阅读说明：本技术 一种反式4-羟基肉桂酸和阿魏酸联合抑制酪氨酸酶活性的方法 (Method for inhibiting tyrosinase activity by combining trans-4-hydroxycinnamic acid and ferulic acid ) 是由 范柳萍 于群 李静 于 2021-01-15 设计创作，主要内容包括：本发明公开了一种反式4-羟基肉桂酸和阿魏酸联合抑制酪氨酸酶活性的方法,属于果蔬精深加工技术领域。本发明所述的方法中采用的酪氨酸酶抑制剂以反式4-羟基肉桂酸和阿魏酸为原料,经过不同配比混合后明显增强了酪氨酸酶活性的抑制能力,混合后的酪氨酸酶抑制率较单一成分提高70％左右。本发明的方法中所述的酪氨酸酶抑制剂中反式4-羟基肉桂酸的浓度为2-12mM,阿魏酸的浓度为2-12mM。本发明的方法或者酪氨酸酶抑制剂可用于抑制果蔬的褐变、延长果蔬产品保质期、制备具有美白功效的化妆品或治疗黑色素分泌过多的药物制剂。(The invention discloses a method for jointly inhibiting tyrosinase activity by trans-4-hydroxycinnamic acid and ferulic acid, belonging to the technical field of deep processing of fruits and vegetables. The tyrosinase inhibitor adopted in the method provided by the invention takes trans-4-hydroxycinnamic acid and ferulic acid as raw materials, the inhibition capacity of the tyrosinase activity is obviously enhanced after the raw materials are mixed according to different proportions, and the inhibition rate of the tyrosinase after mixing is improved by about 70% compared with that of a single component. The concentration of trans-4-hydroxycinnamic acid in the tyrosinase inhibitor in the method is 2-12mM, and the concentration of ferulic acid is 2-12 mM. The method or the tyrosinase inhibitor can be used for inhibiting browning of fruits and vegetables, prolonging the shelf life of fruit and vegetable products, and preparing cosmetics with a whitening effect or pharmaceutical preparations for treating melanin hypersecretion.)

1. A tyrosinase inhibitor characterized by comprising trans 4-hydroxycinnamic acid and ferulic acid.

2. The tyrosinase inhibitor according to claim 1, wherein the trans 4-hydroxycinnamic acid has the formula i:

3. the tyrosinase inhibitor according to claim 1 or 2, wherein the ferulic acid has the formula ii:

4. the tyrosinase inhibitor according to any one of claims 1-3, wherein the concentration of trans 4-hydroxycinnamic acid in said tyrosinase inhibitor is 2-12mM and the concentration of ferulic acid is 2-12 mM.

5. Use of the tyrosinase inhibitor according to any one of claims 1-4 for cosmetic health, for the treatment of pigmented dermatosis, for pest control or for food preservation.

6. A product comprising the tyrosinase inhibitor of any one of claims 1-4.

7. A method of inhibiting tyrosinase activity using trans 4-hydroxycinnamic acid in combination with ferulic acid, which comprises administering a tyrosinase inhibitor according to any one of claims 1 to 4.

8. The method of claim 7, wherein the volume ratio of the tyrosinase inhibitor to the substrate L-DOPA solution is 100-20 μ L: 2.5-3 mL; the concentration of the substrate L-DOPA solution is 2-3 mM.

9. The method as set forth in claim 7, wherein the ratio of the tyrosinase inhibitor to the tyrosinase solution is 100-200: 100-; the concentration of the lactase solution is 0.1-0.3 mg/mL.

10. The method of any one of claims 7 to 9, applied to the deep processing of fruits and vegetables.

Technical Field

The invention relates to a method for jointly inhibiting tyrosinase activity by trans-4-hydroxycinnamic acid and ferulic acid, belonging to the technical field of deep processing of fruits and vegetables.

