CN118178243A

CN118178243A - Skin whitening composition containing semi-licorice isoflavone as active ingredient and application thereof

Info

Publication number: CN118178243A
Application number: CN202410441309.8A
Authority: CN
Inventors: 刘海亮; 吕书瑶
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2024-04-12
Filing date: 2024-04-12
Publication date: 2024-06-14

Abstract

Disclosed is a composition for skin whitening, which comprises a semi-licorice isoflavone as an active ingredient. The invention also discloses application of the composition for whitening skin in preparing a product for whitening skin and lightening spots. The skin whitening composition containing the semi-licorice isoflavone has the capability of remarkably inhibiting melanin formation and higher tyrosinase inhibition activity; has the effect of improving the whiteness of the skin, and can be better used for whitening the skin and lightening the spots. Has no toxic or side effect on kidney, liver and lung, and is safe and effective.

Description

Skin whitening composition containing semi-licorice isoflavone as active ingredient and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a composition for whitening skin, which contains semi-licorice isoflavone as an active ingredient and application thereof.

Background

In the current situation that natural environment is getting worse and life pressure is continuously increasing, with the age, skin darkness, pigmentation, chloasma and other bad symptoms are more and more seen, the improvement of skin color becomes the field of more and more important for modern people, about 15% of population invests in whitening products worldwide, and the research and development of skin care products with whitening effect are also focused; clinicians also apply this to the treatment of pigment-increasing skin disorders, such as: chloasma, post-inflammatory pigmentation, etc. The melanin metabolism of ① as a main factor affecting the skin color; ② Impaired skin barrier, thinning of the stratum corneum or increased transcutaneous water loss (TRANS EPIDERMAL WATER loss, TEWL), dark skin tone; ③ Blood circulation stasis of the skin and dark complexion; ④ The accumulation of oxygen radicals promotes melanin synthesis and accelerates skin aging; ⑤ The inflammatory response induces pigmentation.

Melanin production has been the focus of research for decades. Melanin is synthesized by unique melanosomes in melanocytes and is the end product of melanin formation that determines the color of mammalian skin, hair and eyes. The synthesized melanin is transported to nearby keratinocytes (keratinocyte, KC) through dendrites of melanocytes (melanoncyte, MC) and then diffuses in the epidermis. Melanosomes have a range of mechanisms that regulate melanin synthesis, with the "three enzyme theory" being widely accepted in melanin formation. "Trienzyme" refers to Tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2), wherein TYR is a key enzyme that coordinates melanin formation. In the melanogenesis process, L-tyrosine is first hydroxylated by TYR to L-hydroxyphenylalanine (L-DOPA), then L-DOPA is oxidized to the corresponding o-quinone, which is then oxidized to DOPA chrome, which catalyzes the formation of 5, 6-dihydroxyindole-2 carboxylic acid (DHICA) by the tautomerization reaction of TRP-2. Finally, TRP-1 oxidizes DHICA to carbonylated indoloquinone and produces melanin.

Various extracellular stimuli, such as Ultraviolet (UVR) radiation, 3-isobutyl 1-methylxanthine (IBMX), alpha-melanocyte stimulating hormone (alpha-MSH), wnt, stem Cell Factor (SCF), nitric Oxide (NO), etc., affect melanin formation. The signal pathways common to melanin synthesis are the adenylate cyclase/cAMP dependent protein kinase pathway, the Mitogen Activated Protein Kinase (MAPK) pathway, the phosphatidylinositol 3 kinase/protein kinase B (PI 3K/Akt) pathway, the Wnt/beta-catenin pathway, and the nitric oxide/cyclic guanosine monophosphate/protein kinase G (NO/cGMP/PKG) pathway. The microphthalmia-associated transcription factor (microphthalmia transcription factor, MITF) is the ultimate target for a variety of signaling pathways, the primary regulator of melanogenesis. MITF contains a helix-loop-helix leucine zipper structure that modulates melanin synthesis by activating TYR, TRP1 and TRP2 gene transcription in combination with a promoter sequence containing an M-box or E-box palindromic structure (5 '-TCATGTG-3' or 5 '-CACGTG-3'). Also, studies have shown that MITF is also closely related to reproduction and apoptosis of melanocytes, maturation of melanosomes, and metastasis. Therefore, the expression of MITF is down regulated, which can effectively reduce melanin synthesis and achieve the whitening effect.

