Screening method of tyrosinase inhibitor

Abstract

The invention provides a screening method of a tyrosinase inhibitor. Relates to the technical field of food. Dopamine-modified carbon quantum dots (DA-CDs) are synthesized in the first step, according to the principle that tyrosinase can quench DA-CDs fluorescence, if a component with tyrosinase inhibitory activity exists in a screened sample, enzyme-aminase can be prevented from quenching DA-CDs fluorescence, and DA-CDs still show fluorescence, so that the DA-CDs can be used for detecting the DA-CDs fluorescence. And screening to obtain a test sample with tyrosinase inhibitory activity. And 2, carrying out immobilization treatment on tyrosinase to obtain an immobilized enzyme, and analyzing the test sample screened in the step 1 by using the immobilized enzyme as a chromatography medium and adopting an affinity chromatography to obtain a component with tyrosinase inhibitory activity, namely the tyrosinase inhibitor. According to the method, the tyrosinase inhibitor in a complex sample can be efficiently and accurately screened.

Description

Technical Field

[0001] The invention belongs to the technical field of food screening, and in particular relates to a method for screening tyrosinase inhibitors. Background Art

[0002] Tyrosinase (TYR), also known as polyphenol oxidase (PPO), belongs to class III copper protein and is a copper-containing oxidase that is found in large quantities in bacteria, fungi, animal and plant tissues. Tyrosinase has dual catalytic activities of monophenolase and diphenolase, catalyzing the hydroxylation of monophenol to catechol (monophenolase activity) and the oxidation of catechol to o-benzoquinone (diphenolase activity). Further oxidation and polymerization of o-benzoquinone leads to the formation of melanin. Tyrosinase (TYR) is a key enzyme in the biosynthesis of melanin, and its abnormality is an important sign of skin pigment abnormalities. When the skin is exposed to ultraviolet light (UV), it causes degradation of the extracellular matrix (ECM) and leads to inflammation. Subsequently, melanocytes are triggered to induce tyrosinase-mediated melanin synthesis to protect the skin. The production of melanin depends on tyrosine levels. L-tyrosine and L-dihydroxyphenylalanine (L-DOPA) are both substrates and intermediates in the melanin synthesis pathway, as well as inducers and positive regulators of melanin production. At the same time, the tyrosinase pathway is involved in the enzymatic process of food browning and is the main cause of food spoilage, which is manifested as a decrease in nutritional value and a deterioration in taste, leading to a gradual shortening of the shelf life. Therefore, the development of tyrosinase inhibitors is of vital importance. At present, the widely used tyrosinase inhibitors are mainly kojic acid and arbutin. Natural products extracted from food have become a hot area for the development of tyrosinase inhibitors because of their abundance and cost-effectiveness. However, due to the diverse and complex chemical composition of food, effective screening of bioactive ingredients is extremely difficult. Therefore, it is crucial to develop an efficient and convenient TYR inhibitor screening method that is suitable for food.

[0003] There are colorimetric method, UV-visible spectrophotometry, electrochemical method and fluorescence method for the activity detection of tyrosinase. Among them, fluorescence method is widely used because of its rapid, sensitive and convenient characteristics. Traditional fluorescence detection methods involve organic fluorescein, precious metal clusters, etc. The carbon quantum dots (CODs) involved in this article have the advantages of low ingredients, stable properties, non-toxicity and high yield. Traditional inhibitor screening is time-consuming, inefficient and expensive. In recent years, inhibitor screening methods based on "protein-ligand" interactions have been developed and proved to be an effective and efficient method for screening potential bioactive substances in food. The enzymes used for screening are usually immobilized on different types of carrier materials, such as hollow fiber tubes, multi-walled carbon nanotubes (MWCNTs), silica and magnetic nanoparticles (MNPs). Carbon nanotubes have the advantages of light weight, stable chemical properties, large cavity area, easy modification and large surface area, and are an ideal carrier material. Immobilizing α-glucosidase on carbon nanotubes (MWCNTs) has been used to screen enzyme inhibitors from complex foods.

