CN106955255B

CN106955255B - Pineapple leaf extract and application thereof as tyrosinase inhibitor

Info

Publication number: CN106955255B
Application number: CN201710196026.1A
Authority: CN
Inventors: 纪明慧; 郭飞燕; 陈光英; 吕纪行
Original assignee: Hainan Normal University
Current assignee: Hainan Normal University
Priority date: 2017-03-28
Filing date: 2017-03-28
Publication date: 2020-06-23
Anticipated expiration: 2037-03-28
Also published as: CN106955255A

Abstract

The invention relates to a pineapple leaf extract and application thereof as a tyrosinase inhibitor, and the pineapple leaf extract provided by the invention has a strong inhibition effect on tyrosinase, can be used as a whitening agent in the field of cosmetics, and is natural and non-irritant. The invention effectively solves the problems of waste of pineapple leaf resources, which is a byproduct generated in pineapple processing, and environmental pollution.

Description

Pineapple leaf extract and application thereof as tyrosinase inhibitor

Technical Field

The invention belongs to the field of natural extraction, and particularly relates to an ether extract of a pineapple leaf alcohol extract, a preparation method of the ether extract and application of the ether extract in tyrosinase inhibition.

Background

Melanin (melanin) is mainly produced by melanocytes of the human body, and can reduce the damage of ultraviolet rays to the skin; however, abnormal accumulation of melanin causes hyperpigmentation, which easily causes freckles, age spots, melasma, and the like. Researchers have found that tyrosinase plays a significant role in the biosynthesis of melanin. Tyrosinase plays an important catalytic role in melanin biosynthesis. Tyrosinase can promote L-tyrosine to be converted into L-dopa, L-dopa is converted into dopaquinone through oxidation, and the dopaquinone is polymerized to generate melanin. The inhibition of melanin production is currently achieved mainly by two pathways: one is to prevent the production of melanin (inhibit the activity of tyrosinase); another approach is to facilitate the excretion of the pigment produced. Among them, the former has better effect and is the focus of the current research.

The pineapple leaves have high comprehensive development value, and the fiber extracted from the leaves is an excellent natural antibacterial textile raw material reported in the literature; the residue of the leaves after the fiber extraction can be used for developing feed, methane and organic fertilizer, and realizing comprehensive utilization. However, no report about tyrosinase activity inhibition exists at present, and the tyrosinase activity inhibition is expected to be applied as a whitening agent and applied to the field of whitening cosmetics.

Disclosure of Invention

The invention provides a pineapple leaf extract, which is characterized by being prepared by the following method, wherein the method comprises the following steps:

(1) taking dry pineapple leaves, crushing, adding the crushed pineapple leaves into a solvent, soaking and extracting for 2-4 times at 40-50 ℃, wherein the extraction time is 6-8 hours each time, and the mass-volume ratio (kg/L) of the pineapple leaves to the solvent is 1: 5-20 parts of;

(2) combining the leaching liquor obtained by each soaking extraction in the step (1) to obtain an extracting solution, and filtering to remove solid residues to obtain a filtrate;

(3) concentrating the filtrate obtained in the step (2) under reduced pressure to remove the organic solvent, adding water to disperse the organic solvent, and extracting for 2-4 times by using petroleum ether;

(4) and (4) combining the petroleum ether phases extracted in the step (3), concentrating under reduced pressure, and drying to obtain the pineapple leaf extract.

The solvent in the step (1) is selected from a methanol-ethanol-isopropanol mixed solvent, and the volume ratio of the methanol to the ethanol to the isopropanol is as follows: ethanol: 3-isopropyl alcohol: 6: 1; the amount of water used in the step (3) is 1/3-1/2 of the volume of the organic solvent in the step (1); the dosage of the petroleum ether is 1 to 3 times of the volume of the water; the drying in the step (4) is drying or spray drying.

The pineapple leaf extract provided by the invention has a strong inhibition effect on tyrosinase, can be used as a whitening agent in the field of cosmetics, and is natural and non-irritant. The invention effectively solves the problems of waste of pineapple leaf resources, which is a byproduct generated in pineapple processing, and environmental pollution.

The invention provides a preparation method of a pineapple leaf extract, which is characterized by comprising the following steps:

The invention provides application of a pineapple leaf extract in preparing a whitening agent.

The invention provides application of a pineapple leaf extract in inhibiting tyrosinase.

