitoyl tetrapeptide-7 is a synthetic peptide composed of four amino acids, and can stimulate the generation of laminin IV, V and II type collagen, reduce the generation of inflammatory factor-interleukin (IL-6), reduce the loss of skin collagen and elastic fiber, eliminate inflammation, increase skin elasticity, avoid wrinkles, looseness and uneven skin color, further help to relieve the inflammation remained at the vaccinia part of acne people, and prevent the generation of color sinking after inflammation; The quality of the acetyl dipeptide-1 cetyl ester is preferably 0.01% -1% of the quality of the spot-lightening and seal-lightening synergistic composition; in some embodiments provided herein, the mass of the acetyl dipeptide-1 cetyl ester is specifically 0.02%, 1%, 0.01%, 0.03% or 0.015% of the mass of the spot-lightening, seal-whitening synergistic composition; Acetyl dipeptide-1 cetyl ester can inhibit CGRP and SP (the two mediums can cause micro-vasodilation and plasma extravasation), inhibit vasodilation, reduce the nervous reaction of skin to irritants, improve skin tolerance, be used for soothing and repairing sensitive skin of the face or the body, and in the research process of the invention, the acetyl dipeptide-1 is found to have higher inhibition effect on inflammatory mediums, especially PGE2, and the inhibition effect can reach 74 percent, so the nerve soothing peptide can help to relieve the stimulation caused by high-concentration mandelic acid and phenethyl resorcinol, and also help to lighten the inflammation remained at the vaccinia part, Preventing the generation of color sinking after inflammation; The mass ratio of the phenethyl resorcinol, the palmitoyl tetrapeptide-7 and the acetyl dipeptide-1 cetyl ester is preferably (1-10): (0.01-0.5): (0.01 to 0.5), more preferably 5: (0.01-0.5): (0.01-0.5); in some embodiments provided by the invention, the mass ratio of phenethyl resorcinol, palmitoyl tetrapeptide-7 to acetyl dipeptide-1 cetyl ester is specifically 5:0.02:0.02, 10:1: 1. 1:0.01:0.01, 6:0.03:0.03 or 4:0.15:0.15; the mass ratio of the palmitoyl tetrapeptide-7 to the acetyl dipeptide-1 cetyl ester is preferably 1: (0.5 to 2), more preferably 1: (0.8 to 1.5), and more preferably 1: (0.8 to 1.2), most preferably 1:1, a step of; The emulsifier is an emulsifier well known to those skilled in the art, and is not particularly limited, and one or more of octyl glucoside, arachidyl glucoside, polyglyceryl-10 oleate, polyglyceryl-10 myristate, polyglyceryl-4 oleate, polyoxyethylene hydrogenated castor oil and polysorbate-80 are preferable in the present invention; in the examples provided herein, PEG-40 hydrogenated castor oil is specifically exemplified; the mass of the emulsifier is preferably 3-7% of the mass of the spot-lightening and seal-lightening synergistic composition; in some embodiments provided by the invention, the mass of the emulsifier is specifically 5%, 7%, 3%, 6% or 4% of the mass of the spot-lightening and seal-whitening synergistic composition; The neutral phospholipid is a neutral phospholipid well known to those skilled in the art, and is not particularly limited, and preferably includes one or more of lecithin, soybean phospholipid, hydrogenated soybean phospholipid, dimyristoyl phosphatidylcholine, dioleoyl phosphatidylcholine, phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, cephalin, and sphingomyelin; the mass of the neutral phospholipid is preferably 1-5% of the mass of the spot-lightening and seal-lightening synergistic composition; in some embodiments provided by the invention, the mass of the neutral phospholipid is specifically 3%, 5%, 1%, 4% or 2% of the mass of the spot-lightening and seal-whitening synergistic composition; the oil or fat is one well known to those skilled in the art, and is not particularly limited, and the present invention preferably includes one or more of caprylic/capric triglyceride, palmitic triglyceride, isopropyl palmitate, triglyceride (ethylhexanoate), isopropyl myristate, ethylhexyl cocoate, ethylhexyl palmitate, isopentyl laurate, squalane, olive oil, soybean oil and jojoba seed oil; the mass of the grease is preferably 1-5% of the mass of the spot-lightening and seal-lightening synergistic composition; in some embodiments provided by the invention, the mass of the grease is specifically 3%, 5%, 1%, 4% or 2% of the mass of the spot-lightening and seal-whitening synergistic composition.

According to the invention, the external aqueous phase comprises a exfoliating agent and a polyglycerol ester emulsifier; the exfoliating agent is a exfoliating agent well known to those skilled in the art, and is not particularly limited, and may include other components in addition to mandelic acid, preferably, salicylic acid and/or octanoylsalicylic acid; mandelic acid is also called mandelic acid, belongs to alpha-hydroxy acid fruit acid, and has the unique lipophilic effect. Clinical studies of application of mandelic acid in 1100 patients over 3 years in 1999 by Taylor, indicate that mandelic acid has good effects of inhibiting and improving pigmentation, treating inflammatory acne and photoaging, is relatively mild, and is less likely to produce post-inflammatory pigmentation (compared to glycolic acid, or other pharmaceutical ingredients such as anti-acne medication-retinoic acid, whitening medication-hydroquinone). The mandelic acid is combined with calcium ions in desmosome to destroy the integrity of desmosome protein, so that the connectivity between horny layers is reduced, thereby helping the horny layer to fall off, accelerating the metabolism of the horny layer, and ensuring that pigmentation deposited on the horny layer is fallen off, thereby achieving the whitening effect. The mass of the exfoliating agent is preferably 1% -10% of the mass of the nano composition; in some embodiments provided herein, the exfoliating agent is 1%, 7%, 10%, 8% or 6% of the mass of the spot-lightening, seal-whitening synergistic composition; the polyglyceryl ester emulsifier is a polyglyceryl ester emulsifier well known to those skilled in the art, and is not particularly limited, and the present invention preferably includes one or more of polyglyceryl-10 myristate, polyglyceryl-10 oleate, polyglyceryl-10 dipalmitate and polyglyceryl-10 diisostearate; the mass of the polyglycerol ester emulsifier is preferably 1-5% of the mass of the nano composition; in some embodiments provided herein, the mass of the polyglycerol ester emulsifier is specifically 3%, 5%, 1%, 4% or 2% of the mass of the spot-lightening whitening synergistic composition.

According to the invention, the outer aqueous phase preferably further comprises a second polyol; the second polyol is preferably one or more of glycerol, propylene glycol, butylene glycol, 1, 2-pentanediol, 1, 2-hexanediol, 1, 3-propanediol, dipropylene glycol, PEG-10 propanediol, and polyethylene glycol-400; the mass of the second polyol is preferably 10-20% of the mass of the spot-lightening and seal-lightening synergistic composition; in some embodiments provided herein, the mass of the second polyol is specifically 15%, 20%, 18%, 10% or 12% of the mass of the spot-lightening whitening synergistic composition.

According to the invention, the inner water phase and the outer water phase also comprise water, and the amount of water in the inner water phase and the outer water phase is such that the total amount of the spot-lightening and seal-lightening synergistic composition is 100%; the mass of water in the inner water phase is preferably 5% -20%, more preferably 8% -15%, and even more preferably 10% -15% of the total amount of water in the spot-lightening and seal-lightening synergistic composition.

The outermost layer of the spot-lightening and seal-lightening whitening synergistic composition provided by the invention is a stripping active substance containing high-concentration mandelic acid, and the stripping active substance is released firstly, so that pigmentation formed on the epidermis can be quickly lightened, and the effect of accelerating light black is exerted; the inner layer contains high-concentration phenethyl resorcinol and other whitening active ingredients, can permeate to a target part of melanocytes of a basal layer, and has the effects of blocking blacking and inhibiting source blacking; the skin care concept of highly fitting and accurately releasing is realized through the inner water phase, the oil phase and the outer water phase, so that the skin care composition can help to overcome pigmentation of acne people more efficiently and pointedly; in addition, the dual antiallergic peptide is added into the oil phase, so that the inflammation of the vaccinia skin residue can be reduced, the pigmentation induced by the inflammation can be prevented, the irritation caused by high-concentration mandelic acid and phenethyl resorcinol can be reduced, the secondary irritation to acne skin crowds can be reduced, and further the acne skin can be better reduced.

The invention also provides a preparation method of the mandelic acid-containing spot-lightening and seal-lightening whitening synergistic composition with the targeting transdermal delivery function, which comprises the following steps of: s1) mixing an alpha-MSH antagonist with water to obtain an inner water phase; mixing phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, emulsifier, neutral phospholipid and oil to obtain oil phase; mixing a keratolytic agent and a polyglycerol ester emulsifier with water to obtain an external water phase; s2) dripping the inner water phase into the oil phase for mixing to obtain a water-in-oil system; s3) dropwise adding the water-in-oil system into an external water phase, and mixing to obtain a water-in-oil-in-water emulsion; s4) carrying out micronization treatment on the water-in-oil-in-water emulsion to obtain a micron-sized fraction; s5) carrying out nanocrystallization treatment on the micrometer-sized particles to obtain the spot-lightening, seal-lightening and whitening synergistic composition containing mandelic acid and having the targeted transdermal delivery function.

The sources of all raw materials are not particularly limited, and the raw materials are commercially available; the types and contents of the alpha-MSH antagonist, the phenethyl resorcinol, the palmitoyl tetrapeptide-7, the acetyl dipeptide-1 cetyl ester, the emulsifier, the neutral phospholipid, the grease, the exfoliating agent and the polyglycerol ester emulsifier are all described above, and are not described in detail herein.

