Use of hydroxytyrosol or 3,4-dihydroxyphenylethanol-o-β-d-glucoside in preparing drug for promoting melanin generation or tanning product

Abstract

Use of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside in promoting melanin generation. The effect of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside in the generation of melanin in recombinant human tyrosinase, mouse B16 cells, and AB-strain wild-type Danio rerio has been studied, and the results show that hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can promote the generation of melanin. Hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can be used for tanning, treating hypopigmentation diseases, blackening hair, and the like, to expand its pharmaceutical application.

Description

Application of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside in the preparation of melanin production-promoting drugs or tanning products Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a new application of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside in promoting melanin production. Background Art

[0002] Hydroxytyrosol (chemical name 3,4-dihydroxyphenylethanol, Hydroxytyrosol), molecular formula C8H 10 O3 is a natural phenolic compound found in olive oil. Hydroxytyrosol exhibits anti-inflammatory and antimicrobial properties, as well as anti-cardiovascular, anti-tumor, and neuroprotective effects. Due to its good bioavailability, chemical stability, ease of formulation, and non-toxicity, hydroxytyrosol is considered a promising dietary supplement by the nutraceutical and food industries. However, current research on hydroxytyrosol has not yet addressed its role in promoting melanin production.

[0003] Melanin, a chemically complex, heterogeneous polymer of phenolic and indole substances produced by melanocytes, has multiple functions in biological systems and is the primary component of human skin and hair color. It is widely present in organs such as the skin, mucous membranes, retina, pia mater, and gallbladder. Melanin's structure contains reactive groups such as phenolic hydroxyl, carboxyl, and amino groups, which absorb ultraviolet light, making it a natural, endogenous functional substance. Melanin's structure dictates its diverse activities, including free radical scavenging, antibacterial, metal chelation, anti-tumor, and radiation protection, making research related to melanin production a hot topic and a trending topic.

[0004] Vitiligo, vitiligo, leukoplakia in middle-aged and elderly people, pityriasis simplex and other pigment loss diseases as well as gray hair are all related to melanin production disorders, but there is currently a lack of effective drugs or products to improve their symptoms. Summary of the Invention

[0005] The present invention aims to provide a new application of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside in promoting melanin production. It is the first discovery that hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can promote melanin production, providing a theoretical basis for the subsequent development of tanning products, hair blackening products, and drugs for treating hypopigmentation, and expanding new applications of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The application of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside in the preparation of a drug for promoting melanin production or a tanning product is characterized in that:

[0008] Furthermore, the concentration of hydroxytyrosol required to increase the melanin production in B16 cells by 50% is 7.45 μM, and the concentration of 3,4-dihydroxyphenylethanol-O-β-D-glucoside required to increase the melanin production in B16 cells by 50% is 14.90 μM.

[0009] Furthermore, the hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly increase the melanin production of mouse B16 cells.

[0010] Furthermore, the hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly promote the activity of recombinant human tyrosinase.

[0011] Furthermore, the hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly increase the melanin production of wild-type zebrafish of the AB strain. Beneficial effects

[0012] At present, the relevant research of hydroxytyrosol has not involved the promotion of melanin production. The reason is that the tyrosinase verified by whitening products on the market is basically tyrosinase of plant origin, such as mushroom tyrosinase. The present invention unexpectedly found that hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside is selective for tyrosinase from different sources. Specifically, hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside promotes enzyme activity of animal-derived tyrosinase (such as human tyrosinase) and increases melanin production, which is a tanning effect. However, the two inhibit enzyme activity of plant-derived tyrosinase (such as mushroom-derived tyrosinase), reduce melanin production, and produce a whitening effect. The results of the two are exactly opposite. For the above reasons, those skilled in the art would not think of it as a research on the development of tanning products or melanin-generating drugs.