[0004] Therefore, the present invention aims to construct an innovative fluorescence sensing system and explore carbon nanotubes as TYR carrier materials for efficient screening of TYR inhibitors in food. This study not only helps to gain a deeper understanding of the mechanism of action of TYR, but also provides a powerful technical tool for the field of food safety testing, thereby contributing to protecting public health. Summary of the invention

[0005] In view of this, the method we developed provides an effective strategy for identifying and screening new tyrosinase inhibitors in food. This method can achieve the screening of tyrosinase inhibitors efficiently and accurately.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows: 1. A method for screening tyrosinase inhibitors, comprising the following steps: (1) Screening of samples with TYR inhibitory activity: dopamine and citric acid were used to synthesize dopamine-modified carbon quantum dots by microwave heating. The synthesized quantum dots were diluted with an appropriate amount of purified water, and the pH was adjusted to 7 with 1.0 M sodium hydroxide. After passing through a microporous filter, the solution was placed at -20 °C for use. Tyrosinase and the test sample solution were added to a centrifuge tube and incubated at room temperature for 10 minutes. Then, the diluted DA-CDs were added and incubated again for 30 minutes. Fluorescence measurement was performed. Samples containing potential active ingredients can be screened out based on the change in the maximum fluorescence value. (2) Separation and identification of ingredients with TYR inhibitory activity: affinity chromatography was used to separate the components that can bind to TYR. First, TYR was immobilized to obtain an immobilized enzyme. The immobilized enzyme was placed in a centrifuge tube. After removing the solvent, the sample with TYR inhibitory activity obtained by step (1) was added. After shaking for sufficient reaction for 30 minutes, the unadsorbed supernatant was discarded by centrifugation, and the solution bound to TYR@MWCNTs was eluted with a suitable solvent. The eluate is injected into a liquid chromatograph and a liquid-mass spectrometer for detection, and components that can bind to TYR are obtained and identified. The half-inhibitory concentration of the selected binding components on TYR is determined, and finally a TYR inhibitor is obtained.

[0007] Preferably, in step (1), the molar mass ratio of dopamine to citric acid is 1:2.

[0008] Preferably, in step (2), the immobilization treatment step is: activating the carboxyl-functionalized MWCNTs with EDC and NHS solution for 0.5-4 hours, then adding TYR, placing the mixture in an oscillator and shaking for 2 hours to fix TYR on the surface of MWCNTs, separating the composite mixture by filtration, and washing it with purified water three times to obtain the immobilized enzyme (TYR@MWCNTs).

[0009] Preferably, in step (2), the volume ratio of tyrosinase, test sample solution and DA-CDs solution is 1:1:17.

[0010] Preferably, the concentration of the tyrosinase solution is 30-70 μg / mL.

[0011] Preferably, the method for screening tyrosinase inhibitors described in step (2) is characterized in that: the affinity chromatography method is specifically: the chromatographic column is XBridge C18 (4.6 mm×150 mm, 5 μm), the mobile phase is 0.3% phosphoric acid: methanol-98:2, the column temperature is 40°C, and the flow rate is 0.8 mL / min.

[0012] Preferably, the method for screening tyrosinase inhibitors in step (2) is characterized in that: the LC-MS method is specifically as follows: the chromatographic column is C18 (3.5 μm; 2.1 mm × 50 mm), the mobile phase A is 0.1% formic acid, the mobile phase B is methanol, and the gradient elution program is 0 min-98% A; 8 min-90% A; 15 min-88% A; 18 min-75% A; 28min-55% A; 35 min-45% A; 50 min-10% A; 55 min-98% A; 60 min-98% A;. The nebulizer pressure is 35 psi, the drying gas flow rate is 8 L / min, the gas temperature is 320 °C, the sheath gas temperature is 350 °C, the gas flow rate is 11 L / min, and the mass range is selected as 50-3000 (m / z).

