Compound with tyrosinase inhibitory activity and application thereof in preparation of whitening product

Abstract

The invention discloses a compound with tyrosinase inhibitory activity and application of the compound in preparation of whitening products, and belongs to the technical field of medicinal chemistry. The compound is 5-(4-hydroxyphenyl)-3-O-glucosyl valeric acid, and is a novel compound. The compound can significantly inhibit the activity of tyrosinase, has the ability of blocking tyrosinase from catalyzing tyrosine to be converted into melanin precursor substances, and directly inhibits the generation of melanin, thereby showing better application prospects and values in skin whitening or skin diseases.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a compound with tyrosinase inhibitory activity and its application in the preparation of skin whitening products. Background Technology

[0002] The biosynthesis of melanin is a cascade reaction process strictly regulated by tyrosinase. As a key rate-limiting enzyme, this enzyme catalyzes the conversion of L-tyrosine to dopaquinone through its monophenolase and diphenolase activities, thereby initiating subsequent non-enzymatic reactions and ultimately leading to the formation of melanin polymers.

[0003] While physiological deposition of melanin can protect against UV damage, abnormal metabolism or excessive accumulation of melanin is closely related to the pathogenesis of various skin diseases such as melasma, post-inflammatory hyperpigmentation, and melanoma. At the same time, in the field of beauty, safely and effectively inhibiting melanin production is also a core scientific issue for achieving skin whitening and even skin tone.

[0004] Therefore, targeted inhibition of tyrosinase activity has significant therapeutic and aesthetic applications. Currently, commonly used inhibitors in clinical practice and cosmetics, such as kojic acid and β-arbutin, while showing good efficacy, generally suffer from drawbacks such as insufficient stability, potential skin irritation, or long-term safety concerns. Thus, discovering novel tyrosinase inhibitors from natural products not only holds promise for overcoming the limitations of existing synthetic drugs but also provides a highly promising research direction for developing safer and more effective skin-whitening products and treatments for pigmented diseases. Summary of the Invention

[0005] This invention provides a compound with tyrosinase inhibitory activity, said compound being 5-(4-hydroxyphenyl)-3-O-glucosylvaleric acid, with the following structural formula: .

[0006] This invention provides the use of the above-mentioned compounds in the preparation of drugs with tyrosinase inhibitory activity.

[0007] This invention provides the use of the above-mentioned compound in the preparation of drugs that inhibit melanin production.

[0008] This invention provides the use of the above-mentioned compounds in the preparation of medicaments for treating skin diseases; the skin diseases are related to abnormal pigmentation, particularly those related to abnormal melanin formation.

[0009] This invention provides the application of the above-mentioned compound in the preparation of skin whitening products.

[0010] The present invention provides a dermatological drug containing the above-mentioned compound having tyrosinase inhibitory activity.

[0011] The present invention provides a skin whitening product containing the above-mentioned compound with tyrosinase inhibitory activity.

[0012] This invention provides a method for preparing the above-mentioned compound, the steps of which are as follows: (1) Extraction Star fruit was extracted three times with an ethanol aqueous solution (95%), the extracts were combined, and then concentrated under reduced pressure to remove organic solvents to obtain star fruit extract. (2) Normal phase silica gel column chromatography Star fruit extract was subjected to normal phase silica gel column chromatography, and eluted with elution buffers at a gradient from 100:0 to 60:40 (v / v). The fraction eluted at a v / v ratio of 70:30 was collected. (3) Medium-pressure column chromatography The fraction collected in step (2) was subjected to medium-pressure column chromatography, and eluted with elution buffers in a gradient from a volume ratio of 20:80 to 90:10. The fraction eluted in a volume ratio of 40:60 was collected and then purified by high performance liquid chromatography to obtain the compound.

[0013] In the preparation method of the above compound, in step (2), the eluent is chloroform / methanol.

[0014] In the preparation method of the above compound, in step (3), the eluent is methanol / water; the chromatographic conditions of the high performance liquid chromatography are: 33% methanol, flow rate 6 mL / min, retention time 53 min.

[0015] The beneficial effects of this invention are as follows: This invention provides a novel compound, 5-(4-hydroxyphenyl)-3-O-glucosylvaleric acid, which can significantly inhibit the activity of tyrosinase and has the ability to block tyrosinase from catalyzing the conversion of tyrosine into melanin precursors, thus directly inhibiting melanin production. Therefore, it shows good application prospects and value in skin whitening or skin diseases. Attached Figure Description

[0016] Figure 1 This is the 1H NMR spectrum of compound A.

