Method for rapidly screening and analyzing tyrosinase inhibitor in traditional Chinese medicine
By combining immobilized tyrosinase and liquid technology to apply it to Chinese medicinal materials, the rapid screening and analysis of tyrosinase inhibitors has been solved, and the effect of efficient screening of active ingredients has been achieved.
Patent Information
- Application Number
- CN202510561959.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-07-01
AI Technical Summary
Existing tyrosinase inhibitors are toxic or insufficient in efficacy, making it difficult to effectively treat chronic metabolic diseases and whitening related problems.
Using a method of combining immobilized tyrosinase and liquid technology, tyrosinase inhibitors were quickly screened and analyzed from Chinese herbal medicines to determine their active ingredients.
Rapid screening and analysis of tyrosinase inhibitors was achieved, the complexity and inefficiency of traditional methods were overcome, and 16 potentially active compounds were successfully screened out, and the structure of 9 compounds was determined.
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Figure CN120233025A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of extraction and analysis of active ingredients in traditional Chinese medicine, and particularly relates to a method for rapidly screening and analyzing tyrosinase inhibitors in traditional Chinese medicine, especially a method for rapidly screening and analyzing tyrosinase inhibitors in Bletilla striata. Background Art
[0002] In recent years, the incidence of various chronic metabolic diseases has been increasing continuously, which is mainly due to the dysfunction or overactivity of various functional metabolic enzymes involved in human metabolism. Enzyme inhibitors are important drugs for treating various human diseases. Therefore, inhibiting the catalytic functions of these clinically important enzymes is an important way to treat the above-mentioned disease problems. Tyrosinase, as the key enzyme responsible for melanogenesis, makes it the main target for inhibiting melanin production. Inhibitors acting on tyrosinase have great development potential. Currently, most commercially available whitening agents or cosmetics are based on the inhibitory mechanism of tyrosinase. However, most of the currently known inhibitors have some related toxic effects or insufficient efficacy. For these reasons, the development of tyrosinase inhibitors still needs to be continuously explored.
[0003] Natural active ingredients have occupied a place in today's new drug research and development due to their clear pharmacological effects, diverse skeletal structures, low toxicity, and wide sources. Therefore, the screening of natural active ingredients from traditional Chinese medicines has attracted the attention of researchers. Drug screening at the molecular level is an important means for discovering lead drugs, with advantages such as simple method, clear target, and short time. Currently, the mainly identified drug targets are receptors and enzymes. Traditional Chinese medicine is a complex system, and the compounds it contains usually reach hundreds, and the components are complex, which increases the difficulty of screening active ingredients from it. With the rapid development of technology, a series of methods for screening and identifying active ingredients from complex systems have been developed and applied. Since immobilized tyrosinase can increase the stability and tolerance of the enzyme while quickly and simply separating the enzyme from the solution, it is widely used in inhibitor screening. The affinity-based screening method can directly screen active ligands from complex mechanisms by using the specific recognition ability of the enzyme, which makes this method have significant advantages in the research of natural products. Traditional Chinese medicine Bletilla striata has been shown to have tyrosinase inhibitory activity in many studies, but its specific active ingredients are still unknown. Therefore, the present invention combines immobilized tyrosinase and liquid chromatography-mass spectrometry technology for screening tyrosinase inhibitors in Bletilla striata, and preliminarily determines the active ingredients that play a tyrosinase inhibitory role in Bletilla striata, laying a foundation for the development of whitening drugs. Summary of the Invention
[0004] The object of the present invention is to provide a method for rapidly screening and analyzing tyrosinase inhibitors in traditional Chinese medicines. The method can rapidly screen tyrosinase inhibitors from traditional Chinese medicines by using the immobilized tyrosinase technology;
[0005] Another object of the present invention is to provide a method for rapidly screening and analyzing tyrosinase inhibitors in the extract of Bletilla striata, and to determine the active compounds with tyrosinase inhibitory effect in Bletilla striata.
[0006] In order to achieve the above object of the invention, the present invention adopts the following technical solutions:
[0007] A method for rapidly screening and analyzing tyrosinase inhibitors in traditional Chinese medicines, characterized by comprising the following steps:
[0008] 1) Incubation - separation: Add immobilized tyrosinase to a screening model for co - incubation to obtain an experimental group containing an immobilized tyrosinase - ligand conjugate; separately, add a carrier material with non - immobilized tyrosinase to the same screening model for incubation to obtain a blank control group. Perform a separation operation on the carrier material of immobilized tyrosinase / non - immobilized tyrosinase to make it precipitate, and collect the supernatant to obtain an experimental supernatant and a blank control supernatant;
[0009] 2) LC - MS data collection and analysis: Perform LC - MS scanning and analysis on the experimental supernatant and the blank control supernatant respectively, and preliminarily screen out tyrosinase inhibitors and determine their compound information by comparing the differences in the two groups of experimental data.
