Immobilized tyrosinase complex and its preparation method and application

Abstract

This invention discloses an immobilized tyrosinase complex, its preparation method, and its application. 2-Methylimidazole, 3-methyl-1H-1,2,4-triazole, and 5-methyltetrazolium are dissolved in methanol, and a methanol solution of zinc sulfate heptahydrate is added. The mixture is stirred at room temperature for 2 h, then the solid product is collected by centrifugation, washed, and dried to obtain a multi-component mesoporous metal-organic framework material (mMOF). The mMOF is dispersed in a tyrosinase solution and incubated in a constant temperature shaking incubator. The solid product is then collected by centrifugation and washed with Tris-HCl buffer to obtain the immobilized tyrosinase complex mMOF@Tyrosinase. This invention successfully constructs a highly active and stable immobilized enzyme system and successfully applies it to the rapid and precise screening of tyrosinase inhibitors from the roots of Bletilla striata, achieving high-throughput, low-false-positive target fishing.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and natural drug screening technology, specifically relating to an immobilized tyrosinase complex and its preparation method, as well as its application in screening tyrosinase inhibitors from Bletilla striata roots. Background Technology

[0002] Tyrosinase is a key rate-limiting enzyme in melanin biosynthesis and has become an important target for skin whitening and improving pigmentation disorders. Currently available synthetic tyrosinase inhibitors (such as hydroquinone) are effective, but pose safety risks such as skin irritation and cytotoxicity. Naturally derived tyrosinase inhibitors (such as glycyrrhizin) have attracted widespread attention due to their high safety profile; however, the development cycle for single active ingredients is long and costly, making it difficult to meet market demand. Therefore, the efficient and rapid screening of novel tyrosinase inhibitors from natural plants has significant research value and application prospects.

[0003] Existing screening technologies, such as affinity ultrafiltration and high-resolution bioactivity spectroscopy, while each with its own advantages, generally suffer from low enzyme reusability and poor stability. Target fishing technology based on immobilized enzymes can significantly improve enzyme stability and reusability, but traditional immobilization carriers still need improvement in immobilization efficiency, enzyme activity recovery rate, and mass transfer efficiency. Metal-organic frameworks (MOFs), as novel porous materials, have shown potential in the field of enzyme immobilization, but the microporous structure of conventional MOFs (such as ZIF-8) easily leads to enzyme molecule blockage and impaired mass transfer, and their strongly alkaline microenvironment is not conducive to maintaining enzyme activity.

[0004] Therefore, developing an immobilized tyrosinase system with high enzyme activity recovery rate, excellent mass transfer performance and good stability, and combining it with an efficient target fishing strategy, is of great significance for screening tyrosinase inhibitors from complex natural products. Summary of the Invention

[0005] This invention provides a target fishing strategy based on immobilized tyrosinase from multi-component mesoporous structures for the efficient screening of tyrosinase inhibitors from the rootlets of Bletilla striata. The core of this strategy lies in constructing an immobilized tyrosinase complex with high enzyme activity recovery, excellent mass transfer performance, and good stability, and coupling it with high-resolution mass spectrometry to achieve rapid capture and identification of active components in complex natural products.

[0006] The technical solution of the present invention is as follows: An immobilized tyrosinase complex was prepared by the following method: (1) Construction of multi-component mesoporous metal-organic framework materials 2-Methylimidazole, 3-methyl-1H-1,2,4-triazole and 5-methyltetrazole were dissolved in methanol, and a methanol solution of zinc sulfate heptahydrate was added. The mixture was stirred at room temperature for 2 h, and then the solid product was collected by centrifugation, washed and dried to obtain a multi-component mesoporous metal-organic framework material, denoted as mMOF. The preferred molar ratio of 2-methylimidazolium, 3-methyl-1H-1,2,4-triazole, 5-methyltetrazole, and zinc sulfate heptahydrate is 10:10:10:3; (2) Preparation of immobilized tyrosinase complex The mMOF obtained in step (1) was dispersed in a tyrosinase solution and incubated in a constant temperature shaking incubator. The solid product was then collected by centrifugation and washed with Tris-HCl buffer (to remove unbound enzyme) to obtain the immobilized tyrosinase complex, denoted as: mMOF@Tyrosinase. The tyrosinase solution was prepared by dissolving tyrosinase in 0.05 M phosphate-buffered saline (PBS) at pH 6.5; preferably, the concentration of tyrosinase in the tyrosinase solution was 1.5 mg / mL. The preferred mass-to-volume ratio of mMOF to tyrosinase solution is 10:1, mg / mL; It is preferred to incubate in a constant temperature shaking incubator at 35 ℃ for 60 min.