Background

Tyrosinase, also known as polyphenol oxidase, is an oxidase containing double copper atoms. Tyrosinase can take L-DOPA as a substrate in a human body, melanin is finally generated through multiple oxidation reactions, and excessive melanin accumulation can cause skin diseases such as pigmentation, chloasma, black speck, melanoma and the like; in fruits and vegetables, the browning of fruit and vegetable products can be caused due to catalytic reaction, so that the sensory quality and the nutritional quality of the fruit and vegetable products are reduced. The tyrosinase inhibitor can reduce the adverse effects caused by tyrosinase by inhibiting the activity of tyrosinase. The tyrosinase inhibitors used in the current market are quite limited in types, vitamin C is good in safety but low in inhibition effect, kojic acid is good in inhibition activity but has a carcinogenic effect, and arbutin is good in safety but high in price. Therefore, the development of the tyrosinase inhibitor with high efficiency and low toxicity has important significance and wide application prospect.

In recent years, with the continuous research on tyrosinase inhibitors, phenols have been receiving much attention due to their wide distribution and safety. Synergy or binding is a common method to improve the inhibitory ability of polyphenol tyrosinase. The trans-4-hydroxycinnamic acid and ferulic acid are natural products, have wide sources, are relatively safe and low in toxicity, and have various biological activities. When trans-4-hydroxycinnamic acid or ferulic acid is taken as a tyrosinase inhibitor independently, the problem of poor inhibition effect exists.

Disclosure of Invention

In order to solve at least one problem, the invention provides a method for jointly inhibiting the activity of tyrosinase by trans-4-hydroxycinnamic acid and ferulic acid, which effectively improves the inhibition effect of the trans-4-hydroxycinnamic acid and ferulic acid on the tyrosinase and solves the problem of poor inhibition effect when the trans-4-hydroxycinnamic acid or ferulic acid is used as a tyrosinase inhibitor; in addition, the trans-4-hydroxycinnamic acid and the ferulic acid both belong to natural products, have wide sources, are relatively safe and low in toxicity, and have various biological activities.

The invention provides a tyrosinase inhibitor, which comprises trans-4-hydroxycinnamic acid and ferulic acid, wherein the structural formula of the trans-4-hydroxycinnamic acid is shown as formula I:

the structural formula of ferulic acid is shown as formula II:

in one embodiment of the invention, the concentration of trans-4-hydroxycinnamic acid in the tyrosinase inhibitor is 2-12mM, and the concentration of ferulic acid is 2-12 mM; further preferably, the concentration of trans-4-hydroxycinnamic acid is 6 to 10mM and the concentration of ferulic acid is 6 to 10 mM.

The second purpose of the invention is the application of the tyrosinase inhibitor in the aspects of beauty and health care, treatment of pigmented dermatosis, pest control, food preservation and the like.

A third object of the invention is a product comprising the tyrosinase inhibitor according to the invention.

In one embodiment of the present invention, the product includes a cosmetic having a whitening effect or a pharmaceutical preparation for treating melanin hypersecretion.

The fourth purpose of the invention is to provide a method for inhibiting tyrosinase activity by combining trans-4-hydroxycinnamic acid and ferulic acid, wherein the method adopts the tyrosinase inhibitor disclosed by the invention.

In one embodiment of the invention, the volume ratio of the tyrosinase inhibitor to the substrate L-DOPA solution is 100-20 μ L: 2.5-3 mL.

In one embodiment of the invention, the concentration of the substrate L-DOPA solution is 2-3 mM.

In one embodiment of the present invention, the volume ratio of the tyrosinase inhibitor to the tyrosinase solution is 100-200: 100-200.

In one embodiment of the invention, the concentration of the solution of the aminase is 0.1-0.3 mg/mL.

The fifth purpose of the invention is the application of the method in the aspect of fruit and vegetable deep processing.

In one embodiment of the invention, the application is to inhibit browning of fruits and vegetables or to prolong the shelf life of fruit and vegetable products.

