Dihydrochalcone compound from leucaena leucocephala, preparation method of dihydrochalcone compound and application of dihydrochalcone compound as tyrosinase inhibitor
By extracting the dihydrochalcone compound (±)-4,4'-Methoxy-3,3',β-trihydroxydihydrochalcone from the Leucaena leucocephala plant, the problem of insufficient application of existing tyrosinase inhibitors has been solved, achieving a strong tyrosinase inhibitory effect, which is suitable for applications in multiple fields.
Patent Information
- Application Number
- CN202511509925.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-22
- Publication Date
- 2026-02-10
AI Technical Summary
Existing tyrosinase inhibitors have limited applications in pharmaceuticals, cosmetics, food, and insect control, and are costly, lacking in safety and efficacy, with a lack of effective inhibitors from natural sources.
The dihydrochalcone compound (±)-4,4'-Methoxy-3,3',β-trihydroxydihydrochalcone was extracted from the pods and leaves of the Leucaena leucocephala plant. A compound with significant tyrosinase inhibitory activity was prepared by a multi-step extraction and purification method. It can be used to prepare whitening cosmetics, drugs for treating pigmented skin diseases, insect control agents, or food preservatives.
This compound has a strong inhibitory effect on tyrosinase, with stronger inhibitory activity than kojic acid, and has broad application potential, suitable for whitening cosmetics, treatment of pigmented skin diseases, insect control, and food preservation.
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Figure CN121494714A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of natural medicine technology, specifically relating to a dihydrochalcone compound derived from Albizia julibrissin, its preparation method, and its use as a tyrosinase inhibitor. Background Technology
[0002] Tyrosinase, also known as phenol oxidase or polyphenol oxidase, is widely found in microorganisms, plants, and mammals and is a key enzyme regulating melanin synthesis in organisms. Tyrosinase catalyzes the oxidation of monophenol hydroxyl groups to ortho-diphenols, which are then further oxidized to ortho-diquinones under its catalysis. Following a series of complex non-enzymatic reactions, melanin is ultimately produced. Tyrosinase plays a crucial role in this process.
[0003] As a key enzyme in melanin synthesis, abnormal overexpression of tyrosinase can lead to pigmentation disorders in humans, causing serious health and cosmetic problems such as age spots, dark spots, and freckles. In insects, tyrosinase plays an important role in melanin synthesis, wound healing, and ossification, and is related to tanning during molting, making it an essential enzyme for insect survival. In fruit and vegetable processing and food processing, tyrosinase is the main enzyme causing browning. Its catalytic reaction causes browning in fresh fruits, vegetables, and beverages, thereby reducing their shelf life, nutritional value, and market value.
[0004] Tyrosinase inhibitors can suppress the activity of tyrosinase, thereby inhibiting melanin production and reducing the adverse effects of tyrosinase. Tyrosinase inhibitors can treat common pigmentary skin conditions such as freckles, melasma, and age spots. Because tyrosinase is crucial for insect development, research on its inhibitors also provides guidance for the design of novel biopesticides. Furthermore, by inhibiting tyrosinase activity, tyrosinase inhibitors can prevent or slow down browning during the storage, transportation, and processing of fruits and vegetables, thus showing significant potential as food preservatives.
[0005] Due to the wide range of potential applications of tyrosinase inhibitors, research on tyrosinase inhibitors has been a major focus. However, limited by factors such as efficacy, safety, and cost, only a very small number of tyrosinase inhibitors have been commercially available to date. These include representative examples such as kojic acid, used as a skin whitening agent, and 4-hexylresorcinol, used for food preservation. Therefore, there is an urgent need to develop new tyrosinase inhibitors in fields such as pharmaceuticals, cosmetics, food, and insect control, and tyrosinase inhibitors derived from natural products have broad application prospects.