Oxygen free radicals in the human body mainly comprise hydroxyl free radicals, superoxide anion free radicals, hydrogen peroxide H2O2, singlet oxygen and lipoxygenase free radicals, and can cause peroxidation of membrane lipid, protein modification, DNA damage and other intracellular dysfunction, which are closely related to skin aging. Since the melanin synthesis process is a series of enzymatic oxidation processes, the presence of oxygen is a necessary condition for this process to occur. Research finds that UVR induces oxidative stress reaction to generate ROS, up-regulates expression of Tyrosinase (TYR), and then promotes melanogenesis; after ultraviolet irradiation, KC generates a large amount of oxygen free radicals, and induces organisms to release factors such as prostaglandin-2 (Prostaglandin E, PGE 2) and alpha-melanocyte stimulating hormone (alpha-MSH), so that cAMP is increased and melanin synthesis is increased; oxygen radicals have also been reported to oxidize collagen fibers and make the skin yellow. Thus, antioxidants can retard skin aging and lighten skin color by scavenging oxygen radicals.

Skin damage, such as erythema, DNA damage, etc., caused by excessive UVR radiation results in invisible changes in cellular and genetic levels. These include alterations in immune responses such as increases in mast cells, bursts of keratinocyte cytokines, etc. Inflammatory Pigmentation (PIH) may also be caused. It was found that ultraviolet radiation and immune factors can up-regulate pro-melanoidin (POMC) mRNA, POMC peptide and alpha-MSH receptor levels, bind to MC1R, increase intracellular cAMP levels, stimulate cAMP/PKA signaling pathways, and lead to increased melanin synthesis.

Licorice (licorice) is a traditional medicinal plant with wide application range and has various health care effects.

The Glycyrrhiza isoflavone (semilicoisoflavone B, SFB) is a natural compound isolated from Glycyrrhiza glabra and Glycyrrhiza uralensis. Current research shows that SFB scientists focus on finding that it has a variety of pharmacological activities, can reduce amyloid β (aβ) secretion by inhibiting β -secretase-1 (BACE 1) expression and activity, and can reduce BACE1 expression by increasing pparγ expression and inhibiting STAT3 phosphorylation; can prevent osmotic stress of hyperglycemia; SFB has also been reported to induce apoptosis in oral cancer cells by inducing ROS production, down-regulating MAPK and Ras/Raf/MEK signaling pathways; it is also pointed out that SFB shows a strong tyrosinase inhibitory activity (IC 50: 0.25. Mu. Mol/L) and that it also shows a better tyrosinase inhibitory effect than widely used kojic acid in an enzymatic test.

Heretofore, various whitening ingredients or compositions have been developed, however, in some cases, various whitening compositions fail to exert the intended effects of people and in some cases side effects are generated. For example, some traditional whitening agents such as hydroquinone and kojic acid have proved to have strong whitening efficacy, but with the intensive research, researchers have found that long-term use of hydroquinone can cause brown yellow disease and skin darkness, and even has a risk of carcinogenesis. A series of cosmetic negative reports have made consumers more careful about raw materials used in cosmetics and favour cosmetics containing both safety sources and natural source efficacy. It is well known that safety is a primary consideration for tyrosinase inhibitors. SFB has been shown to be non-cytotoxic to human normal hepatocytes (L-O2), human embryonic kidney cells (HEK 293T), but SFB has been lacking in safety data for skin-related cells and multicellular organisms, and therefore, there is a need for a safe and effective skin whitening product that is free of side effects.

Disclosure of Invention

In view of the above problems, an object of the present invention is to provide a composition for skin whitening comprising as an active ingredient a semi-licorice isoflavone, in which a safe and nontoxic semi-licorice isoflavone which can achieve an excellent whitening effect is used as an active ingredient, and an application thereof.

In order to achieve the above object, in a first aspect, the present invention provides a composition for skin whitening comprising as an active ingredient a semi-licorice isoflavone.