[0013] It can be seen from the above technical scheme that the present invention provides a method for screening tyrosinase inhibitors, and its beneficial effects compared with the prior art are as follows: the present invention first synthesizes dopamine-modified carbon quantum dots, and tyrosinase can quench the fluorescence of DA-CDs in the presence of dopamine-modified carbon quantum dots (DA-CDs), so as to preliminarily screen out samples containing potential active ingredients in the test samples. Then, the tyrosinase is immobilized to obtain an immobilized enzyme, and the immobilized enzyme is used as an affinity chromatography identification medium, and affinity chromatography is used to separate the screened samples containing potential active ingredients, thereby successfully screening out tyrosinase inhibitors. This method can efficiently and accurately realize the screening of tyrosinase inhibitors, with high screening efficiency and low false positive rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0015] Figure 1 Schematic diagram of the screening process of the present invention.

[0016] Figure 2 Validation of the fluorescence activity assay.

[0017] Figure 3 Positive and negative drug validation for the fluorescence sensing method.

[0018] Figure 4 Results of screening for tyrosinase inhibitors in 21 foods, as well as a screened TYR inhibitor and its half-inhibitory concentration IC 50Determination of value. DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of 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.

[0020] The embodiment of the present invention discloses a method for screening tyrosinase inhibitors using fluorescence sensing and affinity chromatography, and records the inhibitory activity of lycium barbarum obtained by screening on tyrosinase. Example 1

[0021] Optimal dilution experiment of quantum dots. Take 8 EP tubes, add 20 μL of the prepared quantum dot solution to each tube, and dilute them into 1:10, 1:20, 1:40, 1:50, 1:60, 1:70, 1:80, and 1:100 solutions in sequence with PBS buffer solution. Record the maximum fluorescence intensity at 375-625 nm under 325 nm excitation wavelength. The results are as follows: Figure 2 As shown in A. The results show that the maximum fluorescence intensity is the highest at a dilution factor of 1:60. Therefore, a dilution factor of 1:60 is selected. Example 2

[0022] TYR linear range and standard curve experiment. Prepare a series of TYR solutions with concentrations of 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, and 70 μg / mL. Take 50 μL of each solution and add 850 μL of DA-CDs solution at the optimal dilution multiple to mix. Incubate at room temperature for 30 minutes. Record the maximum fluorescence intensity at 375-625 nm under an excitation wavelength of 325 nm. The results are shown in Figure 2 The results show that as the concentration of TYR increases, the maximum fluorescence value gradually decreases, that is, the concentration of TYR and the maximum fluorescence value are negatively proportional, and the linear equation is y = -5.83 x + 609.72, R 2 =0.997. Therefore, the TYR concentration can be 30-70 μg / mL. Example 3

[0023] TYR positive inhibitor verification experiment. Prepare a series of arbutin (TYR positive inhibitor, Arbutin) solutions with concentrations of 0 μg / mL, 50 μg / mL, 100 μg / mL, and 500 μg / mL, take 50 μL of each solution, and then add 50 μL, 50 μg / mL TYR, mix each solution and react at room temperature for 10 minutes. Then add 850 μL of DA-CDs solution at the optimal dilution multiple and mix well. Incubate at room temperature for 30 minutes. Record the maximum fluorescence intensity at 375-625 nm under an excitation wavelength of 325 nm. Results are shown in Figure 3 As shown in A. The results show that with the increase of Arbutin concentration, the maximum fluorescence value gradually increases. The above results indicate that arbutin inhibits TYR activity, resulting in an increase in solution fluorescence. That is, the established fluorescence sensing method can be used to screen TYR inhibitors. Example 4

[0024] TYR negative inhibitor verification experiment. Take 50 μL of arbutin, avibactam sodium, norepinephrine and argatroban, and then add 50 μL of 50μg / mL TYR. Mix the solutions and react at room temperature for 10 minutes. Then add 850 μL of DA-CDs solution at the optimal dilution multiple and mix. Incubate at room temperature for 30 minutes. Record the maximum fluorescence intensity at 375-625 nm under 325 nm excitation wavelength. Results are shown in Figure 3 C. The maximum fluorescence intensity of the mixed solution of avibactam sodium, norepinephrine and argatroban was lower than that of the solution of arbutin. The results of negative inhibitors once again confirmed that the established fluorescence sensing method can be used for the screening of TYR inhibitors. Example 5