The mass volume ratio (kg/L) of the pineapple leaves to the solvent is 1: 5-20 refers to that 5-20L of solvent is used for each kilogram of pineapple leaves during single soaking extraction.

Drawings

FIG. 1 is a graph of absorbance versus temperature (A-T) for melanin

FIG. 2 is a graph of absorbance versus reaction time (A-t) for melanin

FIG. 3 is a graph of the absorbance of melanin and the concentration of L-tyrosine (A-C)

FIG. 4 is a graph of absorbance versus L-tyrosine dosage (A-V) for melanin

FIG. 5 is a graph of absorbance of melanin versus potato mass/buffer volume

FIG. 6 GC-MS chart of pineapple leaf extract

FIG. 7 is a graph showing the relationship between the concentration of pineapple leaf extract and the tyrosinase inhibition rate

FIG. 8 is a graph showing the relationship between the concentration of arbutin enzyme and the tyrosinase inhibition rate

Detailed description of the preferred embodiments

The tyrosinase involved in the invention can be extracted from potatoes according to a literature method (the extraction of tyrosinase in plums, luhai swallows, Dongjin dragon. potatoes and the research on the activity thereof [ J ]. Spectroscopy laboratory, 2008, 25 (6): 1040-; or extracting by the following method: cleaning potato, peeling, cutting into pieces, placing in a freezing layer of a refrigerator, and freezing overnight. When needed, the potato is taken out and weighed, and the weight ratio of the potato to the buffer solution [ potato mass: volume of buffer solution is 1: 5(g/mL) ] are put into a stirrer together and stirred; centrifuging the obtained solution at 4000r/min for 5min, and collecting supernatant as experimental sample (i.e. potato extractive solution).

The buffer solution is a mixed phosphate buffer solution with the pH value of 6.86.

The invention extracts ferment from different parts (peel, pulp and leaf) of the pineapple, measures the inhibition effect of the ferment on the tyrosinase activity in the potato, and reflects the inhibition effect by calculating the inhibition rate. Arbutin was used as a positive control to compare the sizes of IC50 (Rehongrong, Yinyingang, Hengchifang, etc.. research on tyrosinase activity inhibition by Nelumbo nucifera extract [ J ]. research and development of natural products, 2011, 23 (06): 1122-1126; filial colour, Gongsheng Zhao, Liangguo Jun, etc.. inhibition by Angelica sinensis extract on tyrosinase [ J ]. daily chemical industry, 2010, 40 (02): 98-100).

The inhibition rate calculation formula is [1- (T2-T1)/(C2-C1) ] × 100%

C1 represents the absorbance value of only the substrate (mixed solution of potato extract and buffer solution), C2 represents the absorbance value containing the substrate and L-tyrosine, and C2-C1 represents the absorbance value of melanin produced after subtracting the absorbance of the substrate. T1 represents the absorbance value containing the substrate and the sample, T2 represents the absorbance value containing the substrate, L-tyrosine and the sample, and T2-T1 represents the absorbance value of melanin remaining after subtracting the absorbance of the sample and the substrate from each other by the inhibition of the sample.

EXAMPLE 1 determination of optimal Activity conditions for tyrosinase to promote L-tyrosine to produce melanin

(1) Effect of temperature on the optimal Activity of tyrosinase

8 clean, dry brown 10mL flasks were taken and divided into 4 groups of 2, designated C1 and C2. The test solutions were added as per table 1:

TABLE 1 composition of the system and amounts of reagents added

The corresponding amount of buffer solution and L-tyrosine solution as the substrate of the reaction system were added to the 4 sets of volumetric flasks according to the above table, then the corresponding amount of potato extract was added, and the 4 sets of solutions were placed at 0 deg.C, 10 deg.C, 30 deg.C, and 40 deg.C, respectively, and after reaction for 35min, the absorbance measurement was immediately carried out (preferably at a measurement wavelength of 475 nm). As in fig. 1.

(2) Effect of System reaction time on the optimal Activity of tyrosinase

Two 100mL clean and dry brown flasks, labeled C1 and C2, were charged with each of the test solutions according to Table 2:

TABLE 2 composition of the system and amounts of reagents added

Adding corresponding amount of buffer solution and L-tyrosine solution into 2 volumetric flasks as substrates of the reaction system according to the table, then adding corresponding amount of potato extract, placing the volumetric flasks in a water bath at 10 ℃, measuring absorbance every 5min, and the total reaction time is 90min (the measurement wavelength is 475 nm). As shown in fig. 2.