[0013] The present invention discovered for the first time that hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly promote the activity of tyrosinase and promote the production of melanin, and can promote the production of melanin at the molecular, cellular and animal levels. This expands the new medical applications of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside and provides new ideas for its tanning, treatment of hypopigmentation diseases, and hair blackening. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Effects of hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside on melanin production in mouse B16 cells. C is the negative control group, and M is the positive control group (M is 8-methoxypsoralen); (a) Effect of hydroxytyrosol on melanin production in mouse B16 cells. The concentrations of hydroxytyrosol are 0.1, 1, 10, 50, and 100 μM, respectively; (b): Effect of 3,4-dihydroxyphenylethanol-O-β-D-glucoside on melanin production in mouse B16 cells; the concentrations of 3,4-dihydroxyphenylethanol-O-β-D-glucoside are 6.25, 12.5, 25, 50, 100, and 200 μM, respectively); (c) EC of hydroxytyrosol 50 ; C1 is the concentration of hydroxytyrosol; (d) EC of 3,4-dihydroxyphenylethanol-O-β-D-glucoside 50 ; C2 is the concentration of 3,4-dihydroxyphenylethanol-O-β-D-glucoside; *P<0.05, **P<0.01, ***P<0.001, compared with the negative control group C.

[0015] Figure 2. Gene sequence alignment of recombinant human tyrosinase. TYR CDS represents the human tyrosinase CDS sequence from the NCBI database. The actual sequence is the tyrosinase sequence obtained after plasmid introduction into E. coli. Blue represents the sequence identity, and red represents the mutated portion.

[0016] Figure 3. Verification of recombinant human tyrosinase activity. C represents the negative control group. TYR-10min, TYR-20min, and TYR-30min represent the melanin production measured by recombinant human tyrosinase at 10, 20, 30, and 45 minutes and at 475 nm. ***P < 0.001, compared with the negative control group C.

[0017] Figure 4 Effects of hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside on human tyrosinase activity; (a) Effect of hydroxytyrosol on human tyrosinase activity, hydroxytyrosol concentrations are 0, 10, 50, 100, 500, 1000, 2000, 3000, and 4000 μM; (b) Effect of 3,4-dihydroxyphenylethanol-O-β-D-glucoside on human tyrosinase activity, 3,4-dihydroxyphenylethanol-O-β-D-glucoside concentrations are 0, 10, 50, 100, 500, and 1000 μM. **P < 0.01.

[0018] Figure 5 Effects of hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside on mushroom tyrosinase activity. M is the positive control (M is 8-methoxypsoralen); (a) Effect of hydroxytyrosol on mushroom tyrosinase activity at hydroxytyrosol concentrations of 10, 50, 100, 500, 1000, 2000, 3000, and 4000 μM; (b) Effect of 3,4-dihydroxyphenylethanol-O-β-D-glucoside on mushroom tyrosinase activity at 3,4-dihydroxyphenylethanol-O-β-D-glucoside concentrations of 0, 10, 50, 100, 500, and 1000 μM. *P < 0.05, **P < 0.01, ***P < 0.001.

[0019] Fig. 6 Effects of hydroxytyrosol (a) and 3,4-dihydroxyphenylethanol-O-β-D-glucoside (b) on melanin production in wild-type zebrafish of the AB strain; ***P<0.001. DETAILED DESCRIPTION

[0020] To make the objects, features, and advantages of the present invention more clearly understood, the present invention is further described below in detail with reference to the accompanying drawings and embodiments. It is apparent that the embodiments described are only a portion of the embodiments of the present invention, rather than all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0021] Unless otherwise specified, the experimental methods described in the following examples are all conventional methods, and the experimental reagents, instruments, and materials used are all commercially available.

[0022] The reagents and materials used in the following experimental protocol mainly include:

[0023] Mouse melanoma cells (B16), recombinant human tyrosinase (prepared with reference to Kong, JN, et al., Characterization of human tyrosinase ectodomain expressed in Escherichia coli. Protein Pept Lett, 2010. 17(8): p. 1026-30.), mushroom tyrosinase, wild-type zebrafish of AB strain, Escherichia coli BL21 (DE3), DMEM, LB medium, E3 culture medium, BCA kit, RIPA lysis buffer, hydroxytyrosol, 3,4-dihydroxyphenylethanol-O-β-D-glucoside, 8-methoxypsoralen, α-MSH, disodium hydrogen phosphate, citric acid, sodium hydroxide, L-DOPA, IPTG, Ni Sepharose 6 Fast Flow filler.

[0024] Example 1 Effects of hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside on melanin production in mouse melanoma cells.