Mixing an alpha-MSH antagonist with water to obtain an internal aqueous phase; the mass of the water is 5-20%, more preferably 8-15%, still more preferably 10-15% of the water content in the spot-lightening and seal-lightening synergistic composition; in the present invention, it is preferable to further add a first polyol; the types and the contents of the first polyol are the same as those described above, and are not described in detail herein; the temperature of the mixing is preferably 40 to 70 ℃, more preferably 40 to 60 ℃, still more preferably 40 to 50 ℃.

Mixing phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, emulsifier, neutral phospholipid and oil to obtain oil phase; the temperature of the mixing is preferably 40 to 70 ℃, more preferably 40 to 60 ℃, still more preferably 40 to 50 ℃.

Mixing a keratolytic agent and a polyglycerol ester emulsifier with water to obtain an external water phase; the temperature of the mixing is preferably 40 to 70 ℃, more preferably 40 to 60 ℃, still more preferably 40 to 50 ℃.

Dropwise adding the inner water phase into the oil phase for mixing to obtain a water-in-oil system; the dropping speed is preferably 1 to 5 drops/s, more preferably 1 to 3 drops/s, still more preferably 1 to 2 drops/s; the rotation speed of the mixing is preferably 10 to 60rpm, more preferably 10 to 40rpm, still more preferably 10 to 30rpm, and most preferably 15 to 20rpm; the temperature of the mixing is preferably 40 to 70 ℃, more preferably 40 to 60 ℃, still more preferably 40 to 50 ℃.

Dropwise adding the water-in-oil system into an external water phase, and mixing to obtain a water-in-oil-in-water emulsion; the dropping speed is preferably 1 to 5 drops/s, more preferably 1 to 3 drops/s, still more preferably 1 to 2 drops/s; the rotation speed of the mixing is preferably 10 to 60rpm, more preferably 20 to 50rpm, still more preferably 20 to 40rpm, most preferably 30rpm; the temperature of the mixing is preferably 40 to 70 ℃, more preferably 40 to 60 ℃, still more preferably 40 to 50 ℃.

Carrying out micronization treatment on the water-in-oil-in-water emulsion to obtain micron-sized particles; the micronization treatment is preferably high-speed shearing; the rotation speed of the high-speed shearing is preferably 4000-10000 rpm, more preferably 6000-10000 rpm, and still more preferably 8000-10000 rpm; the time for high-speed shearing is preferably 1 to 10 minutes, more preferably 1 to 6 minutes, still more preferably 1 to 4 minutes, and most preferably 1 to 2 minutes.

Carrying out nanocrystallization treatment on the micrometer-sized particles to obtain a spot-lightening, seal-lightening and whitening synergistic composition containing mandelic acid and having a targeted transdermal delivery function; the nanocrystallization treatment is preferably a high-pressure homogenization treatment or a high-speed microfluidization treatment; the pressure of the high pressure homogenization treatment is preferably 300-1600 bar, more preferably 500-1600 bar, still more preferably 800-1600 bar, still more preferably 1000-1600 bar, still more preferably 1200-1600 bar, most preferably 1500bar; the number of cycles of the high-pressure homogenizing treatment is preferably 1 to 10, more preferably 1 to 8, still more preferably 2 to 6, and most preferably 2 to 4; the pressure of the high-speed micro-jet treatment is preferably 5000-16000 psi; the number of cycles of the high-pressure microfluidic treatment is preferably 1 to 10, more preferably 1 to 8, still more preferably 2 to 6, and most preferably 2 to 4.

In the invention, the mass of the mandelic acid-containing spot-lightening and seal-lightening whitening synergistic composition with the targeted transdermal delivery function is preferably 0.01-10% of the mass of cosmetics, more preferably 0.01-8%, still more preferably 0.01-5%; the mass concentration of the mandelic acid-containing spot-lightening and whitening synergistic composition with the targeted transdermal delivery function in the cosmetic may be any point value within the above range, specifically, in some embodiments provided by the present invention, the mass of the mandelic acid-containing spot-lightening and whitening synergistic composition with the targeted transdermal delivery function may be 0.01%, 0.04%, 0.1%, 0.2%, 0.5%, 1.5%, 2%, 3%, 5%, 8% or 10% of the cosmetic mass.

In the present invention, the cosmetic is a daily chemical product for application to the skin surface, which is well known to those skilled in the art, and is not particularly limited, and the cosmetic having the effects of whitening and eliminating acne marks is preferable in the present invention.

In the present invention, the cosmetic is preferably a resident skin care product such as essence, lotion, cream, body lotion, etc.

According to the invention, the essence preferably further comprises a humectant; the mass of the humectant is preferably 1-10% of the mass of the essence, more preferably 3-8%, even more preferably 5-8%, and most preferably 6-7%; the humectant is a humectant well known to those skilled in the art, and is not particularly limited, and preferably includes one or more of butanediol, 1, 2-pentanediol, dipropylene glycol, trehalose, panthenol, diglycerol, beta-glucan, glycerin, propylene glycol, 1, 3-propanediol, butanediol, hexanediol, sodium hyaluronate, glycereth-26, and an amino acid humectant.

According to the invention, the concentrate preferably further comprises a chelating agent; the mass of the chelating agent is preferably 0.01-0.1% of the mass of the essence, more preferably 0.02-0.08%, and still more preferably 0.03-0.05%; the chelating agent is a known chelating agent to those skilled in the art, and is not particularly limited, and preferably includes, but is not limited to, one or more of tetrasodium EDTA, disodium EDTA, EDTA and phytic acid.

According to the present invention, the concentrate preferably further comprises an emollient; the mass of the lubricant is preferably 1-10% of the mass of the essence, more preferably 2-8%, even more preferably 2-6%, and most preferably 3-5%; the lubricant is a lubricant well known to those skilled in the art, and is not particularly limited, and preferably includes one or more of dioctyl carbonate, dioctyl ether, coco-caprylate/caprate, octyldodecanol, squalane, tri (ethylhexanoic acid) ester and polydimethylsiloxane.

According to the invention, the concentrate preferably further comprises a thickener; the mass of the thickening agent is preferably 0.01% -1%, more preferably 0.01% -0.5%, still more preferably 0.01% -0.3%, still more preferably 0.05% -0.1%, and most preferably 0.08% of the mass of the essence; the thickener is a thickener well known to those skilled in the art, and is not particularly limited, and preferably includes one or more of carbomer series, acrylic copolymer series, xanthan gum, SEPIGELEG, SIMULGELNS, aristolflex avc and aristolflex HMB.

According to the invention, the concentrate preferably further comprises an emulsifier; the mass of the emulsifier is preferably 0.5-3% of the mass of the essence, more preferably 0.8-2.5%, even more preferably 1-2%, and most preferably 1.5-2%; the emulsifier is well known to those skilled in the art, and is not particularly limited, and preferably includes any one or a combination of at least two of cetyl alcohol, coco glucoside, cetostearyl glucoside (and) cetostearyl alcohol, glyceryl stearate (and) PEG-100 stearate, arachidyl alcohol (and) behenyl alcohol glucoside, PEG-10 polydimethylsiloxane, polysorbate-20, glyceryl stearate, PEG-100 stearate, stearyl polyether-21 sorbitan stearate, cetyl potassium phosphate, sodium methyl cocoyl taurate, methyl glucose sesquistearate, C14-22 alcohol/C12-20 alkyl glucoside, sodium acrylate/sodium acryloyldimethyl taurate copolymer (and) isohexadecane (and) polysorbate-80, ammonium acryloyldimethyl taurate/VP copolymer.

According to the invention, the concentrate preferably further comprises a natural preservative; the mass of the natural preservative is preferably 0.1-0.5% of the mass of the essence, more preferably 0.2-0.4%, and still more preferably 0.3%; the natural preservative is not particularly limited as long as it is a natural preservative well known to those skilled in the art, and the present invention preferably includes but is not limited to p-hydroxyacetophenone.

According to the invention, the concentrate preferably further comprises a pH adjuster; the mass of the pH value regulator is preferably 0.01-0.1% of the mass of the essence, and more preferably 0.01-0.05%; the pH adjustor is not particularly limited as long as it is a pH adjustor well known to those skilled in the art, and the present invention preferably includes, but is not limited to, aminomethylpropanol.

The cosmetic provided by the invention comprises the spot-lightening, seal-lightening and whitening synergistic composition containing mandelic acid and having the targeted transdermal delivery function, and can be used for treating skin problems related to pigmentation.

To further illustrate the present invention, the following examples are provided to describe in detail a mandelic acid-containing spot-lightening, seal-lightening and whitening synergistic composition having a targeted transdermal delivery function, a method for preparing the same and the use thereof.

The reagents used in the examples below are all commercially available; the lecithin used in the examples was obtained from Shanghai Taiwei pharmaceutical Co., ltd; the PBS solution used in the examples was from Gibco and had a pH of 7.4; the lipase used in the examples was 9001-62-1, available from Shanghai Alasdine Biochemical technologies Co., ltd.

The anti-allergic effect of the dual anti-allergic bradykinin is explored. In order to eliminate or reduce the irritation of the phenethyl resorcinol, a plurality of anti-sensitizers are selected, and the effects of the palmitoyl tetrapeptide-7 and the acetyl dipeptide-1 cetyl ester are found to be excellent, especially the effects when the palmitoyl tetrapeptide-7 and the acetyl dipeptide-1 cetyl ester are used together are better. The relevant detection experiments and results are as follows:

1. Preparation of free whitening composition

(1) 0.02% Of nonapeptide-1, 3% of 1, 2-pentanediol, 5% of phenethyl resorcinol, 0.04% of palmitoyl tetrapeptide-7, 5% of PEG-40 hydrogenated castor oil, 3% of lecithin, 3% of caprylic/capric triglyceride, 7% of mandelic acid, 15% of 1, 3-propanediol and 3% of polyglycerol-10 diisostearate are stirred and fully mixed under the water bath condition of 40 ℃ to obtain the free whitening composition 1.

