A uniform skin lightening composition, its preparation method and use

Abstract

The application discloses a kind of even skin tone whitening composition and its preparation method and application, and the even skin tone whitening composition includes: zephyranthes grandiflora extract, north American pinus strobus bark extract, sea freesia extract, synergistic effect exists between composition components, whole-path whitening, improve the efficacy of active substance.The composition inhibits tyrosinase activity, inhibits melanosome transfer to keratinocyte, inhibits melanin release to keratinocyte in this path, and also decomposes melanin into amino acid that can be used by human body by melanocyte autophagy to even skin tone, whitening skin.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of A61K8 / 9789, in particular to a uniform skin color whitening composition and its preparation method and application. BACKGROUND

[0002] The pursuit of whitening skin is an eternal topic for people in the Asia-Pacific region. In order to cater to the increasing whitening market, the efficacy of whitening skincare products has become the focus of researchers and consumers. The color of the skin is mainly determined by the content and distribution of skin pigments, and melanin is the most important determinant. The secretion of melanin by melanocytes is an extremely complex biological process, involving the occurrence of melanocytes, dendrite formation, melanosome maturation, and transport mechanisms, and is closely related to keratinocytes and extracellular matrix, etc.

[0003] At present, general skin whitening agents mainly inhibit tyrosinase activity or block the synthesis pathway of tyrosinase to generate melanin, thereby reducing the production of melanin to achieve the effect of whitening. Such skin whitening agents mainly include arbutin, hydroquinone and its derivatives, L-ascorbic acid and its derivatives, kojic acid and its derivatives, vitamin C ethyl ether and its derivatives, nicotinamide, trisaminic acid, benzenediol and its derivatives, etc.

[0004] CN111388382A discloses a composition for whitening and lightening skin and its application in cosmetics. The composition includes the following components: glabridin carrier, hydroxyethylpiperazine ethane sulfonic acid (HEPES), endothelin antagonist, plant complex essence. The whitening and lightening skin composition can effectively inhibit tyrosinase activity, inhibit melanocyte differentiation, scavenge free radicals, soften keratin, and improve skin resistance, etc. CN106236674B discloses a composition with whitening effect and its application in cosmetics. The composition includes: methyl hydantoin, imide, nicotinamide, thyme extract, citrus peel extract, friedelin, the balance being auxiliary materials and water, and the composition is through inhibiting tyrosinase activity, antioxidant, scavenging free radicals, and lightening skin color. The above-mentioned patent discloses a formula commonly used in most products on the market. The formula realizes the effect of whitening by inhibiting tyrosinase activity or blocking the synthesis pathway of tyrosinase to generate melanin, and the transfer pathway of melanin, while ignoring the transport and degradation pathway of melanin after synthesis. For the generated melanin and pigmented spots, excessive exfoliation of keratin can easily damage the skin barrier, and autophagy mechanism can degrade the generated melanin to reduce pigmentation. Melanocytes can digest and remove excess melanin through autophagy, reducing its excretion to the epidermis, and keratinocytes in the bottom layer can also digest melanin bodies transferred into them through autophagy, reducing their transfer to the epidermis. Activating autophagy to degrade the generated melanin and digest melanin bodies provides a new whitening pathway for skin whitening in addition to inhibiting the synthesis and transfer of melanin.

[0005] To solve the above problems, the present application provides a skin-whitening composition for uniform skin color, which degrades the already generated melanin and melanin transfer pathway through autophagy in addition to inhibiting tyrosinase activity or blocking the synthetic pathway of melanin generated by tyrosinase, thereby achieving an effective whitening effect. SUMMARY

[0006] The present application provides a skin-whitening composition, which comprises a Zantedeschia aethiopica extract, a Pinus strobus bark extract, and a Pancratium zeylanicum extract.

[0007] As a preferred technical solution, the mass ratio of the Zantedeschia aethiopica extract, the Pinus strobus bark extract, and the Pancratium zeylanicum extract is (5-20):(1-5):(0.1-1).

