CN107898732B

CN107898732B - Composition with whitening and spot-fading effects, application thereof and skin care product

Info

Publication number: CN107898732B
Application number: CN201711476629.3A
Authority: CN
Inventors: 刘晓英; 刘光荣; 车飚; 唐健
Original assignee: Infinitus China Co Ltd
Current assignee: Infinitus China Co Ltd
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2020-04-17
Anticipated expiration: 2037-12-29
Also published as: CN107898732A

Abstract

The invention provides a composition with whitening and spot-fading effects, application thereof and a skin care product. The composition provided by the invention comprises hydrolyzed conchiolin, lily extract, dictyophora phalloidea extract, ganoderma lucidum extract and ginger root extract. The invention selects and compounds the lily extract, the dictyophora phalloidea extract, the ganoderma lucidum extract and the ginger root extract with the hydrolyzed conchiolin, the components are reasonably formulated and cooperatively matched, and the obtained composition can effectively protect the skin from being damaged by UVB irradiation and has the effects of obviously whitening and fading spots.

Description

Composition with whitening and spot-fading effects, application thereof and skin care product

Technical Field

The invention relates to the field of cosmetics, and particularly relates to a composition with whitening and spot-fading effects, application thereof and a skin care product.

Background

In recent years, with environmental pollution, fast pace of life, rich night life and the like, more and more people are in a sub-health state for a long time, so that the phenomena of skin darkness, increased color spots, accelerated aging and the like are increasingly aggravated. People who love beauty have, smooth and white skin is the goal of people, especially women, and with the improvement of living standard of substances, people gradually pursue quality of life and pursue natural substances.

At present, skin care products on the market mainly take various natural plant extracts as effective components. Among them, the skin care products for whitening and lightening spots have various types, but have various effects, most of which have insignificant effect on removing melanin and are not ideal in whitening and lightening spots.

Disclosure of Invention

The invention aims to provide a composition with whitening and spot-lightening effects, application thereof and a skin care product.

The invention discloses a composition with whitening and spot-fading effects, which comprises the following components in parts by weight:

preferably, the composition comprises, in parts by weight:

in some embodiments provided herein, the composition comprises:

0.01 part of hydrolyzed conchiolin, 3 parts of lily extract, 5 parts of dictyophora phalloidea extract, 3 parts of lucid ganoderma extract and 1.5 parts of ginger root extract.

In some embodiments provided herein, the composition comprises:

0.05 part of hydrolyzed conchiolin, 3 parts of lily extract, 3 parts of dictyophora phalloidea extract, 5 parts of lucid ganoderma extract and 2 parts of ginger root extract.

In some embodiments provided herein, the composition comprises:

0.1 part of hydrolyzed conchiolin, 0.5 part of lily extract, 1 part of dictyophora phalloidea extract, 1 part of lucid ganoderma extract and 0.2 part of ginger root extract.

Preferably, the preparation method of the hydrolyzed conchiolin comprises the following steps:

step 1, crushing pearls, adding water, stirring to obtain pearl slurry, adding lactic acid, reacting at 95-100 ℃ for 1.5-3 h, filtering, and collecting filtrate and filter residue respectively;

step 2: adding water into filter residues, adjusting the pH value to 6.0-8.0, adding neutral protease, carrying out enzymolysis reaction at 39-41 ℃ for 2.5-4 h, filtering, and collecting filtrate;

and step 3: and (3) combining the filtrates in the step (1) and the step (2), uniformly mixing, centrifuging and freeze-drying to obtain the hydrolyzed conchiolin.

In the step 2, the pH value is preferably adjusted to 7.0, the number of times of the enzymolysis reaction is preferably 2, the temperature of the enzymolysis reaction is preferably 40 ℃, and the enzymolysis time is preferably 3 hours.

Preferably, in the composition of the present invention, the preparation method of the lily extract comprises: lily petals are used as explants, lily callus is obtained through induction culture, subculture and shake flask culture are carried out in sequence, and the lily callus is dispersed in glycerol after being cleaned, so as to obtain the lily extract. Wherein, the lily petals are preferably lily petals, and are preferably induced and cultured by an N6 culture medium to obtain lily callus.

In some embodiments of the present invention, the preparation method of the lily extract comprises:

cutting sterilized lily petals to serve as explants, and placing the explants in an N6 solid culture medium for induction culture to obtain lily callus; selecting a cell mass with a better shape from the callus for subculture, thereby obtaining a loose granular cell mass; filtering, screening out single cell or small cell group as active cell, transferring into shake flask containing culture solution, and shake-flask culturing to obtain free cell. Then placing the free cells on gauze, washing with clear water, removing impurities in the culture solution, and finally dispersing in glycerol to obtain the lily extract.