Further, the composition for skin whitening inhibits melanin formation.

Further, it contains a concentration of 0.1-25. Mu. Mol/L of the semi-licorice isoflavone.

Further, the composition for skin whitening further comprises a solvent selected from one or more of PBS, DMSO, sterilized water, physiological saline, ethanol, propylene glycol, polyethylene glycol, glycerin, or vegetable oil.

Further, it contains a concentration of 6.25-25. Mu. Mol/L of the semi-licorice isoflavone.

Further, the composition for skin whitening is a cosmetic composition or a pharmaceutical composition.

Further, the cosmetic composition is composed of at least one formulation selected from the group consisting of milk, skin softener, toner, astringent, nourishing cream, eye cream, foundation, dry powder, essence, oil, gel, astringent, cosmetic liquid, facial mask foundation, soap, cleansing cream, cleansing foam, and body cleaner;

The pharmaceutical composition is composed of at least one dosage form selected from the group consisting of granules, powders, syrups, elixirs, emulsions, suspensions, tablets, capsules, tinctures, pills, transdermal absorbents, lotions, aerosols, ointments, pasty preparations, lyophilized preparations, suppositories, and injections.

Further, the composition for skin whitening is a topical formulation including a half hay isoflavone, the topical formulation being selected from any one of tincture, cream, paste, ointment, cream, emulsion, oil, mask, gel, spread, aerosol or solution.

In a second aspect, the present invention provides the use of the composition for skin lightening in the preparation of a product for skin lightening and lightening.

Further, the composition for skin whitening is a topical formulation including a half hay isoflavone, the topical formulation being selected from any one of tincture, cream, paste, ointment, cream, emulsion, oil, mask, gel, smear, aerosol or solution; the topical dosage form is used at least twice daily.

Compared with the prior art, the invention has the following technical effects:

(1) The skin whitening composition containing the semi-licorice isoflavone has the capability of remarkably inhibiting melanin formation and has higher tyrosinase inhibition activity.

(2) The composition for skin whitening containing the semi-licorice isoflavone has the effect of improving the whiteness of the skin, and can be better used for skin whitening and spot fading.

(3) The composition for skin whitening containing the semi-licorice isoflavone has no toxic or side effect on kidneys, livers and lungs, is safe and effective, and provides a new raw material selection for skin whitening products.

Drawings

Fig. 1: the cytotoxicity study result graph of the hemicellulosic isoflavone (SFB) on B16 cells in example 1 of the present invention, wherein ns represents no significance and P <0.0001.

Fig. 2: for the statistical graph of melanin content in B16 cells promoted by α -MSH of example 1 of the present invention, wherein CK represents control group, P <0.01.

Fig. 3: a graph showing the results of the melanin content and tyrosinase activity in B16 cells after treatment with the semi-licorice isoflavone (SFB) of example 1 of the present invention; wherein,

Graph a shows a melanin content result graph;

Panel b shows a graph of tyrosinase activity results;

where ns represents no significance, P <0.05, P <0.01, P <0.001, P <0.0001.

Fig. 4: mRNA expression level of Glycyrrhiza isoflavone (SFB) to melanin formation-related factors (MITF, TYR, TRP-1 and TRP-2) in B16 cells in example 1 of the present invention; wherein P <0.001, P <0.0001.

Fig. 5: pictures of skin whitening after UVA-induced mice skin pigmentation model were treated with different concentrations of glabridin (SFB) and Glabridin (GLA) according to example 2 of the present invention; wherein, three mice are selected for photographing in each group.

Fig. 6: skin chromatograms after UVA-induced mice skin pigmentation model were treated with different concentrations of hemicellulosic isoflavone (SFB) and Glabridin (GLA) according to example 2 of the present invention; wherein Model represents UVA-induced negative control mice, P represents UVA-induced positive control mice treated with 50 μg/(kg-day) Glabridin (GLA), M represents UVA-induced medium concentration mice treated with 50 μg/(kg-day) glabridin (SFB), H represents UVA-induced high concentration mice treated with 100 μg/(kg-day) glabridin (SFB), P <0.0001.

