Application of hydroxytyrosol or 3, 4-dihydroxyphenethyl alcohol-O-beta-D-glucoside in preparation of melanogenesis promoting drugs or blackening products

Abstract

The invention discloses a novel application of hydroxytyrosol or 3, 4-dihydroxyphenethyl alcohol-O-beta-D-glucoside in promotion of melanogenesis, and relates to a preparation method of hydroxytyrosol or 3, 4-dihydroxyphenethyl alcohol. Hydroxytyrosol is a phenolic plant chemical substance, has good pharmacological effects in the aspects of microorganism resistance, inflammation resistance, tumor resistance, oxidation resistance, cardiovascular disease and metabolic syndrome treatment, detoxification and the like, and is widely applied to various fields. The pharmacological action of hydroxytyrosol or 3, 4-dihydroxyphenethyl alcohol-O-beta-D-glucoside in promoting melanogenesis is verified by analyzing the melanogenesis condition of hydroxytyrosol or 3, 4-dihydroxyphenethyl alcohol-O-beta-D-glucoside on recombinant human tyrosinase, mouse B16 cells and wild zebra fish melanin of AB strains. The invention finds that the hydroxytyrosol or the 3, 4-dihydroxyphenylethanol-O-beta-D-glucoside can obviously promote the generation of melanin, a new thought can be provided for blackening, treating hypopigmentation diseases, blackening hair and the like by utilizing the new biological activity of the hydroxytyrosol or the 3, 4-dihydroxyphenylethanol-O-beta-D-glucoside, and the new medical application of the hydroxytyrosol or the 3, 4-dihydroxyphenylethanol-O-beta-D-glucoside is expanded.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, 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 has anti-inflammatory and antibacterial properties, anti-cardiovascular disease, anti-tumor, and neuroprotective effects. Due to its good bioavailability, stable chemical properties, easy formulation and non-toxicity, hydroxytyrosol is considered a good food supplement by the nutritional supplement and food industries. At present, the relevant research on hydroxytyrosol has not involved the promotion of melanin production.

[0003] Melanin is a kind of phenolic or indole polymer with extremely complex chemical structure and heterogeneity produced by melanocytes. It has multiple functions in biological systems and is the main component of human skin and hair color. It is widely present in human skin, mucous membranes, retina, pia mater, gallbladder and other organs. Melanin contains active groups such as phenolic hydroxyl, carboxyl, and amino groups in its structure, which can absorb ultraviolet rays and is a natural endogenous functional substance. The structure of melanin determines that it has multiple activities such as scavenging free radicals, antibacterial, chelating metals, anti-tumor, and radiation protection, which makes the research related to melanin production a hot spot and trend.

[0004] Vitiligo, vitiligo, leukoplakia in the middle-aged and elderly, pityriasis simplex and other pigment loss diseases and 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 the production of melanin, and for the first time finds that hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can promote the production of melanin, which provides a theoretical basis for the subsequent development of tanning products, black hair products, and drugs for treating hypopigmentation, and expands the new application of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside.

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

[0007] The use 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]

[0009] Hydroxytyrosol

[0010]

[0011] 3,4-Dihydroxyphenylethanol-O-β-D-glucoside.

[0012] 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.

[0013] Furthermore, the hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly increase the melanin production of mouse B16 cells.

[0014] Furthermore, the hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly promote the activity of recombinant human tyrosinase.

[0015] 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.

[0016] Beneficial Effects

[0017] At present, the relevant research on hydroxytyrosol has not involved the promotion of melanin production. The reason is that the tyrosinases verified by whitening products on the market are basically tyrosinases 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 tyrosinases from different sources, specifically, hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside promotes enzyme activity and increases melanin production for tyrosinase of animal origin (such as human tyrosinase), which is the direction of tanning; while the two inhibit enzyme activity for tyrosinase of plant origin (such as mushroom tyrosinase), reduce melanin production, and produce a whitening effect. The results of the two are exactly opposite. The above reasons lead to the fact that technicians in this field would not think of it as a research on the development of tanning products or melanin-generating drugs.