In the composition of the present invention, the dictyophora indusiata extract and the ganoderma lucidum extract are aqueous extracts, which are well known to those skilled in the art, and can be obtained commercially or by aqueous extraction according to the methods disclosed in the prior art, and the specifications thereof meet national or industrial standards.

Preferably, the dictyophora indusiata extract and the ganoderma lucidum extract are respectively obtained by extracting dictyophora indusiata and ganoderma lucidum with water. More preferably, the dictyophora phalloidea extract and the ganoderma lucidum extract are obtained by respectively extracting dictyophora phalloidea, ganoderma lucidum and ginger root with 20-30 times of water for 2 times, 1-5 hours each time, filtering and concentrating.

In some embodiments provided by the present invention, the preparation method of the dictyophora indusiata extract comprises:

extracting the dictyophora indusiata with 20-30 times of water for 2 times, 2 hours for the first time and 1 hour for the second time, combining the two extracting solutions, filtering and concentrating to constant weight to obtain the dictyophora indusiata extract.

The preparation method of the ganoderma lucidum extract comprises the following steps:

extracting Ganoderma with 20-30 times of water for 2 times, the first time for 5h, and the second time for 1h, mixing the two extractive solutions, filtering, and concentrating to constant weight to obtain Ganoderma extract.

In the composition of the present invention, the ginger root extract is an alcohol extract, which is well known to those skilled in the art, and can be obtained commercially or extracted by a method disclosed in the prior art, and the specification of the ginger root extract meets the national or industrial standard.

Preferably, the ginger root extract is obtained by ultrasonic oscillation extraction of 95% ethanol of ginger root.

In some embodiments provided by the present invention, the preparation method of the ginger root extract specifically comprises:

taking ginger root, carrying out heat treatment at 35-42 ℃ for 1h, crushing, carrying out ultrasonic oscillation extraction on 95% ethanol for 3 times, wherein each time lasts for 1h, combining extracting solutions, filtering, and concentrating filtrate to obtain the ginger root extract, wherein the mass concentration of the filtrate is 18-30 mg/ml.

The temperature of the heat treatment is preferably 40 ℃, and the filtrate is preferably concentrated to a mass concentration of 20 mg/ml.

The invention also provides application of the composition in preparing cosmetics with whitening and spot-lightening effects.

The invention also provides a skin care product with whitening and spot-fading effects, which is prepared from the composition and cosmetically acceptable auxiliary materials.

The composition with the effects of whitening and fading spots comprises the following components in parts by weight: 0.01-1 part of hydrolyzed conchiolin; 0.01-3 parts of lily extract; 0.01-5 parts of bamboo fungus extract; 0.01-5 parts of ganoderma lucidum extract; 0.01-2 parts of ginger root extract. The invention selects and compounds four traditional Chinese medicine extracts of lily extract, dictyophora indusiata extract, lucid ganoderma extract and ginger root extract and hydrolyzed conchiolin, each component is reasonably composed and cooperated, and the obtained composition can obviously reduce the expression quantity of LEF-1 and the activity of tyrosinase, thereby reducing the synthesis of melanin; and the expression level of the Laminin332 and the collagen I is improved so as to improve the barrier capability of the skin. The skin is protected from being damaged by UVB irradiation, the activity of proteasome subunits in natural aging cells can be recovered, the aging is delayed, and the whitening and spot-fading effects are remarkable.

Detailed Description

The invention discloses a composition with whitening and spot-fading effects, application thereof and a skin care product, and a person skilled in the art can realize the whitening and spot-fading effects by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention is further illustrated by the following examples:

in order to further understand the present invention, the composition with whitening and spot-lightening efficacy, the application thereof, and the skin care product provided by the present invention are illustrated below with reference to the following examples, and the scope of the present invention is not limited by the following examples.

The 3D Skin models used in the examples were all purchased from Lab Skin Creations, france; proteasome assay kit (purchased from Sigma).

Example 1 preparation of the composition

(1) Washing Margarita, oven drying, pulverizing, sieving with 100 mesh sieve, weighing 200g Margarita powder, adding small amount of deionized water, stirring in water bath to obtain Margarita slurry, adding 430ml lactic acid when the temperature is 90 deg.C, boiling, reacting at 95-100 deg.C for 2 hr, stirring, filtering after reaction, storing the filtrate separately, adding appropriate amount of deionized water into the residue, and adding Ca (OH)₂Adjusting the pH value to 7.0, adding 0.2g of neutral protease, and reacting at 40 ℃ for 3 h; then adding 0.2g neutral protease, reacting at 40 deg.C for 3h, filtering after reaction, mixing the two filtrates, mixing, ultracentrifuging, and lyophilizing to obtain hydrolyzed conchiolin.