(2) 0.02% Of nonapeptide-1, 3% of 1, 2-pentanediol, 5% of phenethyl resorcinol, 0.04% of acetyl dipeptide-1 cetyl ester, 5% of PEG-40 hydrogenated castor oil, 3% of lecithin, 3% of caprylic/capric triglyceride, 7% of mandelic acid, 15% of 1, 3-propanediol and 3% of polyglycerol-10 diisostearate are stirred and fully mixed under the water bath condition of 40 ℃ to obtain the free whitening composition 2.

(3) 0.02% Of nonapeptide-1, 3% of 1, 2-pentanediol, 5% of phenethyl resorcinol, 0.02% of palmitoyl tetrapeptide-7, 0.02% of acetyl dipeptide-1 cetyl ester, 5% of PEG-40 hydrogenated castor oil, 3% of lecithin, 3% of caprylic/capric triglyceride, 7% of mandelic acid, 15% of 1, 3-propanediol and 3% of polyglycerol-10 diisostearate are stirred and fully mixed under the water bath condition of 40 ℃ to obtain the free whitening composition 3.

2. Patch test

31 Subjects were selected in the experiment and the response of the subjects was recorded. The patch of patch cream filter paper with 10% free whitening composition was placed in a patch tester with normal wells as blank. The sample and the blank are attached to the forearm curved side of the subject, the sample and the blank are uniformly attached to the skin by lightly pressing the palm, the time lasts for 24 hours, the interval is 30 minutes after the spot tester is removed, and the skin reaction is observed after the indentation disappears. The skin reaction was observed after removing the plaque tester for 24 hours and 48 hours, and the results are shown in Table 1. The patch cream comprises the following components: melting 2.0% of PEC-10 polydimethylsiloxane, 1.0% of sucrose stearate, 1.0% of stearyl alcohol, 4.5% of glycerol monostearate, 1.0% of cetostearyl alcohol and 3.0% of jojoba oil in a water bath at 75 ℃ to obtain an oil phase; dissolving 5.0% glycerol, 5.0% propylene glycol, 0.3% triethanolamine, 0.2% carbomer 2020, and 67.0% purified water in 75deg.C water bath to obtain water phase; mixing the oil phase and the water phase under stirring, emulsifying, continuously stirring and cooling to 40 ℃, adding 10% of free whitening composition, and stirring uniformly to obtain the patch cream containing 10% of high-concentration free whitening composition for patch.

TABLE 1 human skin closed Patch test results

The results in Table 1 show that the combination of palmitoyl tetrapeptide-7 and acetyl dipeptide-1 cetyl ester can synergistically improve the anti-sensitization of the phenethyl resorcinol to generate better effects, so that palmitoyl tetrapeptide-7 and acetyl dipeptide-1 cetyl ester are selected as anti-sensitization agents to prepare the spot-lightening and seal-lightening and whitening synergistic composition.

Example 1

(1) 1% Of nonapeptide-1 and 5% of 1, 2-pentanediol were dissolved in a portion of water under water bath conditions of 40℃to obtain phase A (inner water phase). The water used in this step was 10% of the total water usage.

(2) 10% Of phenethyl resorcinol (377), 1% of palmitoyl tetrapeptide-7, 1% of acetyl dipeptide-1 cetyl ester, 7% of PEG-40 hydrogenated castor oil, 5% of lecithin and 5% of caprylic/capric triglyceride are stirred and dissolved under the water bath condition of 40 ℃ to obtain phase B (oil phase).

(3) The phase A was added dropwise to the phase B at a rate of 1 drop/second in a water bath at 40℃with stirring at 15rpm during the addition, and after the completion of the mixing, phase C (water-in-oil) was obtained.

(4) 10% Mandelic acid, 20%1, 3-propanediol, 5% polyglycerol-10 diisostearate were dissolved in the remaining water at 40℃to give phase D (external aqueous phase).

(5) Dropping the phase C into the phase D at a speed of 2 drops/second in a water bath at 40 ℃, controlling the stirring speed to be 30rpm in the dropping process, and obtaining the water-in-oil-in-water system after the mixing is completed.

(6) The water-in-oil-in-water system was emulsified under high shear at 10000rpm for 1min to prepare a micro-sized fraction.

(7) And (3) carrying out high-pressure homogenization treatment on the micro-sized particles under the condition of 1500bar, and circulating for 2 times to obtain the spot-lightening and seal-lightening whitening synergistic composition.

Example 2

(1) 0.01% Of nonapeptide-1 and 1% of 1, 2-pentanediol were dissolved in a portion of water at 40℃in a water bath to obtain phase A (inner water phase). The water used in this step was 10% of the total water usage.

(2) 1% Phenethyl resorcinol (377), 0.01% palmitoyl tetrapeptide-7, 0.01% acetyl dipeptide-1 cetyl ester, 3% PEG-40 hydrogenated castor oil, 1% lecithin, 1% caprylic/capric triglyceride, and stirring and dissolving at 40deg.C in water bath to obtain phase B (oil phase).

(4) 1% Mandelic acid, 10%1, 3-propanediol, 1% polyglycerol-10 diisostearate were dissolved in the remaining water at 40℃to give phase D (external aqueous phase).

Example 3

(1) 0.03% Of nonapeptide-1, 4% of 1, 2-pentanediol was dissolved in a portion of water at 40℃in a water bath to obtain phase A (inner water phase). The water used in this step was 10% of the total water usage.

(2) 6% Of phenethyl resorcinol (377), 0.03% of palmitoyl tetrapeptide-7, 0.03% of acetyl dipeptide-1 cetyl ester, 6% of PEG-40 hydrogenated castor oil, 4% of lecithin and 4% of caprylic/capric triglyceride are stirred and dissolved under the water bath condition of 40 ℃ to obtain phase B (oil phase).

(4) 8% Mandelic acid, 18%1, 3-propanediol, 4% polyglycerol-10 diisostearate were dissolved in the remaining water at 40℃to give phase D (external aqueous phase).

Example 4

(1) 0.015% Of nonapeptide-1, 2% of 1, 2-pentanediol was dissolved in a portion of water at 40℃in a water bath to give phase A (inner aqueous phase). The water used in this step was 10% of the total water usage.

(2) 4% Of phenethyl resorcinol (377), 0.015% of palmitoyl tetrapeptide-7, 0.015% of acetyl dipeptide-1 cetyl ester, 4% of PEG-40 hydrogenated castor oil, 2% of lecithin and 2% of caprylic/capric triglyceride are stirred and dissolved under the water bath condition at 40 ℃ to obtain phase B (oil phase).

(4) 6% Mandelic acid, 12%1, 3-propanediol, 2% polyglycerol-10 diisostearate were dissolved in the remaining water at 40℃to give phase D (external aqueous phase).

Example 5

(1) 0.02% Of nonapeptide-1, 3% of 1, 2-pentanediol was dissolved in a portion of water at 40℃in a water bath to obtain phase A (inner water phase). The water used in this step was 10% of the total water usage.

(2) 5% Phenethyl resorcinol (377), 0.02% palmitoyl tetrapeptide-7, 0.02% acetyl dipeptide-1 cetyl ester, 5% PEG-40 hydrogenated castor oil, 3% lecithin, 3% caprylic/capric triglyceride, and stirring and dissolving at 40deg.C in water bath to obtain phase B (oil phase).

(4) 7% Mandelic acid, 15%1, 3-propanediol, 3% polyglycerol-10 diisostearate were dissolved in the remaining water at 40℃to give phase D (external aqueous phase).

The spot-lightening and seal-lightening synergistic composition comprises mandelic acid which is wrapped in an outer water phase, phenethyl resorcinol, palmitoyl tetrapeptide-7 and acetyl dipeptide-1 cetyl ester which are wrapped in an intermediate oil phase, and nonapeptide-1 which is wrapped in an inner water phase.

Comparative example 1

(1) 7% Of mandelic acid, 5% of phenethyl resorcinol, 5% of PEG-40 hydrogenated castor oil, 3% of lecithin, 15% of 1, 3-propanediol, 3% of polyglycerol-10 diisostearate and 3% of caprylic/capric triglyceride are mixed and dissolved in a water bath at 40 ℃ to obtain phase A (oil phase).

(2) 0.02% Of nonapeptide-1, 0.02% of palmitoyl tetrapeptide-7, 0.02% of acetyl dipeptide-1 cetyl ester and 3% of 1, 2-pentanediol are added into the balance of water, and the mixture is heated and dissolved in a water bath at 40 ℃ to obtain phase B (water phase).

(3) Phase A was added dropwise to phase B at a rate of 2 drops/sec in a water bath at 40℃with stirring at 30rpm during the addition, and phase C (oil in water) was obtained after the completion of the mixing.

(4) Phase C was emulsified by high-speed shearing at 10000rpm for 1min to prepare a micro-sized fraction.

(5) And (3) carrying out high-pressure homogenization treatment on the micro-sized particles under the condition of 1500bar, and circulating for 2 times to obtain the spot-lightening and seal-lightening whitening synergistic composition.

The content of each component in this comparative example was the same as in example 5, mandelic acid and phenethyl resorcinol were coated in the inner oil phase, and nonapeptide-1, palmitoyl tetrapeptide-7 and acetyl dipeptide-1 cetyl ester were coated in the outer aqueous phase.

Comparative example 2

(1) 0.02% Of nonapeptide-1, 7% of mandelic acid, 15% of 1, 3-propanediol and 3% of polyglycerol-10 diisostearate are dissolved in water at 40℃in a water bath to obtain phase A (aqueous phase).