(3) Effect of the quantitative concentration of L-tyrosine on the optimal Activity of tyrosinase

Accurately weighing 0.0181g of L-tyrosine powder by using an electronic balance, dissolving and dissolving the L-tyrosine powder in a 100ml volumetric flask by using a buffer solution, and obtaining 1mmol/L of L-tyrosine solution.

And (3) taking 6 clean and dry volumetric flasks of 10mL, adding the solution into 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL and 6.0mL respectively, and fixing the volume to the scale mark by using a buffer solution to obtain the L-tyrosine solution with the mass concentrations of 0.1mmol/L, 0.2mmol/L, 0.3mmol/L, 0.4mmol/L, 0.5mmol/L and 0.6mmol/L respectively.

12 additional 10mL clean and dry brown flasks were divided into 6 groups of 2, labeled C1 and C2. The test solutions were added as per table 3:

TABLE 3 composition of the system and amounts of reagents added

Corresponding amounts of buffer solution and L-tyrosine solution with different substance amount concentrations are added into 4 groups of volumetric flasks as shown in the table above to serve as substrates of a reaction system, and then corresponding amounts of potato extract are added. The flask was placed in a water bath at 10 ℃ and, after reacting for 35min, the absorbance (measurement wavelength: 475nm) was measured immediately. As shown in fig. 3.

(4) Effect of L-tyrosine dosage on the optimal Activity of tyrosinase

10 clean, dry 10mL volumetric flasks were divided into 5 groups of 2, designated C1 and C2. The test solutions were added as per table 4:

TABLE 4 composition of the system and amounts of reagents added

In the table, x is 1.0mL, 1.5mL, 2.0mL, 2.5mL, 3.0mL, corresponding to y is 8.0mL, 7.5mL, 7.0mL, 6.5mL, 6.0mL, 5 groups of volumetric flasks were charged with corresponding amounts of buffer solution and different amounts of L-tyrosine solution (concentration of 0.3mmol/L) as substrates for the reaction system, and then the corresponding amount of potato extract was added. The flask was placed in a water bath at 10 ℃ and, after reacting for 35min, the absorbance (measurement wavelength: 475nm) was measured immediately. As shown in fig. 4.

(5) Effect of Potato quality versus buffer volume ratio on the optimal Activity of tyrosinase

TABLE 5 composition of the system and amounts of reagents added

The weight ratio (g/mL) of the potatoes in the potato extracting solution to the volume ratio of the buffer solution is 1:1, 1:2, 1:3, 1:4 and 1:5 respectively. Corresponding amounts of buffer solution and L-tyrosine solution (concentration of 0.3mmol/L) were added to 5 sets of volumetric flasks as substrates for the reaction system, and then corresponding amounts of potato extract were added. The flask was placed in a water bath at 10 ℃ and, after reacting for 35min, the absorbance (measurement wavelength: 475nm) was measured immediately. As shown in fig. 5.

The determination of the optimal activity condition of tyrosinase for promoting L-tyrosine to generate melanin: the temperature is 10 ℃, the reaction time is 35min, the concentration of an L-tyrosine substance is 0.3mmol/L, the volume dosage of the L-tyrosine is 2.5mL, and the volume ratio of the potato mass to the buffer solution in the preparation of the potato extracting solution is 1: 4.

example 2

(1) Taking 1kg of dried pineapple leaves, crushing, adding 5L of methanol-ethanol-isopropanol mixed solvent (the volume ratio of the three is 3: 6: 1), soaking and extracting for 4 times at 40-50 ℃, wherein the extraction time is 6 hours each time;

(3) concentrating the filtrate obtained in step (2) under reduced pressure to remove organic solvent, adding 2.5L water for dispersing, and extracting with petroleum ether for 2 times, wherein 5L petroleum ether is used each time;

(4) and (3) combining the petroleum ether phases extracted in the step (3), concentrating under reduced pressure, and spray drying to obtain 3.9g of pineapple leaf extract (GC-MS shown in figure 6).