Fig. 7: for the results of serological tests of liver and kidney function following treatment of UVA-induced mice with different concentrations of Glycyrrhiza isoflavone (SFB) in example 2 of the present invention, CK represents a control group without UVA induction, UV represents a negative control group with UVA induction, and ns represents no significance.

Detailed Description

In order to make the purposes, technical solutions, beneficial effects and significant improvements of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. It is apparent that all of the described embodiments are only some, but not all, embodiments of the invention; 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.

It is to be understood that:

The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances. It should also be noted that the following embodiments may be combined with each other, and the same or similar concepts or processes may not be described in detail in some embodiments.

EXAMPLE 1 Glycyrrhiza isoflavone (SFB) inhibiting production of B16 cell melanin and inhibiting tyrosinase Activity

The specific experimental process is as follows:

1.1 detection of in vitro cytotoxicity of Glycyrrhiza isoflavone (SFB) on B16 cells:

The method comprises the following specific steps:

(1) 1X 10 ³ B16 cells per well were seeded in 96-well plates and incubated for 24h.

(2) Preparing a semi-licorice isoflavone solution: 2mg SFB (Shanghai Tongtian E-2179)

Dissolve in 56

Mu.L DMSO was used as a 100mM stock solution, diluted to a concentration of 6.25. Mu. Mol/L, 12.5. Mu. Mol/L, 25. Mu. Mol/L and 50. Mu. Mol/L with 1640/DMEM medium,

(3) Discarding the culture medium in step (1), and obtaining a concentration of 6.25 in step (2)

μmol/L、

B16 cells in 96-well plates were treated with 12.5. Mu. Mol/L, 25. Mu. Mol/L and 50. Mu. Mol/L of the semi-licorice isoflavone solution for 24h, and the effect of the semi-licorice isoflavone on proliferation and viability of B16 cells was detected using the CCK8 detection kit.

As shown in FIG. 1, the results show that the activities of B16 cells were not affected by the concentration of 6.25. Mu. Mol/L and 25. Mu. Mol/L of the semi-licorice isoflavone, but were affected by the concentration of 50. Mu. Mol/L. The semi-licorice isoflavone has no cytotoxicity to B16 cells at a concentration of not more than 25 mu mol/L. Thus, subsequent cell experiments were performed with both 6.25. Mu. Mol/L and 25. Mu. Mol/L of the semi-licorice isoflavones.

1.2 Determination of the concentration of melanocyte stimulating hormone (α -MSH) which can promote the production of melanin by B16 cells:

The method comprises the following specific steps:

2X 10 ⁵ B16 cells per well were seeded in 12-well plates and incubated for 24h. alpha-MSH (Aladin, M118985) was added at 1. Mu.M and 200nM and incubation continued for 48h. Cells were harvested by pancreatin to give a pellet, which was incubated in 500. Mu.L of 1M NaOH (10% DMSO) in 80℃water bath for 1h and absorbance was measured at 409nm in 96-well plates.

As a result, as shown in FIG. 2, alpha-MSH significantly promoted melanin production by B16 cells at concentrations of both 1. Mu.M and 200 nM. Subsequent experiments were performed with 1. Mu.M of alpha-MSH.

1.3 Detection of the effect of SFB on B16 cell melanin production:

The method comprises the following specific steps:

(1) Two concentrations of 6.25. Mu. Mol/L and 25. Mu. Mol/L of the semi-licorice isoflavone which did not inhibit cell growth in the above experimental procedure 1.1 were selected, and 25. Mu.M of commercially available glabridin (Shanghai Homoda, E-0376) was selected as a positive control to treat B16 cells, respectively, while adding 1. Mu.M of alpha-MSH to promote melanin production by the cells, and culturing of the B16 cells was continued for 48 hours;

(2) Melanogenesis was detected as described in experimental procedure 1.2 above.

As shown in FIG. 3a, the 25. Mu.M concentration of the semi-licorice isoflavone significantly inhibited the production of melanin in cells, whereas the 6.25. Mu.M concentration of the semi-licorice isoflavone had no effect on inhibiting the production of melanin in B16 cells, and the inhibitory effect of the semi-licorice isoflavone on melanin synthesis in B16 cells was concentration-dependent.