[0006] Compound 4,4'-Methoxy-3,3', β-trihydroxydihydrochalcone is a characteristic dihydrochalcone compound, first reported in the literature in 2021 (Chem. Pharm. Bull., 2021, 69(1), 32-39). The literature indicated that this compound was isolated from Japanese persimmon and initially showed potential activity in inhibiting cancer cell growth, but no other functional activities have been reported to date. Our recent study found that this compound can also be isolated from the pods and leaves of the rapidly growing and readily available *Leucaena leucocephala* plant. Of particular note is our first discovery that this compound also possesses significant tyrosinase inhibitory activity, indicating its potential as a natural plant-derived tyrosinase inhibitor with promising application prospects. Summary of the Invention
[0007] The first objective of this invention is to provide a solution from silver acacia ( Leucaena leucocephala Preparation of dihydrochalcone compounds (±)-4,4'-Methoxy-3,3' from plant materials (Lam.) deWit. β The method for preparing trihydroxydihydrochalcone, wherein the compound is isolated and prepared from the pods or leaves of the Leucaena leucocephala plant, includes the following steps:
[0008] a. Preparation of total extract: The dried pods or leaves of Leucaena leucocephala are crushed and extracted with ethanol aqueous solution, ethanol, acetone aqueous solution or acetone. The extract is concentrated to remove ethanol or acetone to obtain crude extract of total extract. The crude extract of total extract is suspended in water and extracted with petroleum ether first, and then with ethyl acetate. The ethyl acetate extract is concentrated to obtain ethyl acetate total extract.
[0009] b. Separation and Purification: The total ethyl acetate extract was subjected to normal-phase silica gel column chromatography with chloroform / methanol as the eluent, eluting sequentially at volume ratios of 97:3, 90:10, 85:15, 70:30, 60:40, and 0:100. The fraction eluted with chloroform / methanol at 90:10 v / v was collected. This fraction was then subjected to medium-pressure reversed-phase silica gel column chromatography with methanol / water as the mobile phase, eluting sequentially at volume ratios of 2:8, 3:7, 4:6, 5:5, 6:4, and 10:0. The fraction eluted with methanol / water at 2:8 v / v was collected. This fraction was then subjected to Sephadex LH-20 column chromatography with methanol as the eluent. The eluent was recrystallized to obtain the target compound (±)-4,4'-Methoxy-3,3'. β -trihydroxydihydrochalcone.
[0010] The compound (±)-4,4'-Methoxy-3,3', βThe structural formula of -trihydroxydihydrochalcone is shown in formula (Ⅰ):
[0011] Preferably, the ethanol aqueous solution or acetone aqueous solution is an ethanol aqueous solution or acetone aqueous solution with a volume fraction of 70% or greater.
[0012] The compound of this invention, (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone, as confirmed by in vitro pharmacological experiments, has a potent inhibitory effect on tyrosinase, with an inhibitory activity (IC50) of [missing value]. 50 =9.45±4.72 μM) was stronger than the positive control kojic acid (IC50, 9.45±4.72 μM). 50 =12.65±8.32 μM). Therefore, this compound has broad application potential as a whitening cosmetic, a drug for treating pigmented skin diseases, an insect control agent, or a food preservative.
[0013] A second object of the present invention is to provide the compound (±)-4,4'-Methoxy-3,3', β Application of β-trihydroxydihydrochalcone in the preparation of tyrosinase inhibitors.
[0014] Preferably, the tyrosinase inhibitor is used in the preparation of cosmetics, pharmaceuticals, insect control agents, or food preservatives.
[0015] A third object of the present invention is to provide a tyrosinase inhibitor comprising an effective amount of the compound (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone or its medicinal salt is used as the active ingredient.
[0016] A fourth object of the present invention is to provide the compound (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone is used as a tyrosinase inhibitor in the preparation of skin-whitening cosmetics, drugs for treating pigmented skin diseases, insect control agents, or food preservatives.
[0017] A fifth object of the present invention is to provide a whitening cosmetic, a medicament for treating pigmented skin diseases, an insect control agent, or a food preservative, comprising an effective amount of the compound (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone or its medicinal salt is used as the active ingredient.
[0018] The compound of this invention, (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone can be combined with common excipients or carriers to prepare various inhibitors or compositions with significant tyrosinase inhibitory activity. These inhibitors or compositions can be used in multiple fields, including: skin whitening cosmetics, drugs for treating pigmented skin diseases, insect control agents, and food preservatives. These inhibitors or compositions can be in the form of wettable powders, pastes, aerosols, etc.; they can also be in controlled-release or sustained-release formulations or nanoformulations known in the modern cosmetics and pharmaceutical industries.