(2) 5% Of phenethyl resorcinol, 0.02% of palmitoyl tetrapeptide-7, 0.02% of acetyl dipeptide-1 cetyl ester, 5% of PEG-40 hydrogenated castor oil, 3% of lecithin, 3% of caprylic/capric triglyceride and 3% of 1, 2-pentanediol are stirred and dissolved under the water bath condition of 40 ℃ to obtain phase B (oil phase).

(3) Phase B was added dropwise to phase A at a rate of 2 drops/sec in a water bath at 40℃with stirring at 30rpm during the addition, and phase C (oil in water) was obtained after the completion of the mixing.

The content of each component in the comparative example is the same as that in example 5, mandelic acid and nonapeptide-1 are wrapped in the outer water phase, and phenethyl resorcinol, palmitoyl tetrapeptide-7 and acetyl dipeptide-1 cetyl ester are wrapped in the inner oil phase.

The spot-lightening, seal-lightening, whitening and synergistic compositions obtained in the examples 1 to 5 and the spot-lightening, seal-lightening, whitening and synergistic compositions obtained in the comparative examples 1 to 2 are light brown yellow to light brown red transparent liquid, have average particle diameters of 10.0 to 100.0nm, and have PDI less than or equal to 0.400. The spot-lightening and whitening synergistic composition obtained in examples 1 to 5 and the spot-lightening and whitening synergistic composition obtained in comparative examples 1 to 2 are placed in a closed container respectively at normal temperature and at 45 ℃ for 3 months, or after freeze thawing experiments (the sample is placed in the closed container and placed at-20 ℃ for 48 hours and then placed at 45 ℃ for 48 hours and circulated twice), no delamination and precipitation phenomena occur, and the particle size is not significantly changed, so that the stability of the spot-lightening and whitening synergistic composition is good.

Comparative example 3

0.02% Of nonapeptide-1, 3% of 1, 2-pentanediol, 5% of phenethyl resorcinol, 0.02% of palmitoyl tetrapeptide-7, 0.02% of acetyl dipeptide-1 cetyl ester, 5% of PEG-40 hydrogenated castor oil, 3% of lecithin, 3% of caprylic/capric triglyceride, 7% of mandelic acid, 15% of 1, 3-propanediol and 3% of polyglycerol-10 diisostearate are stirred and fully mixed under the water bath condition of 40 ℃ to obtain the free whitening composition. The component content in this comparative example was the same as in example 5.

Comparative example 4

The preparation and formulation were the same as in example 5 except that 0.02% of palmitoyl tetrapeptide-7 and 0.02% of acetyl dipeptide-1 cetyl ester were replaced with 0.04% of palmitoyl tetrapeptide-7.

Comparative example 5

The preparation and formulation were the same as in example 5 except that 0.02% of palmitoyl tetrapeptide-7 and 0.02% of acetyl dipeptide-1 cetyl ester were replaced with 0.04% of acetyl dipeptide-1 cetyl ester.

Comparative example 6

The preparation and formulation were the same as in example 5 except that 0.02% of palmitoyl tetrapeptide-7 and 0.02% of acetyl dipeptide-1 cetyl ester were replaced with 0.04% of palmitoyl tripeptide-8.

Comparative example 7

The preparation and formulation were the same as in example 5 except that 0.02% of palmitoyl tetrapeptide-7 and 0.02% of acetyl dipeptide-1 cetyl ester were replaced with 0.04% of acetyl tetrapeptide-15.

Comparative example 8

The preparation and formulation were the same as in example 5 except that 0.02% of palmitoyl tetrapeptide-7, 0.02% of acetyl dipeptide-1 cetyl ester was replaced with 0.02% of acetyl tetrapeptide-15 and 0.02% of palmitoyl tripeptide-8.

Comparative example 9

The preparation and formulation were the same as in example 5, except that the polyglyceryl-10 diisostearate in step (4) was replaced with polyoxyethylene 40 hydrogenated castor oil.

Comparative example 10

The preparation and formulation were the same as in example 5, except that the polyglyceryl-10 diisostearate in step (4) was replaced with coco glucoside.

Test example 1 two-layer encapsulation System and in vitro Release behavior Studies

Microscopic morphology of the spot-lightening and seal-lightening synergistic composition prepared in example 5 is obtained by observation with a transmission electron microscope and is shown in fig. 1. The double-layer structure can be clearly seen through the electron microscope image.

Next, the difference in-vitro release behavior of the spot-lightening and whitening synergistic composition prepared in example 5, the spot-lightening and whitening synergistic compositions prepared in comparative examples 1, 9 and 10, and the free whitening composition prepared in comparative example 3 was investigated using a dialysis bag method. The dialysis bag (molecular weight cut-off 3500 Da) was boiled with ultra-pure water for 5min and soaked with 50% ethanol solution overnight before use. 2g of different spot-lightening and spot-lightening whitening synergistic compositions or dispersion liquid of free whitening compositions are taken and placed in a dialysis bag, two ends of the composition are sealed by dialysis bag clamps, and the composition is placed in a blue mouth bottle containing 80mL of release medium, wherein the release medium is 20% propylene glycol PBS solution (pH 7.4). The 3 groups of samples were shaken in a shaker at 37℃and 100rpm for 12h, 1mL were sampled at 1,2, 4, 6, 8, 10 and 12h, respectively, and then the same volume of release medium was supplemented. The concentration of phenylethanol (377) and mandelic acid in the samples at different time points was measured by HPLC, and the cumulative release percentages of phenylethanol (377) and mandelic acid were calculated and shown in FIGS. 2 to 3.

As can be seen from fig. 2, the release rate of phenethyl toluene diphenol in the spot-lightening and whitening synergistic compositions prepared in example 5, comparative example 1, comparative example 9 and comparative example 10 was significantly slowed, and exhibited significant slow release characteristics, compared to phenethyl resorcinol (377) in comparative example 3. The release rate of mandelic acid in the spot-lightening and whitening synergistic compositions prepared in example 5 and comparative example 1 was slower than that in comparative example 3, exhibiting remarkable slow release characteristics. The mandelic acid in comparative example 1 was in the inner oil phase, i.e., the innermost layer, and the mandelic acid in example 5 was in the outer water phase, i.e., the outermost layer, and the release rate of mandelic acid in the spot-lightening and whitening synergistic composition prepared in comparative example 1 was much slower than in example 5. The release rate of phenethyl resorcinol (377) was faster in the spot-lightening and whitening synergistic compositions prepared in comparative example 9 and comparative example 10 compared to phenethyl resorcinol (377) in example 5. The release rate of mandelic acid was faster in the spot-lightening and whitening synergistic compositions prepared in comparative example 9 and comparative example 10 than in the mandelic acid in example 5. It was demonstrated that the modified release effect of example 5 was less pronounced than that of example 5 when the polyglycerin-10 diisostearate of example 5 was replaced with polyoxyethylene 40 hydrogenated castor oil and coco glucoside of comparative examples 9 and 10.

As can be seen from FIG. 3, the rate of mandelic acid release in example 5 was significantly faster than that of phenethyl resorcinol (377), with a cumulative percent mandelic acid release of 50.9% and a cumulative percent phenethyl resorcinol release of only 14.7% at 2 hours, which also indicated that mandelic acid in the outer aqueous phase released more rapidly than phenethyl resorcinol 377 in the inner oil phase. The result shows that the whitening composition system realizes the grading release effect.

Test example 2 encapsulation efficiency and drug loading test

The encapsulation efficiency of phenethyl resorcinol (377) and mandelic acid in the whitening nano composition is determined by using an ultrafiltration centrifugation method, the spot-lightening and light-printing whitening synergistic composition prepared in example 5, comparative example 9 and comparative example 10 is diluted 5 times by using PBS solution, 0.4mL is placed in an ultrafiltration centrifuge tube (with a molecular weight cut-off of 3 kDa), the mixture is centrifuged for 15min at 5000r/min, and the filtrate is collected, and the contents of phenethyl resorcinol and mandelic acid in the spot-lightening and light-printing whitening synergistic composition are respectively determined by using an HPLC method, namely the content W _F of free phenethyl resorcinol or mandelic acid. And adding methanol into 0.4mL of diluted spot-lightening and seal-lightening whitening synergistic composition to demulsify, centrifuging, and determining the content of phenethyl resorcinol and mandelic acid in the supernatant to obtain the total phenethyl resorcinol or mandelic acid content W _T. The drug loading (Drug loading efficiency, DLE) and encapsulation efficiency (Encapsulation efficiency, EE) of the spot-lightening and seal-lightening synergistic composition were calculated as follows:

W _T: total phenethyl resorcinol or mandelic acid mass, W _F: free phenethyl resorcinol or mandelic acid mass, W _L represents nanocarrier mass.

The HPLC detection shows that the encapsulation rate of the phenethyl resorcinol in the spot-lightening and seal-lightening synergistic composition prepared in the example 5 is 92.4%, and the drug loading rate is 4.62%; the encapsulation efficiency of mandelic acid in the spot-lightening and seal-lightening and whitening synergistic composition prepared in example 5 is 88.9%, and the drug loading rate is 6.22%. The spot-lightening and seal-lightening synergistic composition prepared in comparative example 9 has an encapsulation efficiency of 61.7% of phenethyl resorcinol, a drug loading of 3.09%, an encapsulation efficiency of 54.6% of mandelic acid and a drug loading of 3.82%. The spot-lightening and seal-lightening synergistic composition prepared in comparative example 10 has an encapsulation efficiency of 64.4% of phenethyl resorcinol, a drug loading of 3.22%, an encapsulation efficiency of 57.3% of mandelic acid and a drug loading of 4.01%.