[0007] The immobilized tyrosinase complex described in this invention can be used to screen for tyrosinase inhibitors in the roots of Bletilla striata using a target-fishing strategy. The specific method is as follows: (1) Preparation of Bletilla striata root extract Take the powder of Bletilla striata rootlets and extract it with ultrasonically (40 °C, 40 min) using 70% ethanol aqueous solution. Concentrate the extract under reduced pressure to obtain crude extract. Disperse the crude extract in water and extract it successively with petroleum ether, ethyl acetate and n-butanol. Collect the ethyl acetate extract, concentrate and dry it to obtain the test sample. (2) Target fishing The test sample obtained in step (1) was prepared into a test solution with Tris-HCl buffer. The test solution was incubated with mMOF@Tyrosinase to form an immobilized enzyme-ligand complex. After centrifugation, the sample was washed with Tris-HCl buffer (to remove non-specifically adsorbed components), the ligands bound to the immobilized enzyme were desorbed, and the eluent was collected. Preferred incubation conditions: 35 ℃, 1 h; The specific desorption procedure is as follows: the immobilized enzyme-ligand complex collected by centrifugation and washed is added to methanol (desorbent), soaked for 10 min, and the supernatant is collected as the desorption solution; (3) Analysis of active ingredients The desorption solution obtained in step (2) was analyzed by UHPLC-Q-Orbitrap-MS / MS. Heat-inactivated mMOF@Tyrosinase was used as a control group. Potential inhibitors that specifically bind to tyrosinase were screened based on the calculated binding degree (BD).

[0008] Based on the above-mentioned target fishing method, this invention screened 14 compounds with strong interactions with tyrosinase from the ethyl acetate fraction of Bletilla striata rootlets, with binding rates ranging from 17.83% to 92.89%. Their chemical structures were identified by using precise molecular weight and secondary fragment ion information provided by high-resolution mass spectrometry, combined with comparisons with literature data and a self-built database. These compounds mainly include 13 phenanthrene compounds and 1 bibenzyl compound.

[0009] The tyrosinase inhibitors screened in this invention can be used to prepare skin whitening products or drugs for treating pigmentation disorders. The isolation and activity verification process for potential tyrosinase inhibitors includes: (1) Molecular docking: The three-dimensional structures of the 14 potential tyrosinase inhibitors identified were subjected to molecular docking simulation with the crystal structures of tyrosinase obtained from the protein database to analyze the interaction modes such as binding energy, hydrogen bond, and hydrophobic interaction between the ligand and the enzyme active site. (2) Separation and purification of active substances: Based on the identification results, compounds with high binding degree or representativeness were selected, and three compounds were separated and purified from the rootlets of Bletilla striata by high-speed countercurrent chromatography and semi-preparative high-performance liquid chromatography: 3,4-dimethoxyphenanthrene-2,7-diol, 3,3'-dihydroxy-5-methoxybibenzyl and Bletilla bifenthrin A; (3) In vitro activity verification: In vitro tyrosinase inhibition experiment was conducted with levodopa as substrate and kojic acid as positive control. The half-maximal inhibitory concentration (IC50) of the isolated compound monomers was determined. Among them, Bletilla striata A showed the strongest inhibitory activity, with an IC50 of 5.56 ± 0.61 μM, which was better than the positive control kojic acid (IC50 = 11.01 ± 0.95 μM).

[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention successfully constructs a highly active and stable immobilized enzyme system, which is significantly superior to the existing technology and solves the problems of easy inactivation and difficult recovery of free enzymes.

[0011] 2. The immobilized tyrosinase complex of this invention has been successfully applied to the rapid and accurate screening of tyrosinase inhibitors in the roots of Bletilla striata, achieving high-throughput, low-false-positive target fishing.