[0017] Figure 2 This is the carbon NMR spectrum of compound A.

[0018] Figure 3 Nuclear magnetic resonance of compound A 1 H- 1 H COSY diagram.

[0019] Figure 4 This is the HSQC NMR spectrum of compound A.

[0020] Figure 5 The image shows the nuclear magnetic resonance (HMBC) spectrum of compound A. Detailed Implementation

[0021] The materials used in this invention are as follows: Tyrosinase (from mushroom, EC 1.14.18.1) was purchased from Worthington Pharmaceuticals; L-DOPA was purchased from Guangzhou Weijia Technology Co., Ltd.; L-tyrosine and positive control kojic acid were purchased from Sigma-Aldrich; A375 human melanoma cells were purchased from ATCC; DMEM culture medium, fetal bovine serum (FBS), 0.25% trypsin-EDTA, penicillin + streptomycin (double antibiotic), and phosphate buffer were purchased from Thermo Fisher Scientific, USA; β-arbutin and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, USA.

[0022] This invention provides a novel compound, named 5-(4-hydroxyphenyl)-3-O-glucosylvaleric acid, which can be obtained by the following method: Star fruit was extracted three times with an ethanol-water solution (95%). The extracts were combined and then concentrated under reduced pressure to remove the organic solvent, yielding a star fruit extract. The star fruit extract was subjected to normal-phase silica gel column chromatography, eluted with a chloroform / methanol gradient from 100:0 to 60:40 (v / v). Fraction F10, eluted at a chloroform / methanol volume ratio of 70:30 (v / v), was collected. Fraction F10 was then subjected to ODS-A medium-pressure column chromatography, eluted with a methanol / water gradient from 20:80 to 90:10 (v / v). Fraction F10-3, eluted at a methanol / water volume ratio of 40:60 (v / v), was collected. Fraction F10-3 was purified by high-performance liquid chromatography (33% methanol, flow rate 6 mL / min, retention time 53 min) to obtain compound A.

[0023] Structural identification Compound A is a pale yellow oil, ESI-MS m / z 371.13 [MH] - 395.13 [M+Na] + It is speculated that the molecular formula of compound A is C. 17 H 24 O9.

[0024] One-dimensional nuclear magnetic resonance hydrogen and carbon spectra, such as Figure 1 and Figure 2 As shown, the data is presented in Table 1: Table 1. Proton and carbon NMR spectra data

[0025] Nuclear magnetic resonance 1 H- 1 The two-dimensional correlation spectra of H COSY, HSQC, and HMBC are respectively as follows: Figures 3-5 As shown.

[0026] Based on the above spectral data, compound A can be identified as 5-(4-hydroxyphenyl)-3-O-glucosylpentanoic acid. This compound is a new compound, and its chemical structure is as follows:

[0027] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0028] I. Tyrosinase Inhibitory Activity Test The inhibitory rate (IC50) of the test compound on tyrosinase was determined by colorimetry (Masuda et al., 2005) using a 96-well plate. 50 The determination process is as follows: (1) First, prepare 0.1 M phosphate buffer (PBS, pH=6.8) with ultrapure water; then prepare 46 U / mL tyrosinase solution and 2.5 mM L-DOPA solution with PBS.

[0029] (2) The compound sample and the positive control (kojic acid) were diluted with DMSO in equal proportions to prepare test solutions with initial concentrations of 100, 50, 25, 12.5 and 6.25 µM.

[0030] (3) Add the sample according to the following reaction system (DMSO concentration not exceeding 5% of the total system): Experimental group: 2 μL compound + 118 μL PBS + 40 μL enzyme; Experimental blank group: 2 μL compound + 158 μL PBS; Negative control group: 2 μL DMSO + 118 μL PBS + 40 μL enzyme; Negative control group: 2 μL DMSO + 158 μL PBS; Positive control group: 2 μL kojic acid + 118 μL PBS + 40 μL enzyme; Positive control group: 2 μL kojic acid + 158 μL PBS.

[0031] After adding the sample, mix well and incubate at 25°C for 10 minutes. Then add 40 µL of reaction substrate (L-DOPA solution) to each well and react at 25°C for 10 minutes. Then measure the absorbance (OD) of each well at 475 nm using a microplate reader. Inhibition rate calculation formula: Inhibition rate (%) = [1 − (OD) 样品 - OD 样品空白 ) / (OD 阴性对照 - OD 阴性空白 )]× 100.

[0032] Each sample was replicated at least three times independently, and the results were expressed as mean ± standard deviation.