[0025] Fluorescence sensing was used to screen TYR inhibitors in food. Take 21 EP tubes, add 50 μL, 50 μg / mL TYR, and then add 50 μL food extracts, including wolfberry, pomegranate, grape, potato, persimmon, kiwi, tomato, leek, cucumber, coriander, green pepper, red pepper, bok choy, white radish, apple, citrus, longan, blueberry, carrot, pepino melon and cherry tomatoes. In addition, positive drug arbutin was used as a control. After mixing the above solutions, react at room temperature for 10 minutes. Then add 850 μL of DA-CDs solution at the optimal dilution multiple and mix well. Incubate at room temperature for 30 minutes. Record the maximum fluorescence intensity at 375-625 nm under 325 nm excitation wavelength. Figure 4 As shown in A. The results show that the maximum fluorescence value of the system solution after adding wolfberry fruit increases significantly, indicating that the activity of TYR decreases after reacting with wolfberry fruit extract, that is, wolfberry fruit extract has the effect of inhibiting TYR activity, and there are components in wolfberry fruit extract that can inhibit TYR activity. Example 6

[0026] Preparation and characterization of affinity chromatography carrier material-immobilized enzyme TYR@MWCNTs. 20 mg of carboxyl functionalized MWCNTs were activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide solution for 2 hours, washed with purified water 3 times, and then 2 mg of TYR was added. The mixture was placed in an oscillator for 2 hours to fix TYR on the surface of MWCNTs. The composite mixture was separated by filtration and washed with purified water 3 times to obtain TYR@MWCNTs immobilized enzyme. The properties of TYR, MWCNTs and TYR@MWCNTs were studied through a series of characterization experiments. The surface morphology of TYR@MWCNTs was characterized by transmission electron microscopy (TEM) (TalosF200S); the elemental composition of MWCNTs and TYR@MWCNTs was analyzed by Thermo Fisher K-ALPHA X-ray photoelectron spectrometer (XPS); the surface structure and properties of MWCNTs and TYR@MWCNTs were studied by Bruker Dimension EDGE atomic force microscopy (AFM). TEM results showed that TYR free enzyme and immobilized enzyme had the same basic morphology without significant difference; XPS analysis results showed that the proportion of oxygen and nitrogen elements in the protein on the surface of MWCNTs increased, further proving the fixation of TYR on the surface of MWCNTs. At the same time, AFM images showed that TYR and TYR@MWCNTs were significantly different. The above results show that TYR was successfully immobilized on the surface of MWCNTs, and the TYR@MWCNTs immobilized enzyme affinity chromatography carrier material was prepared. Example 7

[0027] Affinity chromatography positive and negative adsorption verification experiment. Add 200 μL of arbutin (TYR positive inhibitor, Arbutin), avibactam sodium (Avibactam Sodium), norepinephrine (Norepinephrine) and argatroban (Argatroban) to the 4 EP tubes containing TYR@MWCNTs, mix well and place in an oscillator for 30 minutes, let stand, and centrifuge to obtain the supernatant, which is the non-adsorbed liquid. Then add 200 μL of methanol-0.1% phosphoric acid mixed solution (the volume ratio of the two is 1:1) to the remaining solid, mix well and place in an oscillator at room temperature for 30 minutes, centrifuge to obtain the supernatant, which is the eluent. The above drug initial solution, non-adsorbed solution, and eluent were analyzed by high performance liquid chromatography. The results are shown in Figure 3D (adsorption rate), 3B (a: arbutin initial solution; b: arbutin non-adsorbed solution; c: arbutin eluted solution), 3E (a: avibactam sodium initial solution; b: avibactam sodium non-adsorbed solution), 3F (a: norepinephrine initial solution; b: norepinephrine non-adsorbed solution), 3G (a: argatroban initial solution; b: argatroban non-adsorbed solution). The results show that TYR positive inhibitor arbutin has good adsorption effect on TYR@MWCNTs, while the three TYR negative inhibitors have poor adsorption effect on TYR@MWCNTs. That is, TYR@MWCNTs can be used for affinity chromatography screening of TYR inhibitors. Example 8