[0012] 3. This invention has discovered an inhibitor with superior activity compared to the positive control, providing a highly promising candidate compound for the development of novel skin whitening products. Attached Figure Description

[0013] Figure 1 PXRD (A) and ATR-FTIR spectra (B) of mMOF and mMOF@Tyrosinase.

[0014] Figure 2 Field emission scanning electron microscope images of mMOF and mMOF@Tyrosinase.

[0015] Figure 3 XPS spectra of mMOF and mMOF@Tyrosinase; where (A) is the full XPS spectrum and (B) is the fine Zn 2p spectrum.

[0016] Figure 4 Nitrogen adsorption-desorption curves and pore size distribution of mMOF.

[0017] Figure 5 Comparison of mMOF fixation efficiency (B) and enzyme activity recovery rate (A).

[0018] Figure 6 Effect of incubation time (A) and enzyme concentration (B) on immobilization loading rate.

[0019] Figure 7 : Effect of buffer pH (A) and incubation temperature (B) on enzyme activity recovery.

[0020] Figure 8 Cyclic stability (A) and storage stability (B) of mMOF@Tyrosinase.

[0021] Figure 9 Tyrosinase inhibition curves of extracts from different polar parts of Bletilla striata rootlets.

[0022] Figure 10 Screening of tyrosinase inhibitors from the rootlets of Bletilla striata.

[0023] Figure 11 : The structural formulas of the compounds screened and identified.

[0024] Figure 12 Molecular docking diagram of potential tyrosinase inhibitors with tyrosinase.

[0025] Figure 13 : Tyrosinase inhibitory activity curves of nudol, batatasin III, blestriarene A and kojic acid. Detailed Implementation

[0026] The present invention is further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] Example 1: Preparation of multi-component mesoporous metal-organic framework (mMOF) materials and immobilized tyrosinase

[0028] (1) Synthesis of mMOF: 2-methylimidazolium (2-MeIm, 0.8211 g, 10 mmol), 3-methyl-1H-1,2,4-triazole (3-MTZ, 0.8309 g, 10 mmol), and 5-methyltetrazole (5-MTA, 0.8408 g, 10 mmol) were dispersed in 90 mL of methanol. Then, 90 mL of methanol solution containing zinc sulfate heptahydrate (0.8626 g, 3 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the product was collected by centrifugation and washed three times each with methanol and purified water to obtain mMOF.

[0029] (2) Immobilization of tyrosinase: Tyrosinase was dissolved in 0.05 M, pH 6.5 PBS buffer solution to prepare an enzyme solution with a mass concentration of 1.5 mg / mL. 10 mg of the above mMOF was weighed and dispersed in 1 mL of tyrosinase solution, and incubated in a constant temperature shaking incubator at 35℃ for 60 minutes. After incubation, the product was collected by centrifugation and washed three times with 0.05 M, pH 6.5 Tris-HCl buffer solution to obtain the immobilized tyrosinase complex mMOF@Tyrosinase.

[0030] Example 2: Performance Characterization of Immobilized Tyrosinase

[0031] (1) Material Characterization: The functional groups of mMOF, free tyrosinase, and mMOF@Tyrosinase were characterized by ATR-FTIR. PXRD was performed using Cu Kα radiation (wavelength λ = 0.1541 nm) at a working voltage of 40 kV and a current of 40 mA. Diffraction data were acquired in the range of 2θ = 5° – 55° with a step size of 0.0262°. The morphological characteristics of the samples were observed by FE-SEM with an accelerating voltage of 20 kV. The XPS was equipped with a monochromatic aluminum target X-ray source with a working voltage of 15 kV, a current of 3 mA, and an X-ray beam size of 300 × 700 μm2. Charge compensation was performed using a built-in charge neutralization system during the test. The pass energy for the full spectrum scan was set to 160 eV, and the pass energies for the fine spectrum were set to 20 eV, 40 eV, or 80 eV, respectively. The binding energy was corrected using the C-(C, H) component in the C1s peak (BE = 284.8 eV) as the calibration standard, and background correction was performed using the Shirley method. Nitrogen adsorption-desorption tests were performed at -195.80 °C using an ASAP 2010 surface area and pore structure analyzer. Before testing, the samples were degassed at 100 °C for 1 h. Specific surface area and micropore distribution were calculated and analyzed using the Brunauer-Emmett-Teller (BET) model. Results are as follows: Figures 1-4 As shown, this indicates that the material was successfully prepared.