[0033] The test results are shown in Table 2: Table 2. Inhibition rate of the drug at various concentrations

[0034] IC calculation using SPSS software 50 The concentration-inhibition rate data model was established using Probit regression analysis, and the concentration value corresponding to a probability of 0.50 was output.

[0035] Finally, the inhibitory activity (IC50) of compound A against tyrosinase was measured. 50 The concentration was 57.2 µM, and the inhibitory activity (IC50) of the positive control kojic acid against tyrosinase was 57.2 µM. 50 The concentration was 37.7 µM. This indicates that the inhibitory activity of compound A is close to that of kojic acid, demonstrating excellent application prospects and value.

[0036] II. Assay for activity in inhibiting melanin production A375 human melanoma cells in good growth condition were used at a dose of 6 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and cultured for 12 h. After cell attachment, the medium was replaced with 2 mL of serum-free BSA containing different concentrations (400, 200, 100, 50, 25 µM) of β-arbutin and compound A. After culturing for another 24 h, the cells were washed twice with phosphate-buffered saline (pH 7.4), and trypsin-EDTA was added to digest the cells for 6-8 min. Cells were collected in 1.5 mL centrifuge tubes and centrifuged at 1000 rpm for 5 min. The cell pellet was collected, and 1 mL of NaOH solution (1 mol / L, containing 10% DMSO) was added. The mixture was then placed in an 80°C water bath for 30 min to ensure complete cell lysis and dissolution of melanin granules. After centrifugation again, 100 μL of the solution was added to each well of a 96-well plate, and the absorbance was measured at 495 nm using a microplate reader. A control group was used with culture medium containing 0 μg / mL of the drug. Each treatment concentration was tested in triplicate. Melanin production inhibition rate (%) = (A... 495样品 -A 495空白 ) / A 495空白 ×100%.

[0037] The test results are shown in Table 3: Table 3. Inhibition rate of the drug at various concentrations

[0038] The IC50 value was calculated using SPSS software. A concentration-inhibition rate data model was established using Probit regression analysis, and the concentration value corresponding to a probability of 0.50 was output.

[0039] Finally, the inhibitory activity of compound A on melanin production (IC50) was measured. 50 The concentration of β-arbutin was 199.4 µM, and its inhibitory activity against melanin production (IC50) was [missing value]. 50 The concentration was 128.7 µM. This indicates that compound A can significantly inhibit melanin production in A375 human melanoma cells. Although its effect is slightly weaker than β-arbutin, it still demonstrates excellent inhibitory effects, thus showing promising application prospects and value in the fields of melanin production inhibition and skin whitening.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A compound having a tyrosinase inhibitory activity, characterized by, The compound is 5-(4-hydroxyphenyl)-3-O-glucosylpentanoic acid, and its structural formula is as follows: 。 2. Use of the compound of claim 1 in the preparation of a medicament having tyrosinase inhibitory activity.

3. Use of the compound of claim 1 in the preparation of a medicament for inhibiting melanin production.

4. Use of the compound of claim 1 in the preparation of a medicament for treating skin diseases.

5. Use of the compound of claim 1 in the preparation of a whitening product.

6. A dermatological medicament, characterized by, The medicament for treating skin diseases contains the compound of claim 1 having tyrosinase inhibitory activity.

7. A whitening product, characterized by, The whitening product contains the compound of claim 1 having tyrosinase inhibitory activity.

8. A process for the preparation of a compound according to claim 1, characterized in that, The steps are as follows: (1) Immersion extraction Carambola is immersed in an ethanol aqueous solution (95%) for 3 times, and the extract is combined and concentrated under reduced pressure to remove the organic solvent to obtain a carambola extract; (2) Normal phase silica gel column chromatography The carambola extract is subjected to normal phase silica gel column chromatography, eluted with an eluent from a volume ratio of 100:0 to 60:40 by gradient elution, and the eluate at a volume ratio of 70:30 is collected; (3) Medium pressure column chromatography The collected eluate of step (2) is subjected to medium pressure column chromatography, eluted with an eluent from a volume ratio of 20:80 to 90:10 by gradient elution, the eluate at a volume ratio of 40:60 is collected, and then subjected to high performance liquid separation and purification to obtain the compound.

9. The method for preparing the compound according to claim 8, characterized in that, In step (2), the eluent is chloroform / methanol.

10. The method for preparing the compound according to claim 8, characterized in that, In step (3), the eluent is methanol / water; and the chromatographic conditions of the high performance liquid chromatography are as follows: 33% methanol, a flow rate of 6 mL / min, and a retention time of 53 min.

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