By combining the test example 1 and the test example 2, the process can realize the graded release of the double-layer packaging system and obtain higher encapsulation efficiency.

Test example 3 safety test

1. Evaluation of cell safety

The CCK-8 method determines the cellular safety of the nanocomposites. The logarithmic phase HaCaT cells at a density of 1.5X10 ⁴ cells/well, the HSF cells at a density of 1.0X10 ⁴ cells/well were collected, each well was inoculated with 100. Mu.L, and cultured at 5% CO ₂ at 37℃for 24 hours, and 100. Mu.L of DMEM complete medium (manufacturer, gibco) containing the nanocomposites prepared in example 5 or the whitening composition prepared in comparative examples 1 to 3 at different concentrations was added to each well, and the blank group was added with 100. Mu.L of DMEM complete medium alone, and 3 wells were multiplexed. After further culturing for 24 hours, the cell viability was measured by CCK-8 method, and the cell safety results are shown in FIG. 4.

Experimental results show that the spot-lightening and seal-whitening synergistic composition prepared in the example 5, the spot-lightening and seal-whitening synergistic composition prepared in the comparative example 1 and the comparative example 2 or the free whitening composition prepared in the comparative example 3 has no toxicity to HaCaT and HSF cells, and the concentration is in the range of 50-200 mug/mL; at concentrations of 400, 800, 1200 μg/mL, the HaCaT and HSF cell activities of example 5 group were significantly increased over comparative example 3. The results show that the spot-lightening and seal-lightening whitening synergistic composition prepared in the embodiment 5 of the invention can have an attenuation effect on whitening efficacy components.

2. Evaluation of chick embryo allantoic irritation (10% high concentration example safety study)

0.2ML of 10% of the spot-lightening and seal-whitening synergistic composition prepared in example 5, the spot-lightening and seal-whitening synergistic compositions prepared in comparative example 1 and comparative example 2 or the free whitening composition sample prepared in comparative example 3 (prepared by normal saline) was sucked and dropped onto the surface of chorioallantoic membrane, CAM vascular changes were observed within 5min, and the initial time of congestion, bleeding and coagulation of the CAM blood vessels were recorded, and the irritation score IS was calculated.

IS＝[(301-secH)×5+(301-secL)×7+(301-secC)×9]/300

Wherein secH is the initial time of table congestion, s; secL is the initial time of the surface bleeding, s; secC is the initial time of surface coagulation, s.

The mean value of the repeated test is calculated, and the tested object is subjected to the irritation grading according to the size of the mean value, wherein 0 to 0.9, 1.0 to 4.9, 5.0 to 8.9 and 9 to 21.0 are respectively classified into no irritation, slight irritation, medium irritation and serious irritation.

The results show that when the spot-lightening and seal-whitening synergistic composition prepared in the example 5, the spot-lightening and seal-whitening synergistic composition prepared in the comparative example 1 and the comparative example 2 or the free whitening composition prepared in the comparative example 3 is in contact with chick embryo allantois for 300 seconds, capillary vessels have no bleeding, no vascular thawing and no coagulation phenomenon, and the composition belongs to no stimulation. The safety of the spot-lightening and whitening synergistic composition prepared in example 5, the spot-lightening and whitening synergistic compositions prepared in comparative example 1 and comparative example 2 or the free whitening composition prepared in comparative example 3 after 10 times dilution is better.

3. Patch test (10% high concentration example safety study)

31 Subjects were selected in the experiment and the response of the subjects was recorded. The patch filter paper sheet of the patch cream of the spot-lightening and whitening synergistic composition prepared by 10% of the spot-lightening and whitening synergistic compositions prepared by the example 5, the comparative example 1 and the comparative example 2 or the free whitening composition prepared by the comparative example 3 is placed in a patch tester, and the normal hole is a blank control. The patch cream comprises the following components: melting 2.0% of PEC-10 polydimethylsiloxane, 1.0% of sucrose stearate, 1.0% of stearyl alcohol, 4.5% of glycerol monostearate, 1.0% of cetostearyl alcohol and 3.0% of jojoba oil in a water bath at 75 ℃ to obtain an oil phase; dissolving 5.0% glycerol, 5.0% propylene glycol, 0.3% triethanolamine, 0.2% carbomer 2020, and 67.0% purified water in 75deg.C water bath to obtain water phase; mixing the oil phase and the water phase under stirring, emulsifying, continuously stirring and cooling to 40 ℃, adding 10% of the spot-lightening and spot-lightening whitening synergistic composition or the free whitening composition, and stirring uniformly to obtain the spot-pasting cream.

The sample and the blank are attached to the forearm curved side of the subject, the sample and the blank are uniformly attached to the skin by lightly pressing the palm, the time lasts for 24 hours, the interval is 30 minutes after the spot tester is removed, and the skin reaction is observed after the indentation disappears. The skin reaction was observed after removing the plaque tester for 24 hours and 48 hours. The results are shown in Table 2.

The results showed that none of the 31 subjects had erythema, edematous erythema, significant redness, infiltration or pimple and concomitant pimple or bleb, indicating that the whitening composition in this test was non-irritating to human skin.

TABLE 2 cosmetic human skin seal Patch test results

Test example 4 target site Release behavior test

(1) Effect of Lipase on in vitro Release behavior

The effect of lipase on the in vitro release behavior of the spot-lightening and seal-whitening synergistic composition prepared in example 5 was studied by means of a dialysis bag (molecular weight cut-off 3500 Da) which was boiled with ultra-pure water for 5min and immersed overnight in a 50% ethanol solution before use. 2g of different spot-lightening and seal-lightening whitening synergistic compositions are placed in a dialysis bag, two ends of the composition are sealed by dialysis bag clamps, and the composition is placed in a blue mouth bottle containing 80mL of release medium, wherein the release medium is respectively PBS solution containing 20% of propylene glycol and PBS solution containing 0.2% of lipase and 20% of propylene glycol. Samples of the different release media were shaken in a shaker at 37℃and 100rpm for 12h, 1mL were sampled at 1,2, 4, 6, 8, 10 and 12h, respectively, and then the same volume of release medium was replenished. The concentration of phenethyl resorcinol and mandelic acid in the samples at the different time points was determined by HPLC method and the cumulative release percentage of phenethyl resorcinol (377) and mandelic acid was calculated. The experimental results are shown in FIG. 5.

Experimental results show that compared with a release medium without adding lipase, the release medium contains 0.2% of lipase by mass, and has a remarkable promotion effect on the in vitro release of phenethyl resorcinol (377) and mandelic acid in example 5. It is shown that in the presence of lipase, the grease in the spot-lightening and skin-whitening synergistic composition prepared in example 5 is hydrolyzed by lipase to release phenethyl resorcinol (377) and mandelic acid rapidly, wherein the accelerated release of mandelic acid is more remarkable, and the chinese patent publication CN 114177116B also mentions that the lipase activity is increased at the inflammation site of vaccinia, so that in this example, it can be shown that mandelic acid can be released rapidly at high concentration at the inflammation site, and thus can exert its active effect more efficiently, helping to lighten the pigmentation already formed.

Test example 5 in vitro transdermal test

(1) Determination of cumulative penetration and skin retention

The transdermal experiments of the isolated pigskin were performed using the vertical Franz diffusion cell method. The skin was fixed between the receiving chamber and the supplying chamber, and 0.5g each of the free whitening composition prepared in comparative example 3 and the spot-lightening, seal-lightening and whitening synergistic composition prepared in example 5 was placed in the supplying chamber, and was stirred and spread at 37℃with PBS (pH 7.4) as a receiving solution. 0.5mL of the receiving solution was taken at 2, 4, 6, 8, 12 and 24h and an equal amount of constant temperature fresh receiving solution was immediately replenished. HPLC analysis, the cumulative permeation amount per unit area of phenethyl resorcinol (377) and mandelic acid was calculated at different times. After 24 hours, the skin is taken off, washed, sheared and ground into homogenate, a proper amount of receiving liquid is added for centrifugation, and the supernatant liquid is taken for HPLC analysis to calculate the skin storage amount of the phenethyl resorcinol (377) and the mandelic acid in unit area. The experimental results are shown in FIG. 6.

As can be seen from FIG. 6, the cumulative permeation amounts per 24 hours of skin of 377 in comparative example 3 and example 5 were 15.6. Mu.g/cm ² and 43.7. Mu.g/cm ², respectively, and the skin storage amounts were 18.2. Mu.g/cm ² and 66.4. Mu.g/cm ², respectively. As compared with comparative example 3, the skin cumulative permeation per unit area of 377 in example 5 was increased by 180.1%, and the skin storage was increased by 264.8%. The skin cumulative permeation amounts per 24 hours of mandelic acid in comparative example 3 and example 5 were 63.4. Mu.g/cm ² and 132.8. Mu.g/cm ², respectively, and the skin storage amounts were 84.7. Mu.g/cm ² and 276.4. Mu.g/cm ², respectively. As compared with comparative example 3, the cumulative skin permeation per unit area of mandelic acid in example 5 was increased by 109.5%, and the skin storage was increased by 226.3%. The results show that the composition prepared by the process can effectively promote the transdermal absorption and the storage of the functional components in the skin and improve the skin bioavailability.