[0008] As a preferred technical solution, the mass ratio of the Zantedeschia aethiopica extract, the Pinus strobus bark extract, and the Pancratium zeylanicum extract is (10-15):(2-4):(0.1-1).

[0009] As a preferred technical solution, the mass ratio of the Zantedeschia aethiopica extract, the Pinus strobus bark extract, and the Pancratium zeylanicum extract is (10-15):(2-4):(0.1-0.5).

[0010] As a preferred technical solution, the mass ratio of the Zantedeschia aethiopica extract, the Pinus strobus bark extract, and the Pancratium zeylanicum extract is 15:4:0.45 or 15:2:0.5.

[0011] The Zantedeschia aethiopica extract is obtained by extracting Zantedeschia aethiopica flowers and Zantedeschia aethiopica stems.

[0012] The Zantedeschia aethiopica extract can reduce the already generated melanin and inhibit the generation of melanin: induces autophagy of melanin - surrounds the melanin particles with phagocytic vesicles containing LC3I protein, begins to form autophagosomes (LC3I protein is converted into LC3II protein), and the lysosome is combined with the p62 protein on the outer wall of the autophagosome, and the lysosomal enzyme completely decomposes the melanin into individual amino acids; and inhibits the metabolic activity of tyrosine by adjusting the path of melanocyte-stimulating hormone a-MSH.

[0013] The Pinus strobus bark extract can directly and indirectly inhibit melanin generation: directly reduces melanin generation by inhibiting tyrosinase activity; indirectly reduces melanin generation by reducing the expression of endothelin-1 receptors and reducing the PKC pathway between cells; and indirectly reduces melanin generation by reducing the synthesis of stem cell factors and reducing the inducing factors for melanin formation. Meanwhile, the Pinus strobus bark extract can also have antioxidant and protein carbonylation reduction effects, and can maintain the transparency of the skin, thereby achieving a skin lightening effect.

[0014] The sea cyclamen extract can inhibit the expression of pro-opiomelanocortin (POMC), because POMC can promote the release of adrenocorticotropic hormone (ACTH), which is partially converted into tyrosinase kinase (a-MSH) to promote melanogenesis, so it can achieve the effect of inhibiting the most front melanogenesis. At the same time, by reducing the length of the dendrites of melanocytes and inhibiting the synthesis of P substance receptors on the surface of the dendrites, the communication between nerves and melanocytes can be reduced, thereby reducing the release of melanin.

[0015] The inventors unexpectedly found that the composition including the mass ratio of Zephyranthes grandiflora extract, North American Pinus strobus bark extract, and sea cyclamen extract is (5-20):(1-5):(0.1-1), and the components have a synergistic effect, which can enhance the whitening effect. The inventors believe that the possible reason is that the whitening composition acts together to inhibit tyrosinase activity, activate autophagic lysosomes of keratinocytes, and degrade the generated melanin through autophagy. At the same time, by reducing the length of the dendrites of melanocytes and reducing the synthesis of P substance receptors, the communication between nerves and melanocytes can be reduced, thereby reducing the transport and release of melanin.

[0016] The second aspect of the present application discloses a preparation method of the composition, including the preparation of Zephyranthes grandiflora extract; the preparation of North American Pinus strobus bark extract; the preparation of sea cyclamen extract; and the preparation of the composition.

[0017] As a preferred technical solution, the preparation of Zephyranthes grandiflora extract includes: weighing the flowers and stems of Zephyranthes grandiflora, heating and extracting with ethanol to obtain an extract solution, filtering the extract solution with gauze to obtain a primary filtrate, filtering the primary filtrate with a microfiltration membrane to obtain a filtrate, evaporating the solvent in the filtrate, and then adding one or more of deionized water, 1,2-hexanediol, glycerol, ethanol, butanediol, and decyl glucoside to obtain the Zephyranthes grandiflora extract.

[0018] As a preferred technical solution, the ethanol is 20wt%-90wt% ethanol.

[0019] As a preferred technical solution, the ethanol is 80wt% ethanol.