[0025] In this example, mouse melanoma cells were used as an example to explore the effects of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside on melanin production in B16 cells. The specific experimental steps and data calculations are as follows: After digestion and counting of B16 cells, 2×10 5 cells. A negative control group (DMSO 2 μL / well) and a positive control group (100 μmol / L 8-methoxypsoralen, replaced by M in the figure) were set up in the experiment. B16 cells were treated with 0.1, 1, 10, 50, and 100 μM hydroxytyrosol and 6.25, 12.5, 25, 50, 100, and 200 μM 3,4-dihydroxyphenylethanol-O-β-D-glucoside, respectively. Three replicates were set for each concentration. After 48 hours of culture, the cells were washed twice with PBS, and 200 μL of RIPA lysis buffer was added to each well. The cell supernatant was extracted and the total protein concentration was detected by BCA method. 190 μL of 1 M NaOH lysis buffer was added to the precipitate, and the cells were placed in an 80°C water bath for 1 hour. The OD value was measured at a wavelength of 405 nm by a microplate reader. The relative melanin content and melanin production activation rate were calculated according to the following formula: 50 Relative melanin content = OD value of experimental group / protein concentration Melanin production activation rate = (relative melanin content of experimental group / relative melanin content of control group) × 100%

[0026] The results are shown in Figure 1. Hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly increase the melanin production of B16 cells. The EC of hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside on the melanin production of B16 cells 50 They are 7.45μM and 14.90μM respectively.

[0027] Example 2 Effects of Hydroxytyrosol and 3,4-Dihydroxyphenylethanol-O-β-D-glucoside on the Activity of Recombinant Human Tyrosinase, Which Promote the Enzyme Activity of Recombinant Human Tyrosinase and Increase the Production of Melanin in the System

[0028] Tyrosinase is the rate-limiting enzyme in melanin production. In this example, recombinant human tyrosinase was used as an example to investigate the effects of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside on tyrosinase activity. The specific experimental steps are as follows:

[0029] 2.1 Preparation of recombinant human tyrosinase

[0030] The CDS sequence of human tyrosinase (Gene ID 7922) was obtained from NCBI. Using pET-26b(-) as the vector, the target gene was inserted into the plasmid vector by selecting the NdeⅠ-EcoRI restriction site. Figure 2 shows the plasmid sequencing results, which verified that the target gene was correctly introduced into the BL21(DE3) host cells. The method for preparing recombinant human tyrosinase using Escherichia coli as the host was referred to (Kong, JN, et al., Characterization of human tyrosinase ectodomain expressed in Escherichia coli. Protein Pept Lett, 2010.17(8): p.1026-30). The Escherichia coli containing the recombinant plasmid was placed in LB liquid medium containing kanamycin and cultured at 37°C until the OD 600 When the pH value is about 0.5-0.6, IPTG is added to induce protein expression. After centrifugation to collect the bacteria, the supernatant is collected and purified by nickel column and concentrated by ultrafiltration to obtain recombinant human tyrosinase. The activity is then verified and a 1 mg / mL levodopa solution is prepared. 40 μL of recombinant human tyrosinase solution and 100 μL of levodopa substrate are added to a 96-well plate to start the enzymatic reaction, and the reaction is placed at 37°C. A negative control group is set up and each group is repeated 3 times in parallel. The absorbance at 475 nm is detected at 0 min, 10 min, 20 min, and 30 min to reflect the change in melanin production. Melanin production = A475 Xmin -A475 0min

[0031] Where: A475 is the absorbance of melanin at 475nm, X = 10, 20, 30

[0032] The results are shown in FIG3 . The recombinant human tyrosinase plasmid vector was correctly introduced into the E. coli host, and the purified human tyrosinase had good activity.

[0033] 2.2 Verification of the activity of recombinant human tyrosinase by hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside

[0034] Hydroxytyrosol at concentrations of 10, 50, 10, 500, 1000, 2000, 3000, and 4000 μM, 3,4-dihydroxyphenylethanol-O-β-D-glucoside at concentrations of 10, 50, 100, 500, and 1000 μM, and 1 mg / mL L-DOPA were prepared. The enzymatic reaction was initiated by adding 40 μL of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside, 40 μL of recombinant human tyrosinase solution, and 100 μL of L-DOPA substrate to a 96-well plate. After incubation at 37°C for 1 hour, the absorbance of the product, melanin, was measured at 475 nm. A negative control group was included and the experiment was repeated three times.

[0035] The results are shown in Figure 4. Hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly promote the enzymatic activity of recombinant human tyrosinase and increase the production of melanin in the system.

[0036] Example 3 Effects of hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside on the activity of mushroom-derived tyrosinase, which have the ability to inhibit the activity of mushroom tyrosinase.