[0018] The present invention discovers for the first time that hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly promote the activity of tyrosinase and the production of melanin, and can promote the production of melanin at the molecular, cellular and animal levels, thereby expanding the new medical applications of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside and providing new ideas for tanning, treating hypopigmentation diseases, and darkening hair. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 2 Gene sequence alignment of recombinant human tyrosinase. TYR CDS is the human tyrosinase CDS sequence in the NCBI database. The actual sequence is the tyrosinase sequence obtained by sequencing after the plasmid was introduced into Escherichia coli; the blue part is the consistent part of the sequence, and the red part is the mutated part.

[0021] Figure 3 Verification of recombinant human tyrosinase activity. C is the negative control group, TYR-10min, TYR-20min, TYR-30min are the production of melanin detected by recombinant human tyrosinase at 10min, 20min, 30min, 45min and 475nm, ***P<0.001, compared with the negative control group C.

[0022] 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, 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, 1000 μM. **P<0.01.

[0023] Figure 5Effects of hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside on mushroom tyrosinase activity. M is the positive control group (M is 8-methoxypsoralen); (a) Effect of hydroxytyrosol on mushroom tyrosinase activity, the concentrations of hydroxytyrosol are 10, 50, 100, 500, 1000, 2000, 3000, 4000 μM; (b): Effect of 3,4-dihydroxyphenylethanol-O-β-D-glucoside on mushroom tyrosinase activity, the concentrations of 3,4-dihydroxyphenylethanol-O-β-D-glucoside are 0, 10, 50, 100, 500, 1000 μM. *P<0.05, **P<0.01, ***P<0.001.

[0024] Figure 6 Effects of hydroxytyrosol (a) and 3,4-dihydroxyphenylethanol-O-β-D-glucoside (b) on melanin production in wild-type zebrafish of AB strain; ***P<0.001. DETAILED DESCRIPTION

[0025] In order to make the objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods described in the following examples are all conventional methods, and the experimental reagents, instruments and materials used can be obtained commercially.

[0027] The reagents and materials used in the following experimental protocols mainly include:

[0028] Mouse melanoma cells (B16), recombinant human tyrosinase (prepared according 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.

[0029] Example 1 Effects of hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside on melanin production in mouse melanoma cells.

[0030] In this example, mouse melanoma cells were used as an example to explore the effect of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside on the melanin production in B16 cells. The specific experimental steps and data calculation 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 replicate wells were set for each concentration. After 48 hours of culture, the cells were washed twice with PBS, 200 μL RIPA lysis buffer was added to each well, the cell supernatant was extracted, and the total protein concentration was detected by the BCA method. 190 μL 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 using an enzyme reader. The relative melanin content and melanin production activation rate were calculated according to the following formula: 50 .

[0031] Relative melanin content = OD value of experimental group / protein concentration

[0032] Melanin production activation rate = (relative melanin content in the experimental group / relative melanin content in the control group) × 100%

[0033] The results are as follows Figure 1 As shown in the results, 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.

[0034] 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 amount of melanin produced in the system

[0035] Tyrosinase is the rate-limiting enzyme in melanin production. In this example, recombinant human tyrosinase is used as an example to explore the effect of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside on tyrosinase activity. The specific experimental steps are as follows:

[0036] 2.1 Preparation of recombinant human tyrosinase

[0037] The CDS sequence of human tyrosinase (Gene ID 7922) was obtained from NCBI, and pET-26b(-) was used as the vector. The NdeⅠ-EcoRI restriction site was selected to embed the target gene into the plasmid vector. Figure 2 The plasmid sequencing result verifies that the target gene is correctly introduced into the BL21 (DE3) host cell. The method for preparing recombinant human tyrosinase using Escherichia coli as the host is 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 is placed in LB liquid medium containing kanamycin at 37°C for amplification and culture until the OD 600 When the pH value was about 0.5-0.6, IPTG was added to induce protein expression. After centrifugation to collect the bacteria, the supernatant was collected and purified by nickel column and concentrated by ultrafiltration to obtain recombinant human tyrosinase. The activity was then verified and a 1 mg / mL levodopa solution was prepared. 40 μL of recombinant human tyrosinase solution and 100 μL of levodopa substrate were added to a 96-well plate to start the enzymatic reaction, which was placed at 37°C for reaction. A negative control group was set up and each group was repeated 3 times in parallel. The absorbance at 475 nm was detected at 0 min, 10 min, 20 min, and 30 min to reflect the changes in the amount of melanin produced.