1.4 Detection of the effect of SFB on B16 cell tyrosinase activity:

The L-Dopa (Alatine, D111049) is used as a substrate to detect the tyrosinase activity of B16 cells, and the specific steps are as follows:

(1) 2X 10 ⁵ B16 cells per well were seeded in 12-well plates and incubated for 24h.

(2) To step (1), 1. Mu.M and 200nM of alpha-MSH was added and the culture was continued for 48h.

(3) After cells were harvested by pancreatin, 1mL PBS was added and quick frozen at-80℃and reconstituted three times to break up fines

The cells, cell lysates were centrifuged at 13000r/min for 15min at 4℃and the resulting supernatants were used as source of tyrosinase.

(4) Mu.L of the supernatant was mixed with 200. Mu.L of 10.0 mmol/L-DOPA in a 96-well plate,

Incubate at 37℃for 30min. The absorbance of the reaction at 405nm was measured with a microplate reader and used for relative quantification of cell TYR activity and determination of lysate protein concentration using a kit (Bradford assay, siemens (Thermo Scientific)).

As shown in FIG. 3B, the concentration of 25. Mu.M of the semi-licorice isoflavone significantly inhibited the activity of B16 cell tyrosinase, and the effect was concentration-dependent.

1.5 Detection of the effect of SFB on mRNA expression of B16 cell melanin-formation-related factors (MITF, TYR, TRP-1, TRP-2):

(1) Selecting two semi-licorice isoflavones with concentration of 6.25 mu mol/L and 25 mu mol/L which do not inhibit cell growth in the experimental process 1.1 to treat the B16 cells, simultaneously adding 1 mu mol/L of alpha-MSH to promote the B16 cells to generate melanin, and continuously culturing the B16 cells for 48 hours;

(2) mRNA expression levels of melanin formation-related factors MITF, TYR, TRP-1, TRP-2 were detected by fluorescent quantitative PCR. The specific process of fluorescent quantitative PCR comprises the following steps:

1) Total RNA from B16 cells was extracted using Trizol and reverse transcribed to give cDNA.

Reverse transcription system table 1 below shows:

Table 1:

2) Fluorescent quantitative PCR:

A. The fluorescent quantitative PCR reaction system was prepared as shown in Table 2 below:

TABLE 2

Among them, the primers for each melanin-formation-related factor are shown in the following table 3:

table 3:

B. the reaction was carried out in a qPCR instrument according to the procedure shown in table 4 below:

Table 4:

3) Experimental data were collected and data analysis was performed according to method 2 ^-△△CT.

As shown in FIG. 4, the results showed that the expression of B16 cell melanin formation-related factors MITF, TYR, TRP-1 and TRP-2 was significantly inhibited at 25. Mu. MSFB of the concentration of semi-licorice isoflavone, and the effects were concentration-dependent, as compared with the model group.

Example 2 in vivo inhibition of Hemiglycyrrhiza isoflavone melanin increase by UVA irradiation of mouse skin

The specific experimental process is as follows:

(1) A skin pigmentation model was established for UVA-induced mice (BALB/c (line) 6-8 week female mice purchased by Shanghai Ling Biotechnology Co., ltd.): the 12 mice were randomly divided into 4 groups of 3, and were dehaired with a razor at 4x4 cm area of the backs of the mice. Mice were divided into the following experiments:

1. Negative control group (Model): i.e., UVA group, UVA irradiation + spreading solvent (PBS);

2. positive control group (P): 50 μg/(kg. Day) glabridin (Shanghai co-field, E-0376) application +UVA irradiation;

3. Dose-treated group (M) in SFB: local application of 50 μg/(kg. Day) SFB+UVA radiation;

4. SFB high dose treatment group (H): 100 μg/(kg. Day) SFB+UVA radiation was applied topically.

Mice were exposed to 300mJ/cm ² of radiation under a UVA tube for 20 minutes each time, three times a week for four weeks, total radiation amounting to 3600mJ. After the start of UVA irradiation, 200. Mu.L of the therapeutic agent solution (specifically: 50. Mu.g/kg glabridin; 50. Mu.g/kg SFB and 100. Mu.g/kg glabridin) was topically applied to the dorsal skin of mice twice a day for six weeks per day in each treatment group at the above experimental dose. The photographing was performed every week, the photographing result is shown in fig. 5, and the skin colorimetric value was measured using a colorimeter, and the colorimetric value detection result is shown in fig. 6.