[0028] The experiment of screening the components in wolfberry extract that can bind to TYR by affinity chromatography. The wolfberry extract with TYR inhibitory activity obtained by screening in Example 5 was used as the research object. 200 μL of wolfberry extract was added to the EP tube containing TYR@MWCNTs, mixed and placed in an oscillator for 30 minutes, centrifuged to obtain the supernatant (non-adsorbed liquid), and then 200 μL of methanol: 0.1% phosphoric acid-1:1 was added to the remaining solid, mixed and placed in an oscillator at room temperature for 30 minutes, centrifuged to obtain the supernatant (eluent), and the wolfberry extract initial liquid, non-adsorbed liquid, and eluent were respectively analyzed by high performance liquid chromatography. Results are shown in Figure 3 B (a: initial solution of wolfberry extract; b: unadsorbed wolfberry solution; c: wolfberry eluate). The results show that in the HPLC of the wolfberry eluate, a chromatographic peak can be clearly seen, that is, TYR@MWCNTs successfully captured a component in wolfberry that can bind to TYR. Example 9

[0029] Liquid chromatography-mass spectrometry was used to identify the components in wolfberry that can bind to TYR. 200 μL of wolfberry extract was added to the EP tube containing TYR@MWCNTs, mixed and placed in a room temperature oscillator for 30 minutes, centrifuged to obtain the supernatant (unadsorbed liquid), and then 200 μL of methanol-0.1% phosphoric acid mixed solution (the volume ratio of the two was 1:1) was added to the remaining solid, mixed and placed in an oscillator for 30 minutes, centrifuged to obtain the supernatant (eluent), and the wolfberry eluent was analyzed by liquid chromatography-mass spectrometry. The results are shown in Figure 4 C, it was preliminarily determined that the component in wolfberry that can bind to TYR is lycic acid. Example 10

[0030] Liquid chromatography combined with reference substance verification was used to further determine the components in wolfberry that can bind to TYR. Add 200 μL of wolfberry extract to the EP tube containing TYR@MWCNTs, mix well, and place in an oscillator at room temperature for 30 minutes. Centrifuge to obtain the supernatant (unadsorbed liquid). Then add 200 μL of methanol-0.1% phosphoric acid mixed solution (the volume ratio of the two is 1:1) to the remaining solid, mix well, and place in an oscillator for 30 minutes. Centrifuge to obtain the supernatant (eluent). In addition, a lycium barbarum reference substance solution of appropriate concentration was prepared, and the initial solution, unadsorbed solution, eluent and lycium barbarum reference substance solution were respectively analyzed by high performance liquid chromatography. Results are shown in Figure 4 B (a: initial solution of wolfberry extract; b: unadsorbed solution; c: wolfberry eluate; d: lycium barbarum reference solution). The results showed that the chromatographic peak in the eluate was determined to be lycium barbarum. The above results confirmed that the components in wolfberry that can bind to TYR are lycium barbarum. Embodiment 11

[0031] IC50 of TYR inhibitor citric acid 50 Determination experiment. Prepare a series of citric acid reference solutions with concentrations of 100 μg / mL, 250 μg / mL, 500 μg / mL, 600 μg / mL, 800 μg / mL, 1000 μg / mL, 1500 μg / mL, 2000 μg / mL, 2500 μg / mL, 3000 μg / mL, 3500 μg / mL, and 4000μg / mL, take 50 μL of each solution, add 50 μL of 50μg / mL TYR solution, mix well, and react at room temperature for 10 minutes. Then add 850 μL of DA-CDs solution at the optimal dilution multiple and mix well. Incubate at room temperature for 30 minutes. Record the maximum fluorescence intensity at 375-625 nm at an excitation wavelength of 325 nm. Results are shown in Figure 4 D. The results showed that the IC 50 The value was 1.40 mM. The above results further confirmed that lycium barbarum had a good inhibitory effect on TYR activity.