The invention has the beneficial effects that:

(1) the trans-4-hydroxycinnamic acid and the ferulic acid are mixed according to different proportions to prepare the tyrosinase inhibitor, and the effect of the tyrosinase inhibitor prepared by the method is far higher than that of the tyrosinase inhibitor prepared by singly using the trans-4-hydroxycinnamic acid or the ferulic acid.

(2) When the concentration ratio of trans-4-hydroxycinnamic acid to ferulic acid in the tyrosinase inhibitor is respectively increased to 8: 8 and 10: at 10, the inhibition rates of tyrosinase were (79.81 + -4.04%) and (90.44 + -2.34%), respectively.

(3) The tyrosinase inhibitor can be combined with free enzyme or enzyme-substrate complex so as to inhibit the activity of tyrosinase, and KIS (70.97 mu g/mL) > KI (24.44 mu g/mL) indicates that the combination of the tyrosinase inhibitor and the enzyme-substrate complex is dominant.

Drawings

FIG. 1 is a graph obtained by plotting the reciprocal 1/[ S ] of the substrate concentration against the reciprocal 1/v of the reaction rate using the Lineweaver-Burk double reciprocal plot method in example 4.

FIG. 2 is a second plot of the Lineweaver-Burk biperireciprocal plot of example 4 for the inhibition kinetics of tyrosinase for K_IAnd (4) calculating a value.

FIG. 3 is a second plot of the Lineweaver-Burk biperireciprocal plot of example 4 for the inhibition kinetics of tyrosinase for K_ISAnd (4) calculating a value.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

The test method comprises the following steps:

formula for calculation of tyrosinase inhibition: tyrosinase inhibition (%) ([ 1- (B)₂-B₁)/(A₂-A₁)]×100

Wherein A is₁And A₂Denotes the absorbance values of the blank at 0min and 15min, B₁And B₂The absorbance values of the samples at 0min and 15min are shown.

Example 1

A method for inhibiting tyrosinase activity by combining trans-4-hydroxycinnamic acid and ferulic acid, comprising the following steps:

accurately weighing 0.0243g of levodopa L-DOPA powder in a brown reagent bottle by using an electronic balance, and preparing a DOPA solution with the concentration of 2.5mM by using a phosphate buffer solution with the pH of 6.8; then accurately weighing 0.016g of trans-4-hydroxycinnamic acid and 0.019g of ferulic acid in a beaker respectively, fixing the volume to 10mL by using absolute methanol to obtain polyphenol stock solution with the concentration of 10mM, and diluting the stock solution into sample solutions to be detected with the concentrations of 2mM, 4mM, 6mM and 8 mM. Accurately sucking 2.7mL of dopa solution, adding 100 mu L of sample solution to be detected, finally adding 100 mu L of tyrosinase solution with the concentration of 0.2mg/mL, incubating at 30 ℃ for 15 minutes, and determining the inhibition effect of the solution on tyrosinase by a spectrophotometry.

Diluting the solutions after different treatments into different concentrations, and measuring the inhibition effect of the solutions on tyrosinase by a spectrophotometry; the test results are shown in table 1; as can be seen from table 1: the combination of trans-4-hydroxycinnamic acid and ferulic acid can obviously enhance the inhibition effect of the sample on the activity of tyrosinase; when the concentration ratio of trans-4-hydroxycinnamic acid to ferulic acid is increased to 8: 8 and 10: at 10, the inhibition rates of tyrosinase were (79.81 + -4.04%) and (90.44 + -2.34%), respectively.