[0038] Melanin production = A475 Xmin -A475 0min

[0039] Where: A475 is the absorbance of melanin at 475nm, X = 10, 20, 30

[0040] The results are as follows Figure 3 As shown, the recombinant human tyrosinase plasmid vector was correctly introduced into the Escherichia coli host, and the purified human tyrosinase had good activity.

[0041] 2.2 Verification of the activity of recombinant human tyrosinase by hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside

[0042] 10, 50, 10, 500, 1000, 2000, 3000, 4000 μM hydroxytyrosol and 10, 50, 100, 500, 1000 μM 3,4-dihydroxyphenylethanol-O-β-D-glucoside and 1 mg / mL L-DOPA solution were prepared. 40 μL hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside and 40 μL recombinant human tyrosinase solution and 100 μL L-DOPA substrate were added to the 96-well plate to start the enzymatic reaction. After reacting at 37°C for 1 hour, the absorbance of the product melanin was detected at 475 nm. A negative control group was set up and repeated 3 times in parallel.

[0043] The results are as follows Figure 4 As shown, 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.

[0044] 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.

[0045] The specific experimental steps and results are as follows: Buy mushroom-derived tyrosinase, and prepare 100U / mL tyrosinase solution with disodium hydrogen phosphate-citrate buffer (pH6.8) before use, prepare 10, 50, 10, 500, 1000, 2000, 3000, 4000μM hydroxytyrosol and 10, 50, 100, 500, 1000μM 3,4-dihydroxyphenylethanol-O-β-D-glucoside and 1mg / mL levodopa solution. The experiment set up a negative control group and a positive control group (400μmol / L kojic acid, replaced by M in the figure) to add 40μL of the sample to be tested, incubate with 20μL of tyrosinase solution (100U / mL) at 37℃ for 10min, then add 100μL levodopa substrate to start the enzymatic reaction, and then place it at 37℃ for 20min to detect the absorbance of the product melanin at 475nm. The experiment was repeated 3 times in parallel, and the inhibition rate was calculated based on the absorbance value:

[0046] Y=[1-(Aa-Ab) / (Ac-Ad)]C×100%

[0047] Wherein: 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.

[0048] The results are as follows Figure 5 As shown, hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside could significantly inhibit the activity of mushroom tyrosinase.

[0049] Example 4 Effects of hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside on melanin production in wild zebrafish of AB strain.

[0050] In this embodiment, wild-type zebrafish of AB strains 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 strains 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 configured with E3 culture medium and added when the embryo developed to 24hpf, and images were taken by stereomicroscope after 72h of treatment, and the resulting images were processed using imageJ software to count the melanin area.

[0051] The results are as follows Figure 6 As shown, hydroxytyrosol and 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly increase the melanin production in zebrafish.

[0052] The above test results confirm that hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside can significantly promote the production of melanin at the molecular, cellular and animal levels. It provides a new idea for the development of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside as tanning products, hair blackening products and drugs for treating pigmentation loss, which has a good application prospect.

[0053] The above is a detailed introduction to the new 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 ordinary technicians in this technical field, the present invention can also be improved and modified without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside in the preparation of drugs for promoting melanin production or tanning products, It is characterized in that 2. The use according to claim 1, It is characterized in that The tanning products include tanning lotion, tanning spray, tanning essential oil, tanning gel, tanning essence cream, and tanning facial cream.

3. The use according to claim 1, It is characterized in that Application of hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside in preparing medicine for treating pigment deficiency diseases.

4. The use according to claim 3, It is characterized in that The pigment loss diseases are vitiligo, leukoplakia, leukoplakia in the middle-aged and elderly or pityriasis simplex.

5. The use according to claims 1-4, It is characterized in that The dosage forms of hydroxytyrosol or 3,4-dihydroxyphenylethanol-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, and microspheres.

6. The use according to claim 1, It is characterized in that The invention discloses an application of the hydroxytyrosol or 3,4-dihydroxyphenylethanol-O-β-D-glucoside in preparing a hair darkening product.

7. The use according to claim 6, It is characterized in that The products include shampoo, conditioner, scalp care oil, scalp essence, scalp gel, hair care essential oil, scalp scrub, and hair mask.

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