[0032] (2) Enzyme activity recovery and loading rate: 5.0 mg of mMOF@Tyrosinase was accurately weighed and dispersed in 10 mL of 0.8 mM L-DOPA solution (dissolved in Tris-HCl buffer). The reaction system was placed in a constant temperature shaking incubator and reacted for 10 min at 35 ℃ and 200 rpm. After 10 min of reaction, 2 mL of the reaction solution was taken and the absorbance was measured at 475 nm using a microplate reader to calculate the initial reaction rate. To optimize the immobilization conditions, the effects of enzyme solution concentration and incubation time on the immobilization effect were investigated. The immobilization efficiency was evaluated by measuring the mass of tyrosinase immobilized per 10 mg mMOF. In addition, the effects of incubation temperature and buffer pH on the activity during the immobilization process were further studied by comparing the catalytic activity of each 10 mg mMOF@Tyrosinase. Based on this, the enzyme activity recovery rate and loading rate were calculated according to the formulas. The mMOF@Tyrosinase prepared in Example 1 achieved an enzyme activity recovery rate of 87.45% and a loading rate of 92.39% under the optimal conditions of 1.5 mg / mL enzyme concentration, pH 6.5, and 35 ℃. The results are as follows. Figures 5-7 As shown.

[0033]

[0034] Where A represents the activity of mMOF@Tyrosinase and B represents the activity of free tyrosinase.

[0035]

[0036] Where A represents the protein concentration in the tyrosinase solution, and B represents the protein concentration in the supernatant after enzyme fixation.

[0037] (3) Cyclic Stability and Storage Stability: The reusability of the immobilized enzyme was evaluated by conducting multiple catalytic cycles and measuring the residual enzyme activity after each cycle. The immobilized enzyme was washed and recovered after each cycle for the next activity test, with the activity measured at the initial cycle used as a reference. The residual enzyme activity was calculated using a formula, and it was found that it still retained 50.56% of its initial activity after the 10th cycle. Furthermore, the free enzyme and immobilized enzyme were placed separately in Tris-HCl buffer and stored at room temperature. Their residual enzyme activity was measured at set time points to assess their storage stability. After 4 days of storage at room temperature, mMOF@Tyrosinase retained 74.18% of its initial activity, while the free enzyme retained only 50.26% of its initial activity. The results indicate that the immobilized tyrosinase prepared in this invention has excellent reusability and storage stability. The results are as follows: Figure 8 As shown.

[0038]

[0039] Where A represents the activity of mMOF@Tytosinase, and B represents the maximum activity of mMOF@Tyrosinase.

[0040] Example 3: Screening for tyrosinase inhibitors in the roots of Bletilla striata using an immobilized tyrosinase target fishing strategy.

[0041] (1) Preparation and fraction screening of Bletilla striata root extract: 10 g of Bletilla striata root powder was added to 100 mL of 70% ethanol and extracted by ultrasonication at 40 °C for 40 minutes. The crude extract was then concentrated. The crude extract was dispersed in water and extracted successively with petroleum ether, ethyl acetate, and n-butanol to obtain fractions of different polarities. Each dried fraction was sealed and stored in a refrigerator at 4 °C for later use. The ethyl acetate fraction (Fr. EA) showed the strongest tyrosinase inhibitory activity, with an IC50 value of 71.79 ± 8.04 μg / mL. The results are as follows: Figure 9 As shown.

[0042] (2) Target fishing screening experimental procedure: Take 5 mg of Fr. EA sample and prepare a test solution with 1 mL Tris-HCl buffer (2 mg / mL, pH 6.5, containing 10% DMSO). Mix 1 mL of the test solution with 10 mg mMOF@Tyrosinase and incubate at 35℃ for 1 h. After incubation, centrifuge at 12,000 rpm for 5 min, collect the complex, wash 3 times with buffer, and then soak in methanol for 10 min to desorb the bound ligand. Collect the supernatant for UHPLC-Q-Orbitrap-MS / MS analysis. To avoid interference from false positive results, heat-inactivated mMOF@Tyrosinase was set as a control group under the same experimental conditions.