[0010] Preferably, in step 1), the screening model is used for extensive screening of small molecule monomers, natural product monomers, mixtures of natural products, and traditional Chinese medicine extracts. In this patent, the extract of the traditional Chinese medicine Bletilla striata is used as the screening model to obtain drugs with effective inhibition of tyrosinase.
[0011] Preferably, in step 1), the screening model is a solution prepared from a traditional Chinese medicine extract with a concentration of 0.3 - 0.6 mg / mL. The solution used is an ammonium acetate buffer solution with a concentration of 40 - 60 mM and a pH value of 7.3 - 7.6.
[0012] Preferably, in step 1), the temperature of the incubation step is 35 - 40 °C, the incubation time is 20 - 40 min, and the mass of the immobilized tyrosinase used is 5 - 30 mg.
[0013] Preferably, in step 1), the immobilized tyrosinase Fe3O4@UiO - 66 - NH2@tyrosinase is immobilized by a magnetic MOF material, and its separation method is precipitation under the condition of external magnet adsorption.
[0014] Preferably, in step 2), before detection by liquid chromatography-mass spectrometry, arbutin is used as a positive control drug to verify the screening model.
[0015] Preferably, in step 2), potential tyrosinase inhibitors are determined by comparing the peak areas of compounds in the mass spectrometry data obtained from the experimental group and the blank control group, and then these inhibitors are extracted to obtain information such as the molecular weight, retention time, and peak area of the compounds.
[0016] Preferably, the comparison of the peak areas of the experimental group and the blank control group samples is observed through the binding rate, and compounds in the traditional Chinese medicine extract that do not react with tyrosinase are used as internal standard substances. The inhibition rate calculation formula is: I(%) = (1 - A i / A0) × 100%, where I% represents the inhibition rate, and A i and A0 represent the peak areas of the products obtained from the enzymatic reaction under the conditions with and without the inhibitor, respectively.
[0017] Preferably, in step (2), the preliminary screened tyrosinase inhibitors are also subjected to UHPLC-QTOF / MS analysis to determine the structures of the tyrosinase inhibitors.
[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0019] (1) The present invention uses magnetic materials to immobilize tyrosinase for screening potential inhibitors. The introduction of magnetic beads enables the enzyme to be quickly separated from the reaction mixture by means of a magnetic field after participating in the reaction, thereby promptly terminating the enzymatic reaction or the reaction between the enzyme and the inhibitor, facilitating the recycling of the enzyme. Moreover, the immobilized tyrosinase has the advantages of good stability and reusability.
[0020] (2) The method for rapidly screening and analyzing tyrosinase inhibitors in traditional Chinese medicine extracts of the present invention effectively overcomes the drawbacks of the traditional screening mode of separating and then detecting the activity of natural active substances from complex traditional Chinese medicine extracts. It has the advantages of a short experimental period and small workload, and can achieve large-scale and high-throughput screening, which has a positive significance for drug development.
[0021] (3) The rapid screening for tyrosinase inhibitors in the extracts of Bletilla striata of the present invention successfully screened 16 potential active compounds, and 9 compounds were determined by secondary mass spectrometry, among which 8 compounds belong to 2-isobutylmalic acid glucosylbenzyl ester compounds. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, but do not constitute a limitation to the present invention. In the drawings:
[0023] Figure 1 The present invention is a flow chart for screening and identifying natural tyrosinase inhibitors by combining immobilized tyrosinase with ultra-high performance liquid chromatography-time-of-flight mass spectrometry model.
[0024] Figure 2 It is the chromatographic peak of the positive drug arbutin (control group) in the supernatant of the experimental group and the blank control group in the specificity experiment.
[0025] Figure 3 Comparison of total ion current chromatograms of the supernatants of the experimental group (A) and the blank control group (B).
[0026] Figure 4 The mass spectrometer peak area integration of the internal standard substance experimental group (A) and the blank control group (B). DETAILED DESCRIPTION
[0027] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental materials used in the following embodiments, unless otherwise specified, are purchased from conventional biochemical reagent stores.