[0020] As a preferred technical solution, the mass ratio of the total mass of the flowers and stems of Zephyranthes grandiflora to the mass of ethanol is 1:(3-10); preferably 1:5.

[0021] As a preferred technical solution, the extraction process is carried out at a temperature of 10-100℃ for 1-3h.

[0022] As a preferred technical solution, the gauze used is 100-200 mesh.

[0023] As a preferred technical solution, the microfiltration membrane is 1 μm-0.2 μm.

[0024] As a preferred technical solution, the preparation of the North American Pinus strobus bark extract comprises: de-characterizing the North American Pinus strobus bark, performing solid-liquid extraction with deionized water at 40-100 DEG C, then standing and precipitating, centrifuging to obtain a centrifugal liquid, filtering the centrifugal liquid, then adding malt dextrin for vacuum concentration, drying (atomizing) the concentrated liquid to obtain the North American Pinus strobus bark extract.

[0025] As a preferred technical solution, de-characterizing the North American Pinus strobus bark means sterilizing the North American Pinus strobus bark, specifically: sterilizing the white pine bark by dry steam pasteurization (cycle 5-10 minutes, temperature 80-120 DEG C), then vacuum packaging the sterilized raw material until the extraction step.

[0026] As a preferred technical solution, the centrifugation process is completed on a centrifuge at a speed of 3000-5000 r / min for 5-15 min.

[0027] As a preferred technical solution, the drying is spray drying, the inlet air temperature is 120-180 DEG C, the gas flow is 80-100%, and the pump speed is 10-30%.

[0028] As a preferred technical solution, the preparation of the Amaryllis vittatum extract comprises: immersing and extracting the Amaryllis vittatum in solvent A, then clarifying, sterilizing and filtering the clarified liquid to obtain a primary filtrate, adding solvent B to the primary filtrate, then sterilizing and filtering again to obtain the Amaryllis vittatum extract.

[0029] As a preferred technical solution, the solvent A is selected from one or more of deionized water, 1,2-hexanediol, glycerol, ethanol, and butanediol.

[0030] As a preferred technical solution, the solvent B is selected from one or more of deionized water, 1,2-hexanediol, glycerol, ethanol, and butanediol.

[0031] As a preferred technical solution, the filter cloth used for sterilization and filtration has an average pore size of 1 μm-0.3 μm.

[0032] As a preferred technical solution, the preparation of the composition comprises: adding the obtained North American Pinus strobus bark extract and Amaryllis vittatum extract to the Zephyranthes grandiflora extract in proportion, and mixing to obtain the composition.

[0033] The application also provides the use of the composition, which is applied to cosmetics, including but not limited to water, emulsion, spray, cream, mask, and foundation.

[0034] Beneficial effects

[0035] The whitening composition of the present application has the effects of inhibiting the activity of tyrosinase, activating autophagic lysosomes of keratinocytes, and degrading the generated melanin through cellular autophagy. Meanwhile, the communication between nerves and melanocytes is reduced by reducing the length of the dendrites of melanocytes and reducing the synthesis of substance P receptors, thereby reducing the transport and release of melanin. The components have a synergistic effect, act on all pathways of whitening, and can enhance the whitening effect. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is the change test chart of ITA Standard Deviation;

[0037] Figure 2 is the change test chart of facial melanin content before and after use. DETAILED DESCRIPTION

[0038] The raw materials and equipment used in the present application are conventional raw materials and equipment in the art unless otherwise specified, and are all from commercially available products. The methods used in the present application are conventional methods in the art unless otherwise specified.

[0039] There are still other various implementable technical solutions in the present application, which are not listed one by one, and the technical solutions claimed in the claims of the present application are all implementable.