(2) Taking lily, sterilizing, cutting sterilized lily petals to serve as an explant, and placing the explant in an N6 solid culture medium for induction culture to obtain lily callus; selecting a cell mass with a better shape from the callus tissue for subculturing to obtain a loose granular cell mass; filtering, screening out single cell or small cell group as active cell, transferring into shake flask containing culture solution, and shake-flask culturing to obtain free cell. Then placing the free cells on gauze, washing with clear water, removing impurities in the culture solution, and finally dispersing in glycerol to obtain the lily extract.

(3) Extracting Dictyophora Indusiata with 20 times of water for 2 times, 2 hr for the first time, 1 hr for the second time, mixing the two extractive solutions, filtering, and concentrating to constant weight to obtain Dictyophora Indusiata extract.

(4) Extracting Ganoderma with 30 times of water for 2 times, 5 hr for the first time, and 1 hr for the second time, mixing the two extractive solutions, filtering, and concentrating to constant weight to obtain Ganoderma extract.

(5) Taking ginger root, carrying out heat treatment at 40 ℃ for 1h, crushing, weighing 2g of crushed sample, adding 500ml of 95% ethanol, carrying out ultrasonic oscillation extraction at normal temperature for 3 times, each time for 1h, combining extracting solutions, filtering, and concentrating the filtrate in a rotary evaporator to a mass concentration of 20mg/ml at 40 ℃ to obtain the ginger root extract.

Mixing 0.01 part of hydrolyzed conchiolin, 3 parts of lily extract, 5 parts of dictyophora phalloidea extract, 3 parts of lucid ganoderma extract and 1.5 parts of ginger root extract to obtain the composition with the effects of whitening and fading spots.

Example 2 preparation of the composition

(1) Washing Margarita, oven drying, pulverizing, sieving with 100 mesh sieve, weighing 200g Margarita powder, adding small amount of deionized water, stirring in water bath to obtain Margarita slurry, adding 430ml lactic acid when the temperature is 90 deg.C, boiling, reacting at 95-100 deg.C for 2.5h, stirring, filtering after reaction, storing the filtrate separately, adding appropriate amount of deionized water into the residue, adding Ca (OH)₂Adjusting the pH value to 7.0, adding 0.2g of neutral protease, and reacting at 39 ℃ for 4 hours; adding neutral protease 0.2g, reacting at 41 deg.C for 2 hr, filtering after reaction, mixing the two filtrates, mixing, ultracentrifuging, and lyophilizing to obtain the final productObtaining the hydrolyzed conchiolin.

(2) Taking lily, sterilizing, cutting the sterilized lily petals as an explant, and placing the explant in an N6 solid culture medium for induction culture to obtain lily callus; selecting a cell mass with a better shape from the callus, and subculturing the cell mass with the better shape to obtain a loose granular cell mass; filtering the free plant cells, screening single cells or small cell groups as active cells, transferring the active cells into a shake flask filled with a culture solution for shake flask culture to obtain free cells. Then placing the free cells on gauze, washing with clear water, removing impurities in the culture solution, and finally dispersing in glycerol to obtain the lily extract.

Mixing 0.05 part of hydrolyzed conchiolin, 2 parts of lily extract, 3 parts of dictyophora phalloidea extract, 5 parts of lucid ganoderma extract and 2 parts of ginger root extract to obtain the composition with the effects of whitening and fading spots.

Example 3 preparation of the composition

0.1 part of hydrolyzed conchiolin, 0.5 part of lily extract, 1 part of dictyophora phalloidea extract, 1 part of lucid ganoderma extract and 0.2 part of ginger root extract, and the preparation method is the same as example 1, so that the composition with the effects of whitening and fading spots is prepared.

Example 4 preparation of the composition

0.05 part of hydrolyzed conchiolin, 1 part of lily extract, 2 parts of dictyophora phalloidea extract, 2 parts of lucid ganoderma extract and 1 part of ginger root extract, and the preparation method is the same as that of example 1, so that the composition with the effects of whitening and fading spots is prepared.

Example 5 preparation of the composition

The preparation method of the composition with the effects of whitening and fading the spots is the same as that of example 1 except that the hydrolyzed conchiolin 1 part, the lily extract 0.01 part, the dictyophora phalloidea extract 0.01 part, the lucid ganoderma extract 0.01 part and the ginger root extract 0.01 part are adopted.