[0037] The specific experimental procedures and results are as follows: Mushroom-derived tyrosinase was purchased and immediately before use, prepared into a 100 U / mL tyrosinase solution using sodium hydrogen phosphate-citrate buffer (pH 6.8). Hydroxytyrosol (10, 50, 10, 500, 1000, 2000, 3000, and 4000 μM) and 3,4-dihydroxyphenylethanol-O-β-D-glucoside (10, 50, 100, 500, and 1000 μM) were added, along with a 1 mg / mL levodopa solution. Negative and positive controls (400 μmol / L kojic acid, represented by M in the figures) were established. 40 μL of the test sample was added and incubated with 20 μL of the tyrosinase solution (100 U / mL) at 37°C for 10 minutes. The enzymatic reaction was initiated by the addition of 100 μL of levodopa substrate. After incubation at 37°C for 20 minutes, the absorbance of the product, melanin, was measured at 475 nm. The experiment was repeated three times in parallel, and the inhibition rate was calculated based on the absorbance value: Y = [1-(Aa-Ab) / (Ac-Ad)] C × 100%

[0038] Where: Y is the tyrosinase activity inhibition rate; Aa is the absorbance of the sample reaction well; Ab is the absorbance of the sample background well; Ac is the average absorbance of the solvent reaction well; Ad is the average absorbance of the solvent background well.

[0039] The results are shown in FIG5 , and hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly inhibit the activity of mushroom tyrosinase.

[0040] Example 4 Effects of hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside on melanin production in wild zebrafish of the AB strain.

[0041] In this embodiment, wild-type zebrafish of AB strain were used as an example to explore the effect of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside on the amount of melanin produced in zebrafish. The specific experimental steps and data processing are as follows: wild-type zebrafish embryos of AB strain were purchased and collected in six-well plates, 30 per well, and 5mL E3 culture medium was added for culture. The experiment set up a negative control group and a positive control group (50nmol / L of α-MSH), and 0.1, 1, 10μM hydroxytyrosol and 50, 100, 200μM 3,4-dihydroxyphenylethanol-O-β-D-glucoside were prepared with E3 culture medium and added when the embryos developed to 24hpf. After 72h of treatment, images were taken by stereomicroscope, and the resulting images were processed using imageJ software to count the melanin area.

[0042] The results are shown in FIG6 , and hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly increase the melanin production in zebrafish.

[0043] These experimental results confirm that hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside significantly promotes melanin production at the molecular, cellular, and animal levels. This provides a new approach for the development of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside as tanning products, hair darkening products, and medications for treating hypopigmentation, with promising application prospects.

[0044] The above is a detailed introduction to the novel application of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside provided by the present invention in promoting melanin production. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. For those skilled in the art, without departing from the principles of the present invention, the present invention can also be improved and modified, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of hydroxytyrosol or 3,4-dihydroxyphenethyl alcohol - O-β-D-glucoside in the preparation of a drug for promoting melanogenesis or a tanning product, characterized in that, Hydroxytyrosol 3,4-Dihydroxyphenethyl alcohol-O-β-D-glucoside.

2. The application according to claim 1, characterized in that, The self-tanning products include self-tanning lotions, self-tanning sprays, self-tanning essential oils, self-tanning gels, self-tanning essence creams, and self-tanning face creams.

3. The application according to claim 1, characterized in that, Use of hydroxytyrosol or 3,4-dihydroxyphenethyl alcohol-O-β-D-glucoside in the preparation of a medicament for treating pigment deficiency diseases.

4. The application according to claim 3, characterized in that, The pigment deficiency diseases are vitiligo, canities, senile leukoderma or pityriasis simplex.

5. The application according to any one of claims 1-4, characterized in that The dosage forms of hydroxytyrosol or 3,4-dihydroxyphenethyl alcohol-O-β-D-glucoside include creams, oils, lotions, gels, liniments, ointments, mousses, powders, sprays, aerosols, capsules, tablets, granules, powders, tinctures, solutions, suspensions, emulsions, inhalants, gels, microspheres.

6. The application according to claim 1, wherein Use of hydroxytyrosol or 3,4-dihydroxyphenethyl alcohol-O-β-D-glucoside in the preparation of hair darkening products.

7. The application according to claim 6, wherein The products include shampoos, conditioners, scalp care oils, scalp essences, scalp gels, hair care essential oils, scalp scrubs, and hair masks.

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