(2) Laser confocal microscope for observing skin penetration

The transdermal experiments of the isolated pigskin were performed using the vertical Franz diffusion cell method. The skin was fixed between the receiving chamber and the supply chamber, rhoB (0.1% added) was entrapped in the outer aqueous phase as a water-soluble dye, FITC (0.1% added) was entrapped in the intermediate oil phase as a lipid-soluble dye, and the corresponding compositions were prepared as described in example 5 and comparative example 3: rhoB-FITC spot-lightening and whitening synergistic composition and RhoB-FITC free whitening synergistic composition, samples of 0.5g each were placed in a supply chamber, and dispersed under stirring at 37 ℃ using PBS as a receiving solution. After 2h and 4h, the residual sample on the skin was gently wiped off, the skin in the target area was removed, the skin rinsed again, and the residual moisture was wiped off after thorough cleaning. The samples were frozen and the sections were observed by laser confocal microscopy, and representative areas were selected for photography. The results are shown in FIG. 7.

As can be seen from FIG. 7, both RhoB and FITC fluorescence intensities in the skin increased with increasing permeation time. RhoB and FITC in RhoB-FITC free whitening compositions concentrated at the stratum corneum much at 2h, failing to penetrate the stratum corneum barrier. RhoB in the RhoB-FITC spot-lightening and seal-lightening synergistic composition penetrates through the stratum corneum barrier after 2 hours, the fluorescence intensity of the FITC is enhanced compared with that of the free, and the fluorescence intensity and penetration depth of the skin are increased after 4 hours. The results show that the preparation process can promote the rapid transfer of the entrapped components to deep skin tissues.

Test example 6 target cell uptake test

(1) Observing cell uptake behavior by laser confocal microscopy

An appropriate amount of RhoB dye (0.1% of the amount added) was weighed as a fluorescent marker, and a corresponding RhoB spot-lightening and spot-lightening whitening synergistic composition and a RhoB free whitening composition were prepared in the same manner as described in example 5 and comparative example 3. B16F10 cells (target cells of melanin under the basal layer) and HaCaT cells (keratinocytes over the basal layer) in the logarithmic phase were inoculated into 35mm confocal dishes at 3×10 ⁵ cells per dish, respectively, and cultured for 24 hours, and then DMEM medium containing 0.1% rhob free whitening composition or 0.1% rhob spot-lightening, skin-lightening synergistic composition was added thereto, and incubated for 4 hours, respectively. After incubation, the medium was discarded and the cells were washed 3 times with PBS solution, fixed with 4% paraformaldehyde and stained with DAPI solution for 15min each, and photographed under 60-fold objective using a laser confocal microscope. The results are shown in FIG. 8.

As can be seen from FIG. 8, the fluorescence intensity in both B16F10 cells and HaCaT cells was significantly enhanced with prolonged incubation time. When incubated for 2 hours, the fluorescence intensity in B16F10 cells and HaCaT cells of the RhoB free whitening composition (free RhoB solution in FIG. 8) is weak, while the fluorescence of the RhoB spot-lightening and spot-lightening synergistic composition (RhoB nano-carrier in FIG. 8) enters the cells, and the fluorescence intensity is obviously stronger than that of the free RhoB. When incubated for 4 hours, the fluorescence intensity of the RhoB nano-carrier group is further enhanced, and the fluorescence intensity of B16F10 cells (melanin target cells) is stronger than that of HaCaT cells. Experimental results show that compared with the RhoB free whitening composition, the RhoB spot-lightening and whitening synergistic composition can be more ingested by B16F10 cells and HaCaT cells, and compared with the HaCaT cells, the B16F10 (melanin target cells) cells can more ingest, and the spot-lightening and whitening synergistic composition provided by the invention can effectively deliver the entrapped active substances into the skin target cells to exert the whitening effect.

(2) Flow cytometry to detect cell uptake behavior

B16F10 cells and HaCaT cells were seeded in 6-well plates at a density of 2 x 10 ⁵ cells per well and incubated for 24h to allow cell attachment. The old medium was discarded, then the composition prepared in (1) was added to each well, and after further culturing for 2 and 4 hours with untreated cells as negative control, the cells were washed with cold PBS, digested with pancreatin, centrifuged, and the cell pellet was collected and resuspended in 0.5mL of cold PBS solution, and the intracellular fluorescence intensity was measured by flow cytometry. The results are shown in FIGS. 9 and 10.

As can be seen from fig. 9, after incubation for 2h and 4h, the B16F10 cells treated with the RhoB spot-lightening and whitening synergistic composition (RhoB nanocarrier in fig. 9) had average fluorescence intensities of 4832 and 7655, respectively, which were increased by 202.4% and 112.8% respectively, compared to the RhoB free whitening composition (free RhoB in fig. 9). After incubation for 2h and 4h, the average fluorescence intensities of HaCaT cells of the groups treated with the RhoB whitening synergistic composition were 3297 and 5973 respectively, which were increased by 222.9% and 230.7% respectively compared to the RhoB free whitening composition group. Experimental results show that the whitening synergistic composition provided by the invention can remarkably improve the cellular uptake and intracellular accumulation of B16F10 cells and HaCaT cells on the inclusion compound.

As can be seen from FIG. 10, after incubation for 2h and 4h, the average fluorescence intensity of the uptake of B16F10 cells (melanocytes) was stronger than that of HaCaT cells, and the results were substantially consistent with the laser confocal uptake results described above. From this test example, it can be proved that the whitening synergistic composition of the double-layer coating system obtained by the invention can reach the target site of melanocytes of the basal layer, and can better play a role in inhibiting melanin production.

Test examples 1 to 6 show that the invention successfully prepares the spot-lightening and skin-lightening synergistic composition which has a double-layer wrapping system and can be released in a grading manner at a target position and has a targeting transdermal delivery function, and even the composition can penetrate to the target cell position more efficiently, so that the whitening effect can be more efficiently helped.

The following test examples focus on demonstrating from multiple dimensions that the dual-layer encapsulation system prepared according to the present invention has a superior whitening efficacy of the spot-lightening, mark-lightening and whitening synergistic composition with targeted transdermal delivery function, and can help to effectively combat pigmentation such as spots and acne marks.

Test example 7 cell efficacy test

(1) In vitro 2D melanocyte model validation-intracellular tyrosinase activity assay

The activity of intracellular tyrosinase was determined by L-Dopa oxidation. Mouse melanoma cells (B16F 10) were inoculated at 1X 10 ⁵ cells/mL into 24-well plates, 1mL per well, and cultured for 24 hours. The induction was performed by adding 100nM alpha-MSH (except for the normal control group) according to the experimental group, and an alpha-MSH-induced melanin high-expression model was constructed, and then the spot-lightening and seal-whitening synergistic compositions prepared in example 1, example 2 and example 5 were added at a content of 100 mug/mL, and the active concentrations of comparative example 1, comparative example 2 and comparative example 3 were the same as in example 5, with 3 duplicate wells per group. After 48h incubation, the supernatant was discarded, washed 3 times with PBS, 300. Mu.L of PBS buffer containing 1% Triton X-100 was added to each well, frozen in a refrigerator at-80℃for 1h, then thawed at room temperature, and the cell lysate was centrifuged at 12000rpm for 20min to collect the supernatant. 60. Mu.L of the supernatant was placed in a 96-well plate, 140. Mu.L of 0.1% L-Dopa was added thereto, and incubated at 37℃for 1 hour, and absorbance (A) of each well was measured at a wavelength of 490 nm. The results are shown in FIG. 11.

As can be seen from fig. 11, each of comparative examples 1, 2, 3, 1 and 5 significantly reduced tyrosinase activity compared to the model group. The effect of example 1 and example 5 on the reduction of tyrosinase activity on B16F10 cells was more pronounced compared to comparative example 1 (P < 0.01). The effect of example 1 and example 5 on the reduction of tyrosinase activity on B16F10 cells was more pronounced compared to comparative example 2 (P < 0.01). Example 1 and example 5 showed more significant reduction in tyrosinase activity on B16F10 cells (P < 0.01) compared to comparative example 3. The results showed that the tyrosinase activity of the free fraction (comparative example 3) was 57%; mandelic acid, nonapeptide-1 were coated in the outer aqueous phase (i.e., outermost layer), phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester were coated in the inner oil phase (comparative example 2) (i.e., innermost layer) with a tyrosinase activity of 43%; mandelic acid and phenethyl resorcinol are coated in the inner oil phase, and nonapeptide-1, palmitoyl tetrapeptide-7 and acetyl dipeptide-1 cetyl ester are coated in the outer water phase (comparative example 1) and the tyrosinase activity is 50%;1% nonapeptide-1, 10% phenethyl resorcinol, 1% palmitoyl tetrapeptide-7, 1% acetyl dipeptide-1 cetyl ester, 10% mandelic acid (example 1) tyrosinase activity 20%;0.01% nonapeptide-1, 1% phenethyl resorcinol, 0.01% palmitoyl tetrapeptide-7, 0.01% acetyl dipeptide-1 cetyl ester, 1% mandelic acid (example 2) tyrosinase activity was 69%; mandelic acid was entrapped in the outer aqueous phase, phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester was entrapped in the intermediate oil phase, and nonapeptide-1 was entrapped in the inner aqueous phase (example 5) with a tyrosinase activity of 34%, which was 40% lower in example 5 compared to comparative example 3. Under the condition of the same active substance concentration, the mandelic acid is wrapped in an outer water phase, the phenethyl resorcinol, the palmitoyl tetrapeptide-7 and the acetyl dipeptide-1 cetyl ester are wrapped in an intermediate oil phase, and the nonapeptide-1 is wrapped in an inner water phase, so that the effect of whitening functional components can be promoted.