[0040] In the following examples and comparative examples of the present application, the composition having the effects of barrier repair and anti-aging is prepared by the following process:

[0041] (1) Preparation of Zantedeschia aethiopica extract: Zantedeschia aethiopica (flower / stem) is extracted with 80wt% ethanol at 90°C for 3h, the extract is filtered with 100-mesh gauze to obtain a primary filtrate; then the primary filtrate is filtered with a microfiltration membrane of 0.2μm-1μm to obtain a filtrate; then the solvent in the filtrate is evaporated, and then 1,2-hexanediol and decyl glucoside are added, the mass ratio of Zantedeschia aethiopica extract, decyl glucoside and 1,2-hexanediol is 78:20:2, to obtain Zantedeschia aethiopica extract;

[0042] (2) Preparation of Pinus strobus bark extract: Pinus strobus bark is de-characterized, i.e. the white pine bark is sterilized by dry steam pasteurization (cycle: 6 minutes, temperature: 115°C), and then extracted with 60°C deionized water, and then allowed to stand and precipitate, and then centrifuged at 4000r / min for 10min to obtain a centrifugal liquid; the centrifugal liquid is filtered, and then maltodextrin is added to the filtrate and vacuum concentrated; the concentrated liquid is dried (atomized) at an air inlet temperature of 130°C, a gas flow rate of 100% and a pump speed of 20% to obtain Pinus strobus bark extract;

[0043] (3) Preparation of sea daffodil extract: Sea daffodils were soaked in deionized water for extraction and then allowed to stand to clarify. The clarified liquid was sterilized and filtered to obtain the initial filtrate. Deionized water and glycerol were added to the initial filtrate. The mass ratio of sea daffodil extract, glycerol and water was 1:29:70. The mixture was sterilized and filtered again (0.22μm) to obtain sea daffodil extract.

[0044] (4) Add the North American pine bark extract and sea daffodil extract to the calla lily extract in proportion, stir and mix well to obtain a composition with skin whitening effect.

[0045] Table 1 shows the composition ratios of each composition in compositions 1-12 and comparative examples 1-3 (the composition ratios of calla lily extract, North American pine bark extract, and sea daffodil extract are by mass). The compositions and comparative examples were prepared according to Table 1 and the methods described above, and the efficacy of the obtained compositions was tested.

[0046] Table 1

[0047]

[0048] Performance testing

[0049] 1. Cytotoxicity Assay: MTT is a yellow powdered chemical reagent widely used for the detection of cytotoxicity or cell proliferation. Its detection principle is that succinate dehydrogenase in the mitochondria of living cells reduces MTT to water-insoluble blue-purple formazan crystals, which are deposited in the cells. Dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells. The number of living cells is determined based on the measured absorbance value. The specific experimental method is as follows:

[0050] In a 96-well plate, 1×10⁻⁶ ppm per well. 4 100 μL each of DMEM medium containing 10% bovine serum and human immortalized epidermal cells (HaCaT) were seeded at a density of 100 μL and cultured for 24 hours. The culture was then replaced with serum-free medium. The skin-whitening compositions prepared in compositions 1-12 and comparative examples 1-3 were added to the serum-free medium, and the cells were cultured for 24 hours. The medium was then removed, and the cells were treated with 20 μL of MTT solution (phosphate buffer, concentration 5 mg / ml) and cultured for 4 hours. 150 μL of LDMSO was added to the cells after removing the MTT solution, and the mixture was incubated at 37°C for 4 hours until the formazan crystals were completely dissolved. The absorbance was measured at 570 nm, and cell viability was calculated using the following formula.

[0051] Cell viability (%) = (Sample absorbance value / Average absorbance value of control group) × 100%

[0052] The control group did not include any sample in the experiment. Cytotoxicity-related results are shown in Table 2.

[0053] Table 2. Cell viability

[0054]

[0055]

[0056] 2. Assay for Tyrosinase Inhibition: Add phosphate buffer (pH 6.8), compositions 1-12, and the skin-whitening compositions prepared in Comparative Examples 1-3 to the test tubes sequentially as shown in Table 3. Incubate at 35°C for 10 minutes. Then add the tyrosinase solution, mix well, and incubate at 35°C for 30 minutes. Quickly transfer the mixture to a cuvette and measure the absorbance at 475 nm. Zero the absorbance using blank control 1, blank control 2, and blank control 3 for the experimental group, negative control, and positive control, respectively.