[0019] This invention uses the pods and leaves of *Leucaena leucocephala*, a plant widely distributed and rapidly growing in southern my country, as raw materials to extract and isolate a potent tyrosinase inhibitor compound (±)-4,4'-Methoxy-3,3'. β -trihydroxydihydrochalcone is abundant in material. This compound (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone exhibits stronger tyrosinase inhibitory activity than the control compound kojic acid, and therefore is highly likely to be further developed into an effective new whitening cosmetic whitening agent, a drug for treating pigmented skin diseases, an insect control agent, or a food preservative, etc., with significant application development potential. Attached Figure Description
[0020] Figure 1 It is the compound (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone 1 H NMR spectrum.
[0021] Figure 2 It is the compound (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone 13 C NMR spectrum.
[0022] Figure 3 It is the compound (±)-4,4'-Methoxy-3,3', β HSQC spectrum of -trihydroxydihydrochalcone.
[0023] Figure 4 It is the compound (±)-4,4'-Methoxy-3,3', βHMBC spectrum of -trihydroxydihydrochalcone.
[0024] Figure 5 It is the compound (±)-4,4'-Methoxy-3,3', β HR-ESI-MS spectrum of 3-trihydroxydihydrochalcone. Detailed Implementation
[0025] The following embodiments are further illustrations of the present invention, but not limitations thereof.
[0026] Example 1: (±)-4,4'-Methoxy-3,3', β Isolation and preparation of -trihydroxydihydrochalcone
[0027] 1. Sources of plant materials
[0028] Silver acacia for extraction ( Leucaena leucocephala The pods and leaves of the plant (Lam.) de Wit. were collected from the South China Botanical Garden, Chinese Academy of Sciences, and identified by the inventor.
[0029] 2. Extraction and Separation
[0030] The sample (dry sample of pods or leaves of *Leucaena leucocephala*, weighing 2.0 kg) was pulverized and extracted three times with 95% ethanol aqueous solution at room temperature. The extracts were combined and concentrated under reduced pressure to remove the organic solvent ethanol, yielding a crude total extract. The crude total extract was suspended in 500 mL of water and extracted with an equal volume of petroleum ether, followed by three extractions with an equal volume of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to obtain a total ethyl acetate extract (35 g). The total ethyl acetate extract was dissolved in 150 mL of chloroform / methanol at a volume ratio of 1:1. Normal-phase silica gel (80-100 mesh) was added at a weight ratio of 1:1.5, and the mixture was evaporated to dryness. The sample was then dry-packed onto a column (200-300 mesh, 1000 g) and loaded onto the column using a gradient elution with mobile phases of chloroform / methanol = 97:3, 90:10, 85:15, 70:30, 60:40, and 0:100 v / v. Based on thin-layer chromatography, six fractions (F1–F6) were collected sequentially according to their polarity, from smallest to largest. Fraction F2 (1.3 g) eluted from chloroform / methanol at 90:10 v / v was further subjected to reversed-phase medium-pressure column chromatography with MeOH / H2O (2:8, 3:7, 4:6, 5:5, 6:4, 10:0 v / v). Elute with the mobile phase and collect the fraction E eluted by MeOH / H2O 2:8 v / v. 2-2 (66 mg), E 2-2Further purification was achieved by Sephadex LH-20 column chromatography (1500 mm × 25 mm id), eluting with methanol. Recrystallization of the eluted fraction yielded the target compound 1, (±)-4,4'-Methoxy-3,3', as shown in formula (Ⅰ). β -trihydroxydihydrochalcone (7 mg was obtained when dried pods were used as the sample material, and 5 mg was obtained when dried leaves were used as the sample material).