TABLE 1 inhibition of tyrosinase (0.2mg/mL) by tyrosinase inhibitors at different ratios with L-DOPA concentration of 2.5mM

CA：FA	2：0	4：0	6：0	8：0	10：0
						IR(％)	-17.11±1.34	-27.47±1.53	-18.55±1.30	-9.40±0.98	22.17±1.38
CA：FA	0：2	0：4	0：6	0：8	0：10
						IR(％)	-13.16±1.64	-16.46±1.46	-13.92±1.29	-4.56±1.01	12.15±1.59
CA：FA	2：2	2：4	2：6	2：8	2：10
						IR(％)	-17.67±1.27	-14.32±1.08	-5.82±1.02	23.05±1.58	34.68±1.70
CA：FA	4：2	4：4	4：6	4：8	4：10
						IR(％)	-23.76±1.04	-2.49±1.36	16.74±0.98	41.40±1.01	67.87±1.98
CA：FA	6：2	6：4	6：6	6：8	6：10
						IR(％)	-10.09±4.20	15.60±2.99	48.17±3.98	68.58±3.35	70.36±2.47
CA：FA	8：2	8：4	8：6	8：8	8：10
						IR(％)	8.81±2.84	47.80±4.36	71.93±3.87	79.81±4.04	87.47±4.02
CA：FA	10：2	10：4	10：6	10：8	10：10
						IR(％)	43.82±1.02	55.94±1.09	84.62±3.10	88.58±2.67	90.44±2.34

Note: CA: trans 4-hydroxycinnamic acid; FA: ferulic acid; IR: tyrosinase inhibition rate

Example 2

A method for inhibiting tyrosinase activity by combining trans-4-hydroxycinnamic acid and ferulic acid, comprising the following steps:

accurately weighing 0.0292g of L-DOPA powder in a brown reagent bottle by using an electronic balance, and preparing a DOPA solution with the concentration of 3mM by using a phosphate buffer solution with the pH value of 6.8; then accurately weighing 0.016g of trans-4-hydroxycinnamic acid and 0.019g of ferulic acid in a beaker respectively, fixing the volume to 10mL by using absolute methanol to obtain a polyphenol stock solution with the concentration of 10mM, and then diluting the stock solution into tyrosinase inhibitor solutions with different concentrations (6mM, 8mM and 10 mM); accurately sucking 2.7mL of dopa solution, adding 100 mu L of tyrosinase inhibitor solution, finally adding 100 mu L of tyrosinase solution with the concentration of 0.2mg/mL, incubating at 30 ℃ for 15 minutes, and then measuring the inhibition effect of the tyrosinase inhibitor on tyrosinase by a spectrophotometry, wherein the test results are shown in Table 2.

As can be seen from table 2: when the concentration of L-DOPA is increased to 3mM, the concentration ratio of trans-4-hydroxycinnamic acid to ferulic acid is 10: the tyrosinase inhibition rate at 0 (18.56 +/-1.18%), the concentration ratio of trans-4-hydroxycinnamic acid to ferulic acid is 0: the tyrosinase inhibition rate at 10 hours is (7.87 +/-1.03%), the concentration ratio of the trans-4-hydroxycinnamic acid to the ferulic acid is 10: the tyrosinase inhibition rate at 10 was (86.84. + -. 1.02%). The combination of trans 4-hydroxycinnamic acid and ferulic acid significantly enhanced the inhibitory effect of tyrosinase inhibitors compared to example 1, but the enhancement was not as good as the substrate concentration in example 1.

TABLE 2 inhibition of tyrosinase (0.2mg/mL) by tyrosinase inhibitors at different ratios with L-DOPA concentration of 3mM

CA：FA	6：0	8：0	10：0
				IR(％)	-20.53±1.23	-15.12±0.87	18.56±1.18
CA：FA	0：6	0：8	0：10
				IR(％)	-16.90±2.11	-10.16±0.99	7.87±1.03
CA：FA	6：6	6：8	6：10
				IR(％)	42.71±4.02	62.51±4.34	68.19±3.33
CA：FA	8：6	8：8	8：10
				IR(％)	63.93±2.98	70.81±3.03	80.23±4.22
CA：FA	10：6	10：8	10：10
				IR(％)	74.84±3.00	81.85±2.74	86.84±1.02

Note: CA: trans 4-hydroxycinnamic acid; FA: ferulic acid; IR: tyrosinase inhibition rate