Example 6 efficacy testing of the compositions of the invention

The formulations of the compositions of comparative examples 1 to 5 are shown in Table 1, and the preparation method is the same as that of example 1. Wherein, each test adopts a 3D skin model. The dose of UVB radiation treatment in each group is 100mJ/cm²1 time daily for 1 day.

TABLE 1 Experimental groups

The experimental items are as follows: LEF-1 expression (whitening and spot-lightening efficacy), tyrosinase, epidermal melanin (whitening and spot-lightening efficacy), collagen I (anti-aging efficacy) and Laminin332 (moisturizing efficacy).

(ii) skin Barrier Capacity test

The TCF/LEF-1 pathway regulates the barrier function of the skin by regulating epidermal differentiation and fat metabolism. This example uses a 3D skin model to perform a test for the ability to enhance the skin barrier of the composition of the invention.

The treatment was carried out in groups according to Table 1, the effective ingredient of the composition added to each treatment group was 1% of the corresponding composition, and then LEF-1 was labeled by immunohistochemistry, and the expression of LEF-1 was as shown in Table 2.

(II) tyrosinase Activity assay

Tyrosinase is the rate-limiting enzyme in melanin metabolism, and is the most important enzyme in the melanin synthesis process. The activity of tyrosinase was tested using enzymatic kinetics. The treatments were carried out according to the groups of Table 1, each treatment group incorporating a composition having an active ingredient content of 1% of the corresponding composition, according to the following test method:

after completion of the cell incubation, the medium was removed and the human melanocytes were washed with Phosphate Buffered Saline (PBS) and placed in 1% Triton X-100 (Triton X-100) solution (Sigma, France) and incubated for 10 minutes. At 10nM in the absence of Ca²⁺And Mg²⁺The enzymatic reaction was initiated by the addition of L-dopamine (Sigma, France) to the PBS solution.

After incubation for one hour at 37 ℃ in the dark, tyrosinase activity was assessed by measuring absorbance at 475nm using a spectrophotometer.

(III) skin moisturizing ability test

Lamin 332 is mainly distributed on a transparent layer, integrin α 6 β 4 on transmembrane hemidesmosome is a receptor of Laminin 5, the integrin is combined with collagen VII to form an anchor wire, the anchor wire passes through the transparent layer and a compact lower layer and is directly inserted into a papillary layer of dermis to fix the dermis, the anchor wire also forms a covalent complex with Laminin 6 or Laminin 7, and the complex (Lamin 5-6/7) in a basement membrane can directly react with type IV collagen through nestin, so the existence of Laminin332 is a main factor for stabilizing the connection stability of epidermis and dermis.

The treatments were performed according to the grouping shown in Table 1, the corresponding compositions with 1% of the effective component of the composition added to each treatment group were labeled with the immunohistochemical method, and the expression of the treated Lamin 332 was observed, and the results are shown in Table 2.

(IV) testing of collagen I

The anti-aging effect of the composition is observed by detecting the expression quantity of the collagen I, and the collagen I is a main component of the skin extracellular matrix and is also a mark of skin aging. This example uses a 3D skin model to test the composition of the invention.

The treatment was performed according to the grouping shown in Table 1, wherein the effective components of the compositions were added in the same amount (1% of the effective components of the compositions), and then the collagen was labeled by immunohistochemistry, and the expression of collagen I was shown in Table 2.

TABLE 2 analysis of the expression levels of LEF-1, Laminin332 and collagen I and tyrosinase Activity

The results show a significant decrease in LEF-1 positive cell number per epidermal cell length unit (P <0.01) and tyrosinase activity (P <0.001) for the example 1 composition + UVB irradiated group, the example 2 composition + UVB irradiated group, the example 3 composition + UVB irradiated group, the example 4 composition + UVB, the example 5+ UVB irradiated composition group compared to the UVB irradiated group; the number of Laminin332 and collagen I positive cells (P <0.01) is significantly increased.

The number of LEF-1 positive cells per epidermal cell length unit (P <0.01) and tyrosinase activity was significantly reduced (P <0.001) after treatment with the composition of example 1 compared to the composition of comparative examples 1-5; the number of Laminin332 and collagen I positive cells (P <0.01) is significantly increased.

The results show that the composition disclosed in the embodiments 1-5 can obviously reduce the activity of LEF-1 and tyrosinase, improve the expression levels of Lamin 332 and collagen I, protect skin from damage caused by UVB irradiation, and has the effects of whitening and lightening spots. Of these, example 3 added the minimum total amount, but achieved similar results to the other examples. And thus is considered to be the optimum addition ratio.