[0031] 3. Compound 1 is (±)-4,4'-Methoxy-3,3', β Structural identification of -trihydroxydihydrochalcone
[0032] Compound 1 obtained was a yellow oily substance; [α]D 20 0 (c 0.025, CH3OH), according to HR-ESI-MS m / z 341.1001 [M+Na] + (calcd. for C 17 H 18 The molecular formula C of O6Na (341.0996) was determined. 17 H 18 O6 and a molecular weight of 318. 1 H NMR spectrum, 13 The 1C NMR spectrum, HSQC spectrum, HMBC spectrum, and HR-ESI-MS spectrum are respectively as follows: Figures 1 to 5 As shown, corresponding 1 H NMR (500 MHz, CD3OD), 13 CNMR (125 MHz, CD3OD) data are shown in Table 1: Table 1. Nuclear Magnetic Resonance (NMR) Data of Compound 1
[0033] Based on the above spectroscopic data and a comparative analysis with data from the literature (Chem. Pharm. Bull., 2021, 69(1), 32-39), compound 1 can be identified as (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone, whose structural formula is shown in formula (Ⅰ).
[0034]
[0035] Example 2: Assay of tyrosinase inhibitory activity of compound (±)-4,4'-Methoxy-3,3',β-trihydroxydihydrochalcone
[0036] 1. Reagents and Instruments
[0037] Reagents: Tyrosinase was purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis, USA), L-DOPA and kojic acid were purchased from Aladdin Industrial Corporation (USA), Na2HPO4 and NaH2PO4 were used, and the test compounds were prepared for phytochemical analysis.
[0038] Experimental instrument: ELISA reader (Genois microplate reader, Tecan GENios, Swizerland).
[0039] 2. Testing Methods
[0040] a) Preparation of drug solution: Mix the test compound (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone and kojic acid were prepared into 10 mg / mL solutions using dimethyl sulfoxide (DMSO), 67 mmol / L phosphate buffer (prepared with ultrapure water), 46 U / mL tyrosinase solution (prepared with phosphate buffer), and 2.5 mM L-DOPA (prepared with phosphate buffer).
[0041] (b) A colorimetric method was used to determine the half-maximal inhibitory concentration (IC50) of the test compound against tyrosinase in a 96-well cell culture plate. First, the test sample solution was diluted with phosphate buffer at a specific ratio (DMSO volume should be less than 5%, v / v). 120 µL of the sample solution was added to each well to achieve the following final concentrations: 100 µg / mL, 50 µg / mL, 25 µg / mL, 12.5 µg / mL, 6.25 µg / mL, 3.125 µg / mL, 1.56 µg / mL, and 0.78 µg / mL. Then, 40 µL of tyrosinase (46 U / mL) was added to each well, and the reaction was carried out at 23°C for 10 min. Finally, 40 µL of substrate L-DOPA (2.5 mM) was added. After incubation at 23°C for 10 min, the final measurement was performed using a microplate reader at a wavelength of 475 nm. The positive control was kojic acid, and the negative control was phosphate buffer containing DMSO in the same proportion as the sample solution. All blank controls were phosphate buffer of the same volume instead of the enzyme solution. The inhibition rate of the test compound against tyrosinase was calculated using the following formula: Inhibition rate (%) = (OD0.05)0.05 negative control – OD blank ) – (OD test – OD test control ) / (OD negative blank – OD blank )×100%. The half-maximal inhibitory concentration (IC50) of the test compound against tyrosinase is [value missing]. 50 (This is obtained from the dose-response curve.)
[0042] 3. The experimental results are shown in Table 2. Table 2. (±)-4,4'-Methoxy-3,3', β tyrosinase inhibitory activity of β-trihydroxydihydrochalcone
[0043] Leucaena leucocephala is a rapidly growing plant in South my country, with readily available and abundant raw materials. Tyrosinase is a key enzyme in melanin synthesis in organisms, closely related to human aging, wound healing and development in insects, and browning in fruits, vegetables, and foods. Therefore, tyrosinase inhibitors have broad application potential in the preparation of whitening cosmetics, drugs for treating pigmented skin diseases, insect control agents, and food preservatives. The results of this experiment show that the dihydrochalcone compound (±)-4,4'-Methoxy-3,3', isolated from Leucaena leucocephala... β-trihydroxydihydrochalcone has a significant inhibitory effect on tyrosinase, and its inhibitory activity is even stronger than that of the positive control kojic acid. Therefore, it has strong potential for application development and is expected to be further developed into a new whitening cosmetic enhancer, a drug for treating pigmented skin diseases, an insect control agent, or a food preservative, etc., with a wide range of applications.