Example 3

A method for inhibiting tyrosinase activity by combining trans-4-hydroxycinnamic acid and ferulic acid, comprising the following steps:

accurately weighing 0.0243g of L-DOPA powder in a brown reagent bottle by using an electronic balance, and preparing a DOPA solution with the concentration of 2.5mM by using a phosphate buffer solution with the pH of 6.8; then accurately weighing 0.016g of trans-4-hydroxycinnamic acid and 0.019g of ferulic acid in a beaker respectively, fixing the volume to 10mL by using absolute methanol to obtain a polyphenol stock solution with the concentration of 10mM, and then diluting the stock solution into tyrosinase inhibitor solutions with different concentrations (6mM, 8mM and 10 mM); accurately sucking 2.7mL of dopa solution, adding 100 mu L of tyrosinase inhibitor solution, finally adding 100 mu L of tyrosinase solution with the concentration of 0.25mg/mL, incubating at 30 ℃ for 15 minutes, and then measuring the inhibition effect of the tyrosinase inhibitor on tyrosinase by a spectrophotometry, wherein the test results are shown in Table 3.

As can be seen from table 3: when the concentration of the tyrosinase solution is increased to 0.25mg/mL, the concentration ratio of trans-4-hydroxycinnamic acid to ferulic acid is 10: the tyrosinase inhibition rate at 0 (15.64 +/-1.12%), the concentration ratio of trans-4-hydroxycinnamic acid to ferulic acid is 0: the tyrosinase inhibition rate at 10 hours is (12.37 +/-2.22%), the concentration ratio of trans-4-hydroxycinnamic acid to ferulic acid is 10: the tyrosinase inhibition at 10 was (84.46. + -. 4.67%). Compared with the examples 1 and 2, the combined action of the trans-4-hydroxycinnamic acid and the ferulic acid can enhance the inhibition effect of the tyrosinase inhibitor on tyrosinase, but the enhancement effect is not as good as the condition in the example 1, and is closer to the effect in the example 2.

TABLE 3 inhibition of tyrosinase (0.25mg/mL) by tyrosinase inhibitors at different ratios with L-DOPA concentration of 2.5mM

CA：FA	6：0	8：0	10：0
				IR(％)	-22.35±2.65	-14.64±3.20	15.64±1.12
CA：FA	0：6	0：8	0：10
				IR(％)	-19.65±3.24	-10.23±1.00	12.37±2.22
CA：FA	6：6	6：8	6：10
				IR(％)	40.01±4.06	60.34±4.28	70.36±3.69
CA：FA	8：6	8：8	8：10
				IR(％)	65.13±2.02	71.29±4.03	78.35±4.45
CA：FA	10：6	10：8	10：10
				IR(％)	70.14±3.40	78.02±3.95	84.46±4.67

Note: CA: trans 4-hydroxycinnamic acid; FA: ferulic acid; IR: tyrosinase inhibition rate

Example 4

The inhibition type of the tyrosinase inhibitor on the tyrosinase is researched by taking L-DOPA as a substrate, the reaction system for evaluating the inhibition activity of the tyrosinase is continuously used, the concentration of the tyrosinase is fixed to be 0.2mg/mL, the concentration of the substrate is changed, and the influence of adding different concentrations of the tyrosinase inhibitor on the activity of the tyrosinase is measured.

As shown in FIGS. 1-3, by using a Lineweaver-Burk double reciprocal plot method, the reciprocal 1/[ S ] of the substrate concentration is plotted against the reciprocal 1/v of the reaction speed, and a group of straight lines with different slopes and intersecting with the second quadrant can be obtained, which indicates that the tyrosinase inhibitor is a mixed inhibitor, i.e., the tyrosinase inhibitor can be combined with both free enzyme and enzyme-substrate complex, thereby inhibiting the activity of tyrosinase. And KIS (70.97 μ g/mL) > KI (24.44 μ g/mL), indicating that inhibitor binding to enzyme-substrate complex predominates.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.