[0028] Example 1: Preparation of Chinese herbal medicine extract
[0029] Weigh 50g of Bletilla striata medicinal material and crush it. Use 80% ethanol to reflux and extract it three times, each time for 1h. Mix the filtrates obtained from the three times and concentrate them into a viscous liquid at 50℃. Transfer the liquid to a watch glass and dry it to obtain a brown extract. Accurately weigh 10mg of the extract and dissolve it with 1mL of methanol by ultrasonic. Then take 250μL of the alcohol solution and dissolve it in 4mL of PBS buffer solution and make up to 5mL. Centrifuge the obtained aqueous solution of Bletilla striata extract at 12000g for 10min, collect the supernatant as the test solution for inhibitor screening experiment, and store it at 4℃ for later use.
[0030] Example 2: Specificity verification of immobilized tyrosinase
[0031] Accurately take two identical portions of 5 mg of the buffer solution of immobilized tyrosinase. Heat one portion at 100 °C for 1 hour to inactivate it as the enzyme-inactivated control group. After uniformly dispersing the two portions of immobilized tyrosinase in the buffer solution, use an external magnet to adsorb the immobilized tyrosinase to remove the supernatant. Then, add a 0.1 mg / mL arbutin solution to each of them, vortex for 2 min to mix well, and incubate at 37 °C for 30 min. After the incubation, use an external magnet to adsorb the Fe3O4@UiO-66-NH2-immobilized tyrosinase and the carrier material Fe3O4@UiO-66-NH2, take the supernatant for liquid phase analysis, and repeat each experiment three times.
[0032] It can be seen from Figure 2 that the HPLC peak area of arbutin in the supernatant after incubation of the inactivated group is basically the same as that of the control group, while the peak area of the supernatant after incubation of the active immobilized tyrosinase is significantly smaller than that of the control group, indicating that arbutin has a specific binding with the immobilized tyrosinase.
[0033] Example 3: Inhibitory effect of Bletilla striata extract on tyrosinase
[0034] Add a mixture of the substrate L-tyrosine and the Chinese herbal medicine extract to a centrifuge tube containing 5 mg of immobilized tyrosinase and incubate at 37 °C for 15 min. The final concentration of the Chinese herbal medicine is 5 mg / mL, and the concentration of the substrate L-tyrosine is 0.2 mg / mL. Then, use an external magnet to adsorb and separate the immobilized tyrosinase, and perform liquid phase determination on the supernatant. Calculate the inhibition rate by comparing the peak areas of the product L-DOPA in the supernatants of the blank group and the Chinese herbal medicine group.
[0035]
[0036] where I% represents the inhibition rate, and A i and A0 represent the peak areas of the products obtained from the enzymatic reactions under the conditions with and without the inhibitor, respectively. The inhibition rate of Bletilla striata on tyrosinase was calculated to be 30.1 ± 1.4%.
[0037] Example 4: Screening and identification of inhibitors in Bletilla striata extract
[0038] Accurately take 30 mg of the immobilized tyrosinase and the carrier material, and uniformly disperse them in 1 mL of Bletilla striata extract, respectively, and incubate at 37 °C for 30 min. After the incubation, use an external magnet to adsorb the immobilized tyrosinase and the carrier material, and take the supernatant for UHPLC-QTOF analysis. The obtained characteristic chromatogram is as Figure 3As shown, in the characteristic spectra of the experimental group and the blank control group, the compound at a retention time of 22.97 min was used as the internal standard substance, which did not react with tyrosinase. Therefore, by comparing the integration of its peak area, it was determined that the initial concentrations of the blank group and the experimental group were the same, as Figure 4 shown. The integral of the internal standard substance in the immobilized tyrosinase group was 4.38e 6 , while the integral of the internal standard substance in the blank control group was 4.42e 6 . The influence of the carrier material Fe3O4@UiO-66-NH2 on the compound content was less than 1%, so it could be ignored. From Figure 3 , it was found that the contents of multiple ligand components decreased significantly. They were recorded, and it was speculated that they might be active components with tyrosinase inhibitory effects. A mass spectrometry database was established through relevant data to analyze and identify these active components. A total of 16 compounds were found to have significantly decreased contents. Nine of these compounds were identified through comparison. The detailed information such as their first-order and second-order mass spectra is shown in Table 1. Seven of these compounds were not detected and remained to be further studied.