(2) In vitro 2D melanocyte model validation-effect on melanogenesis in cells

The content of black in the cells was determined by NaOH cleavage. B16F10 cells were seeded at 5X 10 ⁴ cells/mL in 6-well plates with 2mL per well and cultured for 24h. The induction was performed by adding 100nM alpha-MSH (except for the normal control group) according to the experimental group, and an alpha-MSH-induced melanin high-expression model was constructed, and then the spot-lightening and seal-whitening synergistic compositions prepared in example 1, example 2 and example 5 were added at a content of 100 mug/mL, and the active concentrations of comparative example 1, comparative example 2 and comparative example 3 were the same as in example 5. After 48h incubation, the supernatant was discarded, and after 3 washes with PBS, 300. Mu.L of 1.0mmol/L NaOH solution (containing 10% DMSO) was added to each well, the cells were lysed well at 80℃for 1h, and the absorbance (A) of each well was measured at 405 nm. The results are shown in FIG. 12.

As can be seen from fig. 12, each of comparative examples 1,2, 3, 1,2 and 5 significantly reduced the intracellular melanin content compared to the model group. The effect of example 1 and example 5 on reduction of intracellular melanin content of B16F10 cells was more remarkable (P < 0.01) compared to comparative example 1. The effect of example 1 and example 5 on reduction of intracellular melanin content of B16F10 cells was more remarkable (P < 0.01) compared to comparative example 2. The effect of example 1 and example 5 on the reduction of intracellular melanin content of B16F10 cells was more remarkable (P < 0.01) compared to comparative example 3. The results showed that the free fraction (comparative example 3) had a melanin content of 69% and that mandelic acid, nonapeptide-1 were coated in the outer aqueous phase and that phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester were coated in the inner oil phase (comparative example 2) and that the melanin content was 54%; mandelic acid and phenethyl resorcinol are coated in the inner oil phase, and nonapeptide-1, palmitoyl tetrapeptide-7 and acetyl dipeptide-1 cetyl ester are coated in the outer water phase (comparative example 1) and the melanin content is 66%; mandelic acid was coated in the outer aqueous phase, phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester was coated in the intermediate oil phase, 1% nonapeptide-1, 10% phenethyl resorcinol, 1% palmitoyl tetrapeptide-7, 1% acetyl dipeptide-1 cetyl ester, 10% mandelic acid (example 1) melanin content was 24%;0.01% nonapeptide-1, 1% phenethyl resorcinol, 0.01% palmitoyl tetrapeptide-7, 0.01% acetyl dipeptide-1 cetyl ester, 1% mandelic acid (example 2) melanin content 75%; the encapsulation of the nonapeptide-1 in the inner aqueous phase (example 5) resulted in a melanin content of 43% and a 38% reduction in the melanin content of example 5 compared to comparative example 3. Under the condition of the same active substance concentration, the oil mandelic acid is wrapped in an outer water phase, the phenethyl resorcinol, the palmitoyl tetrapeptide-7 and the acetyl dipeptide-1 cetyl ester are wrapped in an intermediate oil phase, and the effect of the whitening functional components can be promoted by wrapping the nonapeptide-1 in an inner water phase.

(3) In vitro 2D inflammatory cell model verification-cell inflammatory factor determination

RAW264.7 cells were inoculated into 24-well cell culture plates at a density of 1.0X10 ⁵/mL, 0.5mL per well was used, after 24 hours of culture, 1. Mu.g/mL of LPS was added to each of the cells except for the blank group to establish a cell inflammation model, only 1. Mu.g/mL of LPS was added to the model group cells to establish a cell inflammation model, 1. Mu.g/mL of LPS and 0.5mL of the comparative examples 4 to 8 and the comparative example 5 (test concentration: 0.01%) were added to each of the sample experimental groups to prepare a spot-and whitening synergistic composition, and incubation was continued at 37℃for 24 hours. The upper culture solution was collected and the expression levels of IL-6 and TNF-. Alpha.were detected according to the instructions of ELISA kit. The results are shown in Table 3.

As can be seen from Table 3, the expression levels of IL-6 and TNF- α were significantly increased (p < 0.01) in the model group after LPS addition, compared with the blank group; compared with the model group, the spot-lightening and seal-lightening synergistic compositions prepared in comparative examples 4 to 8 and example 5 can obviously reduce the expression quantity of IL-6 and TNF-alpha (p < 0.01). The effect of the spot-lightening and whitening synergistic composition prepared in example 5 on inhibiting IL-6 and TNF-alpha secretion of Raw264.7 cells is more remarkable than that of comparative examples 4 to 8, which shows that the effect obtained in terms of inhibiting inflammation is optimal when 0.02% of palmitoyl tetrapeptide-7 and 0.02% of acetyl dipeptide-1 cetyl ester are contained in the spot-lightening and whitening synergistic composition, and is superior to that of spot-lightening and whitening synergistic compositions containing 0.02% of acetyl tetrapeptide-15 and 0.02% of palmitoyl tripeptide-8, and it is presumed that the two peptides contained in example 5 have better antiallergic and anti-inflammatory effects.

TABLE 3 influence on the expression level of inflammatory factors

Note that: ^## p <0.01 compared to the placebo group; ^** p <0.01 compared to model group; ^&& p <0.01 compared to example 5;

Test example 8 whitening efficacy test of in vitro 2D innovative zebra fish model

Screening based on whitening efficacy: T/SHRH036-2021 method for cosmetic melanin inhibition test-test of zebra fish embryo. 24-tail 8h large zebra fish embryos are exposed to 0.1% test concentration example 5 sample solution, while a blank control group, a 100% melanin inhibiting model group (30 mg/L phenylthiourea) and a positive control group (2.5 g/L kojic acid) are set, and after 48h exposure, fish embryos are subjected to microscopic photographing to measure melanin signal intensity and are subjected to statistical analysis. The results are shown in FIG. 13.

As is clear from fig. 13, example 5 has a remarkable melanin production-inhibiting effect on zebra fish embryos, and the melanin production-inhibiting rate is 21%, which is comparable to the melanin production-inhibiting effect of kojic acid having a 2.5-fold concentration. Therefore, the spot-lightening and seal-lightening whitening synergistic composition of the double-layer coating system prepared by the invention has a whitening effect.

Test example 9 whitening efficacy test of 3D skin melanin model

3D melanin skin modelPlaced in 6-well plates and incubated in a CO ₂ incubator. Negative control, positive control (kojic acid 500. Mu.g/mL) and sample group (spot-lightening and seal-lightening synergistic composition prepared in example 5, content of 5mg/mL, active substance concentration of comparative example 1, comparative example 2 and comparative example 3 same as example 5) were subjected to UVB irradiation treatment daily (50 mJ/cm ²), and blank control was not subjected to UVB irradiation. The positive control group and the sample group were administered twice on days 3 and 5, respectively, in a volume of 10. Mu.L. After 7 days of continuous model culture, the apparent brightness (L values) and melanin content of the 3D skin melanin model were tested. The results are shown in FIG. 14.

As can be seen from fig. 14 (a), the apparent brightness of each group was significantly reduced (P < 0.01) after UVB irradiation treatment compared to the blank group, and the apparent brightness was significantly improved in each of comparative example 1, comparative example 2, comparative example 3 and example 5 compared to the model group. The apparent brightness improvement of example 5 was more pronounced (P < 0.01) compared to comparative example 1. The apparent brightness improvement of example 5 was more pronounced (P < 0.01) compared to comparative example 2. The apparent brightness improvement of example 5 was more pronounced (P < 0.01) compared to comparative example 3.

As can be seen from fig. 14 (B), each group had significantly increased melanin content (P < 0.01) after UVB irradiation treatment compared to the blank group, and each of comparative example 1, comparative example 2, comparative example 3 and example 5 had significantly reduced melanin content compared to the model group. The melanin content was reduced more significantly (P < 0.01) in example 5 compared to comparative example 1. The melanin content was reduced more significantly (P < 0.01) in example 5 compared to comparative example 2. The melanin content reduction was more pronounced in example 5 (P < 0.01) compared to comparative example 3.

The results showed that the free component (comparative example 3) had a value of 61% L and a melanin content of 26 μg/mL; mandelic acid, nonapeptide-1 in the outer aqueous phase, phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester in the inner oil phase (comparative example 2) with a L value of 65% and a melanin content of 21 μg/mL; mandelic acid and phenethyl resorcinol are coated in an inner oil phase, and nonapeptide-1, palmitoyl tetrapeptide-7 and acetyl dipeptide-1 cetyl ester are coated in an outer water phase (comparative example 1) with a L-value of 63% and a melanin content of 23 mug/mL; mandelic acid was coated in the outer aqueous phase, phenethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester in the middle oil phase, and nonapeptide-1 in the inner aqueous phase (example 5) had a L-value of 71% and a melanin content of 18 μg/mL, which was a 16% improvement in L-value and a 31% reduction in melanin content in example 5 compared to comparative example 3. The oil mandelic acid is wrapped in the outer water phase, the phenethyl resorcinol, the palmitoyl tetrapeptide-7 and the acetyl dipeptide-1 cetyl ester are wrapped in the middle oil phase, and the effect of the whitening efficacy components can be promoted by wrapping the nine peptide-1 in the inner water phase.