Example 7 proteasome Activity assay

This example demonstrates the anti-aging spot-lightening efficacy of the composition of the present invention by testing on proteasomes. Proteasomes are one of the mechanisms by which misfolded proteins are cleared from mammalian cells, and accumulation of misfolded proteins directly results in cellular senescence.

The extraction method of proteasome comprises the following steps:

(1) three proteasome subunits were isolated from standard aged cells (donor age: 62 years). The cell extract was centrifuged at 4 ℃ for 16 hours (1000 rcf). The pellet was dissolved in Tris-HCl buffer (25mM, pH7.5) and injected into hydrogen bromide agarose (CNBrSepharose column) containing monoclonal antibodies capable of direct purification of proteasome subunits, which had been equilibrated with buffer (Tris-HCl 25mM, pH 7.5). The column was then washed with the same buffer, and the proteasome was eluted with a Tris-HCl solution containing 2M sodium chloride (NaCl) (pH 8) and then dialyzed at 4 ℃ for 16 hours (or injected into a gel filtration column (PD10 Sephadex)).

(2) Purified protease sample was mixed with denaturing loading buffer (SDS 0.1%) and incubated at 100 ℃ for 5 minutes. The proteins were then separated by acrylamide gel (12%) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The migration of proteins at room temperature is completed within 30 minutes of 80V and 2 hours of 120V of stabilization.

(3) A final volume of 200. mu.l of a mixture containing 50. mu.g of the crude homogenate of protein or 3. mu.g of the purified proteasome (at Tris-HCl25mM pH7.5) was incubated with the lower layer at a temperature of 37 ℃ for 30 minutes. The reaction was stopped using 100. mu.l of acid or ethanol. After adding 2ml of distilled water, the fluorescence of MCA was measured using a microplate reader, and the excitation light and emission light wavelength were set to 350/440 nm.

The experiment sets up substrate group, experiment group 1~ 5: proteasome + composition group of example 1, proteasome + composition group of example 2, proteasome + composition group of example 3, proteasome + composition group of example 4, proteasome + composition group of example 5. The effective component content of each of the experimental groups 1-5 is 1% by mass of the corresponding composition.

The test method comprises the following steps: after the cells were directly added to the cell culture medium and cultured for 24 hours according to the concentration to be tested, the proteasome was extracted as described above, and the MCA fluorescence was detected using a microplate reader. Wherein proteasome activity is defined as: difference in total activity and residual activity after addition of 20 μ M proteasome inhibitor. The results are shown in Table 3.

TABLE 3 proteasome Activity assays

Note: denotes p < 0.05 compared to the substrate group.

The results show that the cells treated with the compositions of examples 1 to 5 of the present invention restored proteasome subunit activity in naturally senescent cells (donor age: 62 years), and they were significantly different from the substrate group (p < 0.05). Of these, example 3 added the minimum total amount, but achieved similar results to the other examples. And thus is considered to be the optimum addition ratio. The comparative examples 1 to 5 have no significant difference from the substrate group.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The composition with the effects of whitening and fading spots is characterized by comprising the following raw materials in parts by weight:

0.1-1 part of hydrolyzed conchiolin;

0.01-1 part of lily extract;

0.01-2 parts of bamboo fungus extract;

0.01-2 parts of ganoderma lucidum extract;

0.01-1 part of ginger root extract;

the preparation method of the lily extract comprises the following steps:

lily petals are used as explants, lily callus is obtained through induction culture, subculture and shake flask culture are carried out in sequence, and the lily callus is dispersed in glycerol after being cleaned to obtain a lily extract;

the dictyophora phalloidea extract and the lucid ganoderma extract are respectively obtained by extracting dictyophora phalloidea and lucid ganoderma with water;

the ginger root extract is obtained by extracting ginger root with 95% ethanol.

2. The composition according to claim 1, characterized by consisting of the following raw materials:

0.1 part of hydrolyzed conchiolin;

0.5 part of lily extract;

1 part of bamboo fungus extract;

1 part of ganoderma lucidum extract;

0.2 part of ginger root extract.

3. The composition according to claim 1 or 2, wherein the hydrolyzed conchiolin is prepared by a method comprising:

4. The composition as claimed in claim 1, wherein the extraction of ginger root extract is ultrasonic vibration extraction.

5. Use of the composition according to any one of claims 1 to 4 for preparing a cosmetic with whitening and spot-lightening effects.

6. A skin care product with whitening and spot-fading effects is characterized by being prepared from the composition as claimed in any one of claims 1 to 4 and cosmetically acceptable auxiliary materials.