[0044] The embodiments of this invention are merely illustrative of the technical solutions of this invention and are not intended to limit the scope of protection of this invention. Any equivalent modifications or substitutions made based on the technical solutions of this invention, as long as they do not depart from the spirit and scope of the technical solutions of this invention, should be included within the scope of protection of the claims of this invention.
Claims
1. A dihydrochalcone compound (±)-4,4'-Methoxy-3,3', β The method for preparing -trihydroxydihydrochalcone is characterized by, The compound was prepared by isolating from the pods or leaves of the Leucaena leucocephala plant, and the specific steps are as follows: a. Preparation of total extract: The dried pods or leaves of Leucaena leucocephala are crushed and extracted with ethanol aqueous solution, ethanol, acetone aqueous solution or acetone. The extract is concentrated to remove ethanol or acetone to obtain crude extract of total extract. The crude extract of total extract is suspended in water and extracted with petroleum ether first, and then with ethyl acetate. The ethyl acetate extract is concentrated to obtain ethyl acetate total extract. b. Separation and Purification: The total ethyl acetate extract was subjected to normal-phase silica gel column chromatography with chloroform / methanol as the eluent, eluting sequentially at volume ratios of 97:3, 90:10, 85:15, 70:30, 60:40, and 0:
100. The fraction eluted with chloroform / methanol at 90:10 v / v was collected. This fraction was then subjected to medium-pressure reversed-phase silica gel column chromatography with methanol / water as the mobile phase, eluting sequentially at volume ratios of 2:8, 3:7, 4:6, 5:5, 6:4, and 10:
0. The fraction eluted with methanol / water at 2:8 v / v was collected. This fraction was then subjected to Sephadex LH-20 column chromatography with methanol as the eluent. The eluent was recrystallized to obtain the target compound (±)-4,4'-Methoxy-3,3'. β -trihydroxydihydrochalcone.
2. The preparation method according to claim 1, characterized in that, The compound (±)-4,4'-Methoxy-3,3', β The structural formula of -trihydroxydihydrochalcone is shown in formula (Ⅰ):
3. The preparation method according to claim 1, characterized in that, The ethanol-water solution or acetone-water solution is an ethanol-water solution or acetone-water solution with a volume fraction of 70% or greater.
4. Compound (±)-4,4'-Methoxy-3,3', β The use of trihydroxydihydrochalcone in the preparation of tyrosinase inhibitors, characterized in that the compound (±)-4,4'-Methoxy-3,3', β The structural formula of -trihydroxydihydrochalcone is shown in formula (Ⅰ):
5. The application according to claim 4, characterized in that, The tyrosinase inhibitor is used in the preparation of cosmetics, pharmaceuticals, insect control agents, or food preservatives.
6. A tyrosinase inhibitor, characterized in that, Contains an effective amount of the compound (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone or its medicinal salt is used as the active ingredient.
7. Compound (±)-4,4'-Methoxy-3,3', β The application of β-trihydroxydihydrochalcone as a tyrosinase inhibitor in the preparation of skin-whitening cosmetics or drugs for treating pigmented skin diseases is characterized by, The compound (±)-4,4'-Methoxy-3,3', β The structural formula of -trihydroxydihydrochalcone is shown in formula (Ⅰ):
8. Compound (±)-4,4'-Methoxy-3,3', β The application of β-trihydroxydihydrochalcone as a tyrosinase inhibitor in the preparation of insect control agents, characterized by, The compound (±)-4,4'-Methoxy-3,3', β The structural formula of -trihydroxydihydrochalcone is shown in formula (Ⅰ):
9. Compound (±)-4,4'-Methoxy-3,3', β The application of β-trihydroxydihydrochalcone as a tyrosinase inhibitor in the preparation of food preservatives is characterized by, The compound (±)-4,4'-Methoxy-3,3', β The structural formula of -trihydroxydihydrochalcone is shown in formula (Ⅰ):
10. A whitening cosmetic, a drug for treating pigmented skin diseases, an insect control agent, or a food preservative, characterized in that, Contains an effective amount of the compound (±)-4,4'-Methoxy-3,3', β -trihydroxydihydrochalcone or its medicinal salt is used as the active ingredient.