[0043] (3) Analysis and identification of active ingredients: LC-MS / MS analysis was performed using a Q-Exactive high-resolution mass spectrometer coupled with a Dionex Ultimate 3000 ultra-high performance liquid chromatography system. Chromatographic separation was performed using an H&E SP-ODS-A column (4.6 × 250 mm, 5 μm). The mobile phase consisted of 0.1% formic acid aqueous solution (phase A) and acetonitrile (phase B). Elution gradient: 0 min, 5% B; 2 min, 5% B; 5 min, 10% B; 15 min, 20% B; 20 min, 30% B; 35 min, 35% B; 50 min, 40% B; 60 min, 50% B; 63 min, 100% B; 70 min, 100% B; 71 min, 5% B; 75 min, 5% B. The column oven temperature was 30 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL. Mass spectrometry detection was performed using an electrospray ionization (ESI) source in negative ion mode. The relevant parameter settings were as follows: spray voltage 3000 V; collision gas: nitrogen (N2); sheath gas flow rate 40 L / min; auxiliary gas flow rate 10 L / min; sweep gas flow rate 0 L / min; capillary temperature 350 °C. Data acquisition was performed using Full MS-ddMS. 2 Scan mode. MS 1 With MS 2 The scanning range is set to m / z 80-1200, of which MS 1 The resolution is 70,000, MS 2 The resolution was 17,500, the TopN was 5, and the collision energies were 20, 40, and 60 eV, respectively. Data acquisition and processing were performed on an Xcalibur workstation. The results are as follows: Figure 10As shown in Table 1, the binding degree (BD) was calculated by comparing the chromatographic peak areas of the sample group and the heat-inactivated enzyme control group. A total of 14 potential tyrosinase inhibitors with BD values ​​between 17.83% and 92.89% were screened. Based on mass spectrometry fragmentation patterns and literature comparison, 13 phenanthrene compounds and 1 bibenzyl compound were identified. Specific results are shown in Table 1 and... Figure 11 .

[0044] Table 1. Identification of potential tyrosinase inhibitors in the roots of Bletilla striata by UHPLC-Q-Orbitrap-MS / MS

[0045] Example 4: Isolation and Activity Verification of Potential Tyrosinase Inhibitors

[0046] (1) Molecular docking simulation: The interaction between the 13 potential inhibitors screened and tyrosinase was analyzed using molecular docking. The docking results are shown in Table 2. The results show that all candidate compounds can form relatively stable binding conformations with tyrosinase, with predicted binding energies between -7.6 kcal / mol and -11.7 kcal / mol, indicating that the enzyme-ligand complex has good binding stability. To further elucidate its binding mode, the docking conformations were visualized. Figure 12 As shown.

[0047] Table 2 Molecular docking binding energy

[0048] (2) Separation and purification: The target compound was separated by high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography. The high-speed countercurrent chromatography solvent system was n-hexane-ethyl acetate-methanol-water (containing 0.03 M Na2CO3 and 0.07 M NaHCO3) (4:6:4:6, v / v), eluted at a flow rate of 2 mL / min and a rotation speed of 800 rpm. The semi-preparative HPLC used water (phase A) and acetonitrile (phase B) as the mobile phase, eluted with a gradient program of 0 min, 30% B; 30 min, 40% B; 50 min, 45% B; 60 min, 100% B. Chromatographic separation was performed at a constant flow rate of 3.0 mL / min, and the target fraction was collected manually. Finally, 3,4-dimethoxyphenanthrene-2,7-diol (15 mg), 3,3'-dihydroxy-5-methoxybibenzyl (497 mg) and Bletilla striata bifenthrin A (51 mg) were isolated and their structures were confirmed by NMR and HRMS. The corresponding spectroscopic data are shown in Table 3.