From the results of FIGS. 5 and 6, it can be seen that different doses (50. Mu.g/kg and 100. Mu.g/kg) of SFB treated UVA induced mice resulted in significantly reduced melanin content and dose dependency compared to the negative control; in addition, the positive control (P) in this example was selected from the whitening factor, glabridin (GLA), which is currently in wide use; SFB of 50. Mu.g/kg and 100. Mu.g/kg reduced melanin content better than the positive control.

(2) Mice were sacrificed at the end of the experiment and liver function was detected by serum: glutamic-oxaloacetic transaminase GOT/ALT, glutamic-oxaloacetic transaminase AST; renal function: creatinine Cr and urea nitrogen BUN levels, SFB was observed for toxic side effects on kidney, liver and lung, and the results are shown in FIG. 7, wherein CK means a control group not subjected to UVA irradiation, UV means a negative control group subjected to UVA irradiation, and 50 μg/kg/day GLA means a positive control group of 50 μg/kg/day glabridin.

As can be seen from FIG. 7, there was no significant difference in the detection of glutamic-oxaloacetic transaminase GOT/ALT, glutamic-oxaloacetic transaminase AST, creatinine Cr, and urea nitrogen BUN levels in serum of mice treated with 100. Mu.g/kg/day of semi-licoflavone (SFB) compared with the control group, indicating that licoflavone (SFB) is non-toxic to both liver and kidney, so that it is safe and non-toxic to use licoflavone for skin whitening.

The preferred embodiments of the present invention have been described above. It is to be understood that the invention is not limited to the specific embodiments described above, wherein devices and structures not described in detail are to be understood as being implemented in a manner common in the art; any person skilled in the art can make many possible variations and modifications to the technical solution of the present invention or modifications to equivalent embodiments without departing from the scope of the technical solution of the present invention, using the methods and technical contents disclosed above, without affecting the essential content of the present invention. Therefore, any simple modification, equivalent variation and modification of the above embodiments according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A skin whitening composition comprising as an active ingredient a semi-licorice isoflavone.

2. The composition for skin whitening according to claim 1, wherein the composition for skin whitening inhibits melanin formation.

3. The composition for skin whitening according to claim 1, comprising a concentration of 0.1 to 25 μmol/L of a hemicelluloses.

4. The composition for skin whitening according to claim 1, further comprising a solvent selected from one or more of PBS, DMSO, sterilized water, physiological saline, ethanol, propylene glycol, polyethylene glycol, glycerin, or vegetable oil.

5. The composition for skin whitening according to claim 1, comprising a concentration of 6.25 to 25 μmol/L of a hemicelluloses.

6. The composition for skin whitening according to claim 1, wherein the composition for skin whitening is a cosmetic composition or a pharmaceutical composition.

7. The composition for skin whitening according to claim 1, wherein the cosmetic composition is composed of at least one formulation selected from the group consisting of milk, skin softener, toner, astringent, nourishing cream, eye cream, foundation, dry powder, essence, oil, gel, astringent, cosmetic liquid, mask, facial mask foundation, soap, cleansing cream, cleansing foam, and body cleanser;

8. The composition for skin whitening according to claim 1, wherein the composition for skin whitening is a topical formulation comprising a half-dry grass isoflavone, the topical formulation being selected from any of tincture, cream, paste, ointment, cream, emulsion, oil, mask, gel, spread, aerosol, or solution.

9. Use of the composition for skin whitening according to any one of claims 1 to 8 for preparing a product for skin whitening and spot lightening.

10. The use according to claim 9, wherein the composition for skin whitening is a topical formulation comprising a half-dry grass isoflavone, the topical formulation being selected from any of tincture, cream, paste, ointment, cream, emulsion, oil, mask, gel, spread, aerosol or solution; the topical dosage form is used at least twice daily.