Test example 10 peel test

After the pigskin is thawed and washed, the skin is fixed between a supply chamber and a receiving chamber of the Franz cell diffusion cell, the skin cuticle faces the supply chamber, and one side of the dermis faces the receiving chamber; after 7.0mL (PBS, pH 7.4) of receiving solution was added to the receiving chamber and the skin of the suckling pig was tightly fixed, 1.5mL of receiving solution (PBS) was added to the receiving chamber by a sampler, and air was purged to bring the dermis layer of the skin into close contact with the receiving solution. To the skin surface in the supply chamber were added 50. Mu.L of 100mg/mL salicylic acid (PBS as solvent, positive control sample), 50. Mu.L of the spot-lightening and whitening synergistic composition prepared in example 5 (mandelic acid concentration: 70 mg/mL), 50. Mu.L of the free whitening composition prepared in comparative example 3 (mandelic acid as active substance, the concentration of which was the same as in example 5) and 50. Mu.L of PBS buffer (blank control sample), respectively, and then the above samples were uniformly spread radially from the center of the skin to the edge, and the exposed area of the sample was 3.14cm ². 3 replicates of each sample were run in parallel, a constant temperature water bath was maintained at (32.+ -. 1) ℃ and the water bath sandwich was ensured to be bubble free. After incubation for 30min, the finger glove was put on, the sample application area was rubbed for two minutes, and after kneading, 0.5mL of a washing liquid (0.1% Triton X) was added to the supply chamber, and keratinocytes detached from the skin surface were blown off. The wash solution was transferred to a 1.5mL centrifuge tube and BCA protein assay was performed. Total protein content assay was performed according to BCA protein assay kit instructions. After the sample treatment, the skin surface is rubbed and washed, the eluted keratinocytes are lysed, and the total protein content in the lysate is detected, and the results are summarized in table 15.

As can be seen from fig. 15, the total protein content of the positive control exfoliated keratinocytes was significantly increased compared with the blank control group, which means that the number of exfoliated keratinocytes was significantly increased, and the test positive control detection was effective. Compared with the blank control group, the total protein content of the exfoliating keratinocytes on the skin surface of the example 5 and the comparative example 3 is obviously increased (P < 0.01), which shows that the number of the exfoliating keratinocytes is obviously increased, and the exfoliating effect of the example 5 and the comparative example 3 can be achieved by increasing the exfoliating of the keratinocytes, and the protein exfoliating lifting rate is 205.77% and 232.69%, respectively, so that in the preparation process of the invention, the exfoliating effect of the outermost layer in the example 5 can still be exerted though the outermost layer is immediately subjected to the encapsulation treatment, and the exfoliating effect of the free mandelic acid is not much different. Therefore, the double-layer coated spot-lightening and seal-lightening whitening synergistic composition prepared by the invention can well lighten melanin deposited on the epidermis horny layer, and can help the acne people to exert the effect of accelerating blackness.

Application example1 human whitening test (test concentration 1% and 3%)

The whitening composition was prepared into a whitening essence according to the formulation frame in table 4, and the human body whitening test was scheduled. 33 subjects 23 to 39 years old (27.18+/-2.95) were tested, people with facial spots and acne marks were subjected to random use of the test product for 28 days in the morning and evening on the left and right half faces of the face, the spots and the acne marks were observed, the melanin fading condition was mainly monitored, and the melanin content change was mainly monitored, and the test results are shown in table 5. From table 5, the long-term thin acne mark effect of example 5 was studied, and the effect of lightening the melanin at the acne mark and the stain was found to be improved gradually with the increase of the use time after 28 days at the two concentrations of example 5, and thus the effect of lightening the dark brown acne mark and the stain was found to be exhibited by example 5.

Table 4 whitening essence formula

Table 5 summary of 28 day whitening test results

The preferred embodiments of the present invention disclosed above are only for the purpose of helping to illustrate the present invention, but the present invention is not limited thereto. It will be understood by those skilled in the art that, within the scope of the technical concept of the present invention, the technical solutions of the present invention may be modified or some technical features may be combined in any other manner, and these modifications or combinations should not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the present invention, and should be regarded as the disclosure of the present invention and all fall within the protection scope of the present invention.

Claims

1. A spot-lightening, scar-lightening and whitening synergistic composition containing mandelic acid and having a targeted transdermal delivery function, characterized in that it is formed by an inner water phase, an oil phase and an outer water phase;

The internal aqueous phase includes an α-MSH antagonist;

The oil phase includes phenylethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, emulsifier, neutral phospholipids and oil;

The external aqueous phase includes a keratin exfoliant and a polyglycerol ester emulsifier;

The exfoliating agent includes mandelic acid;

The preparation method of the spot-lightening, scar-lightening and whitening synergistic composition containing mandelic acid and having a targeted transdermal delivery function comprises the following steps:

S1) mixing an α-MSH antagonist with water to obtain an inner aqueous phase;

Mixing phenylethyl resorcinol, palmitoyl tetrapeptide-7, acetyl dipeptide-1 cetyl ester, an emulsifier, a neutral phospholipid and oil to obtain an oil phase;

Mixing a keratin exfoliant, a polyglycerol ester emulsifier and water to obtain an external aqueous phase;

S2) adding the inner water phase dropwise to the oil phase and mixing to obtain a water-in-oil system;

S3) adding the water-in-oil system dropwise to the external aqueous phase and mixing to obtain a water-in-oil-in-water emulsion;

S4) micronizing the water-in-oil-in-water emulsion to obtain a micron-sized dispersion;

S5) subjecting the micron-sized dispersion to nano-treatment to obtain a spot-lightening, scar-lightening, whitening and synergistic composition containing mandelic acid and having a targeted transdermal delivery function;

The emulsifier is selected from polyoxyethylene hydrogenated castor oil;

The polyglycerol ester emulsifier is selected from one or more of polyglyceryl-10 myristic acid ester, polyglyceryl-10 oleic acid ester, polyglyceryl-10 dipalmitate and polyglyceryl-10 diisostearate;

The micronization process in step S4) is high-speed shearing;

The nano-processing in step S5) is a high-pressure homogenization process or a high-pressure microfluidization process.

2. The spot-lightening, mark-lightening and whitening synergistic composition according to claim 1, characterized in that the mass of the α-MSH antagonist is 0.01% to 1% of the mass of the spot-lightening, mark-lightening and whitening synergistic composition;

The mass of the phenethyl resorcinol is 1% to 10% of the mass of the spot-lightening and whitening synergistic composition;

The mass of the palmitoyl tetrapeptide-7 is 0.01% to 1% of the mass of the spot-lightening and whitening synergistic composition;

The mass of the acetyl dipeptide-1 cetyl ester is 0.01% to 1% of the mass of the spot-lightening and whitening synergistic composition;

The mass of the keratin peeling agent is 1% to 10% of the mass of the spot-lightening, scar-lightening and whitening synergistic composition.

3. The spot-lightening and whitening synergistic composition according to claim 1, characterized in that the mass ratio of phenethyl resorcinol, palmitoyl tetrapeptide-7 and acetyl dipeptide-1 cetyl ester is (1-10): (0.01-0.5): (0.01-0.5).

4. The spot-lightening and whitening synergistic composition according to claim 1, characterized in that the α-MSH antagonist is selected from one or more of nonapeptide-1, undecylenoyl phenylalanine and hexapeptide-2;

The keratolytic agent also includes salicylic acid and/or capryloyl salicylic acid.

5. The spot-lightening and whitening synergistic composition according to claim 1, characterized in that the inner aqueous phase further comprises a first polyol; the outer aqueous phase further comprises a second polyol;

The first polyol and the second polyol are each independently selected from one or more of glycerol, propylene glycol, butylene glycol, 1,2-pentanediol, 1,2-hexanediol, dipropylene glycol, PEG-10 propylene glycol and polyethylene glycol-400;

The mass of the first polyol is 1% to 5% of the mass of the spot-lightening and whitening synergistic composition;

The mass of the second polyol is 10% to 20% of the mass of the spot-lightening and whitening synergistic composition.

6. The spot-lightening and whitening synergistic composition according to claim 1, characterized in that the inner aqueous phase further comprises a first polyol; the outer aqueous phase further comprises a second polyol;

The first polyol and the second polyol are each independently selected from one or more of glycerol, 1,3-propylene glycol, butylene glycol, 1,2-pentanediol, 1,2-hexanediol, dipropylene glycol, PEG-10 propylene glycol and polyethylene glycol-400;

7. The spot-lightening, mark-lightening, whitening and synergistic composition according to claim 1, characterized in that the mass of the emulsifier is 3% to 7% of the mass of the spot-lightening, mark-lightening, whitening and synergistic composition;

The mass of the neutral phospholipid is 1% to 5% of the mass of the spot-lightening and whitening synergistic composition;

The neutral phospholipid is selected from one or more of lecithin, soybean lecithin, hydrogenated soybean lecithin, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, phosphatidylethanolamine, dioleoylphosphatidylethanolamine, cephalin, and sphingomyelin;

The mass of the oil is 1% to 5% of the mass of the spot-lightening, mark-lightening, whitening and synergistic composition;

The oil is selected from one or more of caprylic/capric triglyceride, palmitic triglyceride, isopropyl palmitate, triethylhexanoin, isopropyl myristate, ethylhexyl cocoate, ethylhexyl palmitate, isoamyl laurate, squalane, olive oil, soybean oil and jojoba seed oil;

The mass of the polyglycerol ester emulsifier is 1% to 5% of the mass of the spot-lightening and whitening synergistic composition.

8. The spot-lightening and whitening synergistic composition according to claim 1, characterized in that the dripping speed in step S2) and step S3) is independently 1-5 drops/s; the mixing speed in step S2) and step S3) is independently 10-60 rpm;

The high-speed shearing speed is 4000-10000 rpm; the high-speed shearing time is 1-10 min;

The pressure of the high-pressure homogenization treatment is 300-1600 bar; the number of cycles of the high-pressure homogenization treatment is 1-10 times; the pressure of the high-pressure microfluidization treatment is 5000-16000 psi; the number of cycles of the high-pressure microfluidization treatment is 1-10 times.

9. A cosmetic, characterized in that it comprises the spot-lightening, scar-lightening and whitening synergistic composition containing mandelic acid and having targeted transdermal delivery function as claimed in any one of claims 1 to 8.

10. The cosmetic according to claim 9, characterized in that the mass of the spot-lightening, scar-lightening and whitening synergistic composition containing mandelic acid and having targeted transdermal delivery function is 0.01% to 10% of the mass of the cosmetic.