[0057] The concentration of the composition and arbutin solution used in the experiment was 1 mg / mL.

[0058] Tyrosinase inhibition rate = [(AB) / A] × 100%

[0059] A represents the absorbance value of the negative control, and B represents the absorbance value of the experimental group or the positive control.

[0060] Table 3

[0061]

[0062] The inhibition rates of tyrosinase by compositions 1-12 and comparative examples 1-3 are shown in Table 4.

[0063] Table 4

[0064]

[0065]

[0066] Table 4 shows the experimental results:

[0067] (1) The compositions of compositions 1-12 and comparative examples 1-3 all have the ability to inhibit tyrosinase activity;

[0068] (2) Comparative compositions 1-12 and comparative examples 1-3 show that there is a synergistic effect among the components of calla lily extract, North American pine bark extract and sea daffodil extract;

[0069] (3) The inhibition rate of tyrosinase showed a certain dose-response relationship with all three raw materials. After the mass fraction increased to a certain range, the upward trend slowed down. Comparing compositions 1-4, it was found that the inhibition rate of tyrosinase in compositions 3 and 4 was not significantly different, indicating that the effect of calla lily extract did not increase significantly after the mass fraction was greater than 150. Comparing compositions 3, 5, and 6, it was found that the inhibition rate of tyrosinase in compositions with a mass fraction of 2 and 4 of North American pine bark extract was not significantly different. Comparing compositions 6, 9, and 12, it was found that the inhibition rate of tyrosinase in compositions with a mass fraction of 0.45 and 0.5 of sea daffodil extract was not significantly different. Therefore, considering the inhibitory ability of tyrosinase and economic benefits, compositions 9 (15:4:0.45) and 11 (15:2:0.5) are preferred.

[0070] In summary, different proportions of the three raw materials correspond to different tyrosinase inhibitory effects. The preferred mass ratio of calla lily extract, North American pine bark extract, and sea daffodil extract is (10-15):(2-4):(0.1-0.5). More preferably, the mass percentages of calla lily extract, North American pine bark extract, and sea daffodil extract are 15:4:0.45 and 15:2:0.5.

[0071] 3. Autophagy Activity Assay: GFP fluorescent protein was labeled onto LC3 protein as a marker for autophagy activity. A GFP-LC3 cell line (B16F1 / GFP-LC3) was obtained from the B16F1 melanoma cell line. In the autophagy mechanism, LC3 is dissolved in the cytoplasm, also known as LC3-I. LC3-I is activated by Atg7, translocates to Atg3, and eventually binds to PE, forming LC3-PE. This lipid-modified form of LC3 is also known as LC3-II. LC3-II is located on autophagosomes. LC3-II is present throughout the entire process from autophagosome formation to lysosomal binding, and is only degraded by acidic hydrolases in the lysosome after fusion. Therefore, LC3-II is widely recognized as a marker for monitoring autophagosomes and autophagy activity.

[0072] Compositions 1-12 and Comparative Example 1 were used to treat B16F1 / GFP-LC3 cells at a concentration of 100 ppm. The AU reference value is the fluorescence intensity value of untreated B16F1 / GFP-LC3 cells. The higher the AU value, the more effectively the produced melanin is broken down into individual amino acids. On the other hand, if ineffective, AU represents a reference value of 1. The specific results are shown in Table 5.

[0073] Table 5

[0074]

[0075] Test results show that:

[0076] (1) Arbutin has no effect on autophagy;

[0077] (2) Experimental results of compositions 1-12 show that the skin whitening compositions have a significant autophagy effect. Considering cell autophagy capacity and economic benefits, the preferred examples are compositions 9 and 11.

[0078] 4. Safety Test: Add 20 μl of the test solution containing the composition to the patch applicator, with a blank control (pure water) in the control well. Apply the patch applicator containing the test substance to the flexor side of the subject's forearm, gently pressing it with the palm to ensure even application to the skin, and leave it on for 24 hours. Observe skin irritation and sensitization according to Table 4 at 30 min, 24 h, and 48 h after removing the patch applicator, and record the results. The grading criteria for skin reaction in the closed patch test are shown in Table 6.