[0049] Table 3 Spectral data of the compounds

[0050] (3) In vitro activity verification: An in vitro enzyme inhibition experiment was conducted, using kojic acid as a positive control, to determine the inhibitory effect of the isolated compound on tyrosinase. The specific steps were as follows: 10 μL of the test compound solution, 90 μL of PBS buffer, and 50 μL of tyrosinase solution (71.4 U / mL) were added sequentially to a 96-well plate. After pre-incubation at 30 °C for 10 min, 50 μL of levodopa solution (5 mM) was added to initiate the enzymatic reaction. The absorbance of each well was monitored at 475 nm using a microplate reader. The results are as follows: Figure 13 As shown, Bletilla striata-methyl-2,7-diol, 3,3'-dihydroxy-5-methoxybibenzyl, and 3,4-dimethoxyphenanthrene-2,7-diol all exhibited tyrosinase inhibitory activity, with IC50 values ​​of 5.56 ± 0.61 μM, 81.25 ± 4.99 μM, and 188.05 ± 26.01 μM, respectively. Among them, Bletilla striata-methyl-2,7-diol showed superior activity compared to the positive control kojic acid (IC50 = 11.01 ± 0.95 μM).

Claims

1. An immobilized tyrosinase complex, characterized in that, It is prepared as follows: (1) Construction of multi-component mesoporous metal-organic framework materials 2-Methylimidazole, 3-methyl-1H-1,2,4-triazole and 5-methyltetrazole were dissolved in methanol, and a methanol solution of zinc sulfate heptahydrate was added. The mixture was stirred at room temperature for 2 h, and then the solid product was collected by centrifugation, washed and dried to obtain a multi-component mesoporous metal-organic framework material, denoted as mMOF. (2) Preparation of immobilized tyrosinase complex The mMOF obtained in step (1) was dispersed in a tyrosinase solution and incubated in a constant temperature shaking incubator. The solid product was then collected by centrifugation and washed with Tris-HCl buffer to obtain the immobilized tyrosinase complex, denoted as mMOF@Tyrosinase.

2. The immobilized tyrosinase complex of claim 1, wherein, In step (1), the molar ratio of 2-methylimidazole, 3-methyl-1H-1,2,4-triazole, 5-methyltetrazole, and zinc sulfate heptahydrate is 10:10:10:

3.

3. The immobilized tyrosinase complex of claim 1, wherein, In step (2), the tyrosinase solution is obtained by dissolving tyrosinase in 0.05 M phosphate buffer at pH 6.5; the concentration of tyrosinase in the tyrosinase solution is 1.5 mg / mL.

4. The immobilized tyrosinase complex of claim 1, wherein, In step (2), the mass-to-volume ratio of mMOF to tyrosinase solution is 10:1, mg / mL.

5. The immobilized tyrosinase complex according to claim 1, characterized in that, In step (2), incubate in a constant temperature shaking incubator at 35 ℃ for 60 min.

6. The application of the immobilized tyrosinase complex as described in claim 1 in screening tyrosinase inhibitors in Bletilla striata roots using a target fishing strategy.

7. The application as described in claim 6, characterized in that, The method is as follows: (1) Preparation of Bletilla striata root extract Take the powder of Bletilla striata rootlets and extract it with ultrasonically using a 70% (v / v) ethanol aqueous solution. Concentrate the extract under reduced pressure to obtain a crude extract. Disperse the crude extract in water and extract it successively with petroleum ether, ethyl acetate and n-butanol. Collect the ethyl acetate extract, concentrate and dry it to obtain the test sample. (2) Target fishing The test sample obtained in step (1) was prepared into a test solution with Tris-HCl buffer. The test solution was incubated with mMOF@Tyrosinase to form an immobilized enzyme-ligand complex. After centrifugation, the sample was washed with Tris-HCl buffer, the ligands bound to the immobilized enzyme were desorbed, and the eluent was collected. Incubation conditions: 35 ℃, 1 h; The desorption procedure is as follows: the immobilized enzyme-ligand complex collected by centrifugation and washed is added to methanol, soaked for 10 min, and the supernatant is collected as the desorption solution; (3) Analysis of active ingredients The desorption solution obtained in step (2) was analyzed by UHPLC-Q-Orbitrap-MS / MS. Heat-inactivated mMOF@Tyrosinase was used as a control group. Potential inhibitors that specifically bind to tyrosinase were screened based on the calculated binding degree (BD).

8. The application as described in claim 7, characterized in that, The tyrosinase inhibitors obtained through screening were: 3,4-dimethoxyphenanthrene-2,7-diol, 3,3'-dihydroxy-5-methoxybibenzyl, and leucophenanthrene A; the obtained tyrosinase inhibitors are used to prepare skin whitening products or drugs for treating pigmentation disorders.

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