Example 3

(1) Taking 3kg of dried pineapple leaves, crushing, adding 60L of methanol-ethanol-isopropanol mixed solvent (the volume ratio of the three is 3: 6: 1), soaking and extracting for 2 times at 40-50 ℃, wherein the extraction time is 8 hours each time;

(3) concentrating the filtrate obtained in step (2) under reduced pressure to remove organic solvent, adding 20L water to disperse, and extracting with petroleum ether for 4 times, wherein 20L petroleum ether is used each time;

(4) and (4) combining the petroleum ether phases extracted in the step (3), concentrating under reduced pressure, and performing spray drying to obtain 13.7g of pineapple leaf extract.

Example 4

According to the literature (Chinese herbal medicine, 46 th vol, 7 th 949-954)

Weighing 20kg of dried pineapple leaves, crushing, soaking and extracting with 95% ethanol (5L) at room temperature for 3 times, wherein the soaking time is 5, 7 and 7 days respectively, combining extracting solutions, concentrating under reduced pressure to obtain 1.0kg of ethanol extract, dispersing the ethanol extract with water (5L), extracting with petroleum ether for 4 times (5L of petroleum ether is used each time) and ethyl acetate for 4 times (5L of ethyl acetate is used each time), concentrating and drying the petroleum ether phase and the ethyl acetate phase, namely an aqueous phase respectively to obtain 32g of petroleum ether part, 310g of ethyl acetate part and 105g of aqueous phase part.

Example 5

The acetone extract of pineapple leaf is obtained according to the method of patent CN 104523463A.

500.0g of cleaned, dried and crushed pineapple leaves are treated with 10L of acetone and ultrasonic treatment at 50 ℃ for 90min, wherein the power is 144W, and the frequency is 25 Hz. Filtering, extracting the residue for 2 times, mixing the 3 filtrates, concentrating at 50 deg.C under reduced pressure, and freeze drying the concentrated solution to obtain 11.21g dried extract.

According to HPLC analysis, the peak conditions of the HPLC diagrams of the pineapple leaf extracts prepared in the examples 2 and 3 are consistent with 93%, the peak conditions of the pineapple leaf extracts are poor in consistency with those of the petroleum ether part prepared in the example 4, the similarity is only 52%, and the consistency with the ethyl acetate part and the water phase part is even worse and is only 8% -15%; the peak identity with the acetone extract of pineapple leaf of example 5 was also poor, with only 33% similarity.

Example 6

The pineapple leaf extract prepared in example 2 was tested for its effect on tyrosinase activity under the conditions determined in example 1 for optimal activity of tyrosinase to promote L-tyrosine to produce melanin, and arbutin was used as a positive control.

(1) Inhibition of tyrosinase activity by pineapple leaf extract

A25 mg sample of the pineapple leaf extract solid of example 2 was accurately weighed into 1 25mL clean and dry volumetric flask, and dissolved in a buffer solution and fixed in the volumetric flask to obtain a sample solution with a concentration of 1 mg/mL.

Taking 6 clean and dry volumetric flasks of 10mL, respectively adding 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL and 3.0mL of the sample solution into each volumetric flask, fixing the volume to the scale mark by using a buffer solution to obtain 6 flasks with mass concentrations of 0.05mg/mL, 0.1mg/mL, 0.15mg/mL, 0.20mg/mL, 0.25mg/mL and 0.30mg/mL, and labeling the flasks.

Then 24 clean and dry volumetric flasks of 10mL are taken and divided into 6 groups of 4 bottles, which are respectively marked as C₁、C₂、T₁、T₂. The test solutions were added as per table 6:

TABLE 6 composition of the system and amounts of reagents added

Corresponding amounts of buffer solution and L-tyrosine solution (concentration of 0.3mmol/L) as substrates of the reaction system were added to 6 groups of volumetric flasks according to the above table, then corresponding amounts of potato extract (the ratio of the mass of the potatoes to the volume of the buffer solution is 1:4) were added, and equal amounts of sample solutions of different concentrations were added to T1 and T2 in different groups. The flask was placed in a water bath at 10 ℃ and, after reacting for 35min, the absorbance (measurement wavelength: 475nm) was measured immediately. As shown in FIG. 7, the inhibition rate of the petroleum ether extract on tyrosinase was gradually increased when the concentration of the petroleum ether extract was increased from 0.05mmol/L to 0.20mmol/L, and the inhibition rate of the petroleum ether extract on tyrosinase was 90% when the concentration of the petroleum ether extract was increased to 0.20mmol/L, but when the concentration of the petroleum ether extract was more than 0.2mmol/L, the inhibition rate of the petroleum ether extract on tyrosinase was not increased with the increase of the concentration of the petroleum ether extract, and the half inhibition concentration was 0.08mg/mL, i.e., IC₅₀＝0.08mg/mL。

(2) Inhibitory Effect of arbutin on tyrosinase Activity

Taking 1 clean and dry volumetric flask with 25mL, accurately weighing 0.25g of arbutin solid sample, dissolving and dissolving in a buffer solution and fixing in the volumetric flask to obtain a sample solution with the concentration of 10 mg/mL.