[0079] Table 6. Grading Criteria for Skin Reactions in Closed Patch Tests

[0080]

[0081] Experimental Results: Human skin patch tests were conducted on the compositions obtained in proportions 9 and 11, and the results are shown in Table 7. The human skin patch test results showed that no adverse skin reactions were observed in 30 participants, indicating that compositions 9 and 11 were safe and non-irritating.

[0082] Table 7

[0083]

[0084] 5. Human testing: To test the whitening and skin-evening effect of the composition in the serum, Sample 1 and Sample 2 were applied to the left and right cheeks of 15 female volunteers, respectively.

[0085] 1. Facial ITA Standard Deviation was measured using a C-Cube (PIXIENCE, France) in a laboratory environment at 21±1℃ and 50±5% relative humidity. A lower ITA Standard Deviation indicates a more even skin tone. Test results are as follows: Figure 1 .

[0086] Test results showed that the ITA Standard Deviation of Sample 1 and Sample 2 was reduced by 15.21% and 16.58% respectively, with a significant effect on even out skin tone.

[0087] 2. Using a Mexameter (CK, Germany), the facial melanin content of volunteers was tested before and 28 days after product use. The higher the melanin value, the higher the melanin content in the skin; the lower the melanin content, the whiter the skin.

[0088] Test results are as follows Figure 2 .

[0089] The results showed that after four weeks of use, the melanin content in the face of samples 1 and 2 was significantly reduced, demonstrating a noticeable whitening effect.

[0090] Content not described in detail in this application specification is common knowledge to those skilled in the art. The foregoing description illustrates and describes several preferred embodiments of this application; however, as mentioned above, it should be understood that this application is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the inventive concept described herein through the foregoing teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of this application should be within the protection scope of the appended claims.

Claims

1. A skin-whitening composition for even skin tone, characterized in that, Including calla lily extract, North American pine bark extract, and sea daffodil extract; The preparation of the calla lily extract includes: weighing the calla lily flowers / stems, heating and extracting them with a 20-90 wt% ethanol aqueous solution to obtain an extract, filtering the extract with gauze to obtain a primary filtrate; filtering the primary filtrate with a microfiltration membrane to obtain a filtrate, evaporating the solvent in the filtrate, and then adding one or more of deionized water, 1,2-hexanediol, glycerol, ethanol, butylene glycol, and decyl glucoside to obtain the calla lily extract; The preparation of the North American pine bark extract includes: decharacterizing the North American pine bark, performing solid-liquid extraction with deionized water at 40-100℃, then allowing it to settle, centrifuging to obtain a centrifuged liquid, filtering the centrifuged liquid, adding maltodextrin for vacuum concentration, and drying the concentrate to obtain the North American pine bark extract; the decharacterizing of the North American pine bark is achieved by sterilizing the pine bark with dry steam pasteurization; Sea daffodils were extracted by soaking in deionized water and then clarified. The clarified liquid was sterilized and filtered to obtain the initial filtrate. Solvent B was added to the initial filtrate and then sterilized and filtered again to obtain sea daffodil extract. The solvent B is selected from one or more of deionized water, 1,2-hexanediol, glycerol, ethanol, and butanediol; The mass ratio of the calla lily extract, the North American pine bark extract, and the sea daffodil extract is 20:1:0.1, 15:4:0.1, 15:1:0.45, 15:4:0.45, 15:2:0.45, 15:1:0.5, 15:2:0.5, or 15:4:0.

5.

2. The method for preparing the composition according to claim 1, characterized in that, This includes the preparation of calla lily extract; the preparation of North American pine bark extract; the preparation of sea daffodil extract; and the preparation of compositions.

3. The preparation method according to claim 2, characterized in that, The preparation of the composition includes: adding the obtained North American pine bark extract and sea daffodil extract to calla lily extract in proportion, and mixing well to obtain the composition.

4. The application of the composition according to claim 1, characterized in that, The composition is used in cosmetics.

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