[0032] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0033] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for screening tyrosinase inhibitors, comprising the following steps: (1) Screening of samples with tyrosinase (TYR) inhibitory activity: dopamine-modified carbon quantum dots (DA-CDs) were synthesized by microwave heating using dopamine and citric acid. The synthesized DA-CDs were diluted with an appropriate amount of purified water, the pH was adjusted to 7 with 1.0 M sodium hydroxide, and the mixture was filtered through a microporous filter and stored at -20°C for later use. Tyrosinase and the test sample solution were added to a centrifuge tube and incubated at room temperature for 10 minutes. The diluted DA-CDs were then added and incubated again for 30 minutes. The fluorescence was measured and the samples with tyrosinase inhibitory activity were screened out based on the change in the maximum fluorescence value. (2) Separation and identification of components with TYR inhibitory activity: affinity chromatography was used to separate components that can bind to TYR. First, TYR was immobilized to obtain an immobilized enzyme. The immobilized enzyme was placed in a centrifuge tube. After removing the solvent, the sample with TYR inhibitory activity obtained by step (1) was added. After sufficient oscillation for 30 minutes, the unadsorbed supernatant was discarded by centrifugation. A suitable solvent was used to elute the solution bound to TYR@MWCNTs. The eluate is injected into a liquid chromatograph and a liquid-mass spectrometer for detection, and components that can bind to TYR are obtained and identified. The half-inhibitory concentration of the selected binding components on TYR is determined, and finally a TYR inhibitor is obtained.

2. The method for screening tyrosinase inhibitors according to claim 1, characterized in that: In step (1), the molar ratio of dopamine to citric acid is 1:

2.

3. The method for screening tyrosinase inhibitors according to claim 1, characterized in that: In step (2), the immobilization treatment step is: activating the carboxyl-functionalized multi-walled carbon nanotubes (MWCNTs) with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide solution for 0.5-4 hours, then adding TYR, placing the mixture in an oscillator and shaking for 2 hours to fix TYR on the surface of MWCNTs, separating the composite mixture by filtration, and washing with water 3 times to obtain the immobilized enzyme (TYR@MWCNTs).

4. The method for screening tyrosinase inhibitors according to claim 1, characterized in that: In step (2), the volume ratio of tyrosinase, test sample solution and DA-CDs solution is 1:1:

17.

5. The method for screening tyrosinase inhibitors according to claim 1, characterized in that: The concentration of the tyrosinase solution is 30-70 μg / mL.

6. The method for screening tyrosinase inhibitors according to claim 1, characterized in that: The affinity chromatography method is specifically as follows: chromatographic column: XBridge C18 (4.6 mm×150 mm, 5 μm), mobile phase: 0.3% phosphoric acid: methanol-98:2, column temperature: 40°C, flow rate: 0.8 mL / min.

7. The method for screening tyrosinase inhibitors according to claim 1, characterized in that: The LC-MS method is as follows: the chromatographic column is C18 (3.5 μm; 2.1 mm × 50 mm), the mobile phase A is 0.1% formic acid, the mobile phase B is methanol, and the gradient elution program is 0 min-98% A; 8 min-90% A; 15 min-88% A; 18 min-75% A; 28 min-55% A; 35 min-45% A; 50 min-10% A; 55 min-98% A; 60 min-98% A;. The nebulizer pressure is 35 psi, the drying gas flow rate is 8 L / min, the gas temperature is 320 °C, the sheath gas temperature is 350 °C, the gas flow rate is 11 L / min, and the mass range is selected as 50-3000 (m / z).

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