Taking 9 clean and dry volumetric flasks of 10mL, adding 0.5mL, 0.8mL, 1.0mL, 1.5mL, 1.8mL, 2.0mL, 2.5mL, 3.0mL and 3.5mL of the solution into each volumetric flask, fixing the volume to the scale mark by using a buffer solution to obtain 9 flasks with mass concentrations of 0.5mg/mL, 0.8mg/mL, 1.0mg/mL, 1.5mg/mL, 1.8mg/mL, 2.0mg/mL, 2.5mg/mL, 3.0mg/mL and 3.5mg/mL respectively, and labeling the flasks.

Then, 36 10mL clean and dry volumetric flasks were divided into 9 groups of 4 bottles, each labeled C1, C2, T1 and T2. The test solutions were added as per table 7:

TABLE 7 composition of the system and amounts of reagents added

Corresponding amounts of buffer solution and L-tyrosine solution (concentration of 0.3mmol/L) as substrates of the reaction system were added to the 9 groups of volumetric flasks according to the above table, then corresponding amounts of potato extract (the ratio of the mass of the potatoes to the volume of the buffer solution is 1:4) were added, and equal amounts of sample solutions of different concentrations were added to T1 and T2 in different groups. The flask was placed in a water bath at 10 ℃ and, after reacting for 35min, the absorbance (measurement wavelength: 475nm) was measured immediately. As shown in FIG. 8, under the optimal condition of tyrosinase activity, the inhibition rate of the sample on the enzyme activity increases with the increase of the concentration when the concentration is between 0.5mg/mL and 2.0mg/mL, and reaches the maximum when the concentration is 2.0mg/mL, and the inhibition rate is 61.68%; when the concentration is between 2.0mg/mL and 3.5mg/mL, the inhibition rate decreases with increasing concentration. In the portion where the inhibition rate was increased, the half-inhibitory concentration was 1.55mg/mL, i.e., IC₅₀＝1.55mg/mL。

Example 7

According to the method in the embodiment 6 of the invention, the petroleum ether part, the ethyl acetate part and the water phase part prepared in the embodiment 4 and the pineapple leaf acetone extract prepared in the embodiment 5 are tested for tyrosinase inhibition activity, and the result shows that the inhibition rate of the water phase part and the pineapple leaf acetone extract on tyrosinase is less than 20% when the concentration is 5 mg/mL; when the concentration of the petroleum ether part is 5mg/mL, the inhibition rate of the petroleum ether part on tyrosinase is less than 50 percent; and when the concentration of the ethyl acetate part is 1mg/mL, the ethyl acetate part shows a certain activation effect on tyrosinase, and the activation rate is 15%.

Claims

1. A pineapple leaf extract, characterized in that the pineapple leaf extract is prepared by a method comprising the steps of:

(1) taking dry pineapple leaves, crushing, adding the crushed pineapple leaves into a solvent, soaking and extracting for 2-4 times at 40-50 ℃, wherein the extraction time is 6-8 hours each time, and the mass volume ratio kg/L of the pineapple leaves to the solvent is 1: 5-20 parts of;

(4) combining the petroleum ether phases extracted in the step (3), concentrating under reduced pressure, and drying to obtain the pineapple leaf extract;

the solvent in the step (1) is selected from a methanol-ethanol-isopropanol mixed solvent, and the volume ratio of the methanol to the ethanol to the isopropanol is as follows: ethanol: 3-isopropyl alcohol: 6: 1;

the amount of water used in the step (3) is 1/3-1/2 of the volume of the organic solvent in the step (1); the amount of petroleum ether is 1-3 times of water volume.

2. The pineapple leaf extract of claim 1, wherein the drying in the step (4) is oven drying or spray drying.

3. Use of the pineapple leaf extract of any one of claims 1 to 2 for the preparation of a whitening agent.

4. Use of the pineapple leaf extract of any one of claims 1 to 2 for the preparation of a tyrosinase inhibiting whitening agent.