[0039] Table 1 Identification of the chromatographic peaks of sixteen tyrosinase inhibitors in the total ion current chromatogram of the Bletilla striata extract
[0040]
[0041] Through the analysis of the liquid chromatography-mass spectrometry data, we know that for the nine identified compounds, except for compound 4, the rest belong to the class of 2-isobutylmalic acid glucosyloxybenzyl esters, which are mainly represented by Militarine in Bletilla striata. In recent years, studies have found that this class of compounds has relatively novel chemical compositions and has been verified to have pharmacological activities such as neuroprotective function, anti-inflammatory, and intelligence-enhancing effects. This class of compounds is mainly composed of 2-isobutylmalic acid, glucose, and benzyl linked by an ester bond. Therefore, when cleaved, this class of compounds mainly breaks preferentially at the ester bond connection and the glycosidic bond connection.
[0042] In this study, through rapid screening at the molecular level, it was considered that they also had tyrosinase inhibitory effects and could be used in the subsequent development of whitening products and food preservation, etc. In addition, the compound Coelonin screened in this study was also screened as a tyrosinase inhibitor in the experiments of Chu et al. At the same time, in the further in vitro half-inhibitory concentration test experiment, it was confirmed that the inhibitory effect of this compound was stronger than that of arbutin, which also indicated the success of this screening model and enhanced the credibility of the screening results.
Claims
1. A method for rapid screening and analysis of tyrosinase inhibitors in traditional Chinese medicine, characterized in that: The steps include: 1) Incubation-separation: adding tyrosinase immobilized by metal skeleton material to the screening model for incubation to obtain an experimental group containing immobilized tyrosinase-ligand conjugate; adding carrier material without immobilized tyrosinase to the same screening model for incubation to obtain a blank control group, performing separation operation on the experimental group / blank control group to precipitate, collecting the supernatant, and obtaining the supernatant of the experimental group and the supernatant of the blank control group; 2) LC-MS data acquisition and analysis: LC-MS scanning and analysis were performed on the supernatant of the experimental group and the supernatant of the blank control group, respectively, and the differences in the experimental data of the two groups were compared to preliminarily screen out tyrosinase inhibitors and determine their compound information.
2. The method for rapid screening and analysis of tyrosinase inhibitors in traditional Chinese medicine according to claim 1, characterized in that: In step 1), the screening model is for small molecule monomers, natural product monomers, mixtures of natural products or traditional Chinese medicine extracts.
3. The method for rapid screening and analysis of tyrosinase inhibitors in traditional Chinese medicine according to claim 2, characterized in that: In step 1), the screening model is the extract of medicinal material Bletilla striata.
4. The method for rapid screening and analysis of tyrosinase inhibitors in traditional Chinese medicine according to claim 3, characterized in that: In step 1), the concentration of the Bletilla striata extract is 0.3-0.6 mg / mL, and the solution used is an ammonium acetate buffer solution with a concentration of 40-60 mM and a pH value of 7.3-7.
6.
5. The method for rapid screening and analysis of tyrosinase inhibitors in traditional Chinese medicine according to claim 1, characterized in that: In step 1), the incubation temperature is 35-40° C., the incubation time is 20-40 min, and the mass of the immobilized tyrosinase used is 5-30 mg.
6. The method for rapid screening and analysis of tyrosinase inhibitors in traditional Chinese medicine according to claim 1, characterized in that: In step 1), the immobilized tyrosinase is Fe3O4@UiO-66-NH2@tyrosinase that can be adsorbed by a magnet.
7. The method for rapid screening and analysis of tyrosinase inhibitors in traditional Chinese medicine according to claim 1, characterized in that: In step 2), before the detection by liquid chromatography-mass spectrometry, arbutin was used as a positive control drug to verify the screening model.
8. The method for rapid screening and analysis of tyrosinase inhibitors in traditional Chinese medicine according to claim 1, characterized in that: In step 2), the potential tyrosinase inhibitor is determined by comparing the peak area of the compound in the mass spectrometry data obtained from the experimental group and the blank control group, and the compound is extracted to obtain the molecular weight, retention time and peak area of the compound.
9. The method for rapid screening and analysis of tyrosinase inhibitors in traditional Chinese medicine according to claim 8, characterized in that: The peak area comparison of the experimental group and the blank control group samples was observed by binding rate, and the compound in the Chinese medicine extract that does not react with tyrosinase was used as the internal standard substance. The inhibition rate was calculated by the formula: I (%) = (1-A i / A0)×100%, where 1% represents the inhibition rate, A i and A0 represent the peak areas of the products obtained by the enzymatic reaction with and without inhibitors, respectively.
10. The method for rapid screening and analysis of tyrosinase inhibitors in traditional Chinese medicine according to claim 1, characterized in that: Step (2) also includes a step of subjecting the preliminarily screened tyrosinase inhibitor to MS / MS analysis to determine the structure of the tyrosinase inhibitor.