CN117018094A

CN117018094A - Traditional Chinese medicine compound phenolic extract and application thereof in preparation of tyrosinase inhibitor

Info

Publication number: CN117018094A
Application number: CN202311130240.9A
Authority: CN
Inventors: 张丽先; 陈飞; 纵伟; 李飞飞; 王学方; 马艳妮; 李晓; 王伟; 宁二娟; 陈玲; 王建军; 崔炜
Original assignee: Henan Napu Biotechnology Co ltd; Henan Academy of Sciences
Current assignee: Henan Napu Biotechnology Co ltd; Henan Academy of Sciences
Priority date: 2023-09-04
Filing date: 2023-09-04
Publication date: 2023-11-10

Abstract

The application relates to the technical field of medicines, and particularly discloses a traditional Chinese medicine compound phenol extract, which is prepared by adopting an ultrasonic-assisted enzymolysis method and a macroporous adsorption resin method separation and purification method, wherein the extract of multiple sections of components can be extracted from materials of a five-flavor disinfection drink, and the extraction rate of polyphenol can be improved to the greatest extent by determining the optimal extraction conditions of enzyme mass fraction, feed liquid ratio, enzymolysis temperature and ultrasonic time. Experiments prove that the selected extracted polyphenol extract has strong tyrosinase inhibition capability, can be used as a potential efficient tyrosinase inhibitor, and is effectively applied to the industries of cosmetics and medicines.

Description

Traditional Chinese medicine compound phenolic extract and application thereof in preparation of tyrosinase inhibitor

Technical Field

The application belongs to the technical field of medicines, and particularly relates to a traditional Chinese medicine compound phenol extract and application thereof in preparation of tyrosinase inhibitors.

Background

Melanin is the primary pigment of pigmentation of human skin, hair and eyes, and is produced by melanocytes. Melanogenesis and skin pigmentation are the most important photoprotective factors of the human body against solar ultraviolet radiation damage and skin photo carcinogenesis. However, excessive synthesis and accumulation of these pigments can lead to a number of skin disorders including chloasma, freckles, periorbital pigmentation, risk of skin cancer, and the like.

Tyrosinase is a biological enzyme containing a plurality of copper ions in living bodies, and plays an important role in the processes of melanin generation and enzymatic browning. Tyrosinase inhibitors can be used as whitening agents in the cosmetic and pharmaceutical industries. Natural anti-tyrosinase inhibitors isolated from plants have low toxicity and good bioavailability, and are particularly suitable for the application in the fields of foods, cosmetics and medicaments, and have been attracting more attention in recent years. Therefore, screening for the development of natural tyrosinase inhibitors is an important approach to the treatment of melanin excessive deposition and thus lesions. Many plant extracts have tyrosinase inhibitory activity and are an important source for finding novel anti-tyrosinase compounds in natural plants.

The polyphenol compounds are main active ingredients of the five-flavor disinfection drink, have remarkable antioxidation and anti-inflammatory effects, and the effect of the existing tyrosinase inhibitor on treating melanin excessive deposition and then lesions is insufficient at present, so that the application is developed based on the fact.

Disclosure of Invention

In order to overcome the defects in the prior art, the application provides a traditional Chinese medicine compound phenolic extract which has good tyrosinase inhibition effect and can be used for preparing tyrosinase inhibitors.

The application also provides application of the traditional Chinese medicine compound phenolic extract in preparing tyrosinase inhibitors.

In order to achieve the above purpose, the present application provides the following technical solutions:

a preparation method of a traditional Chinese medicine compound phenol extract comprises the following steps:

step 1) ultrasonic-assisted enzymatic crude extraction of polyphenol compounds:

mixing flos Lonicerae, flos Chrysanthemi Indici, herba Violae, herba Taraxaci, and radix Semiaquilegiae, adding cellulase with different amounts, adding distilled water according to different feed liquid ratios, adjusting pH to 4.5-5.5, and performing ultrasonic enzymolysis;

performing solid-liquid separation after ultrasonic enzymolysis, extracting filter residues by using ethanol, combining the two filtrates, and concentrating under reduced pressure to obtain concentrated solution;

step 2) purifying the polyphenol extract by macroporous adsorption resin:

and (3) adding the concentrated solution to a macroporous adsorption resin column, sequentially flushing the macroporous adsorption resin column with pure water, 30% ethanol solution, 60% ethanol solution and 90% ethanol solution until the eluent is colorless to obtain corresponding target I section component, target II section component, target III section component and target IV section component eluents, concentrating the target section component eluents under reduced pressure until the target section component eluents are viscous, and freeze-drying the target section component eluents to obtain the macroporous adsorption resin column.

Specifically, in the step 1), the addition amount of the cellulase can be 0.2-0.4% of the sum of the mass of honeysuckle, wild chrysanthemum, viola philippica, dandelion and radix semiaquilegiae.

Specifically, in the step 1), 28-32g of honeysuckle, 10-12g of wild chrysanthemum, 10-12g of herba violae, 10-12g of dandelion and 10-12g of semiaquilegia root are taken. Further preferably, the Chinese medicinal prescription five-flavor disinfection drink is used, namely 30g of honeysuckle, 12g of wild chrysanthemum, 12g of herba violae, 12g of dandelion and 12g of semiaquilegia root are taken and mixed uniformly.

Specifically, in step 1), the ratio of the feed to the liquid may be 1g: distilled water was added in 55-65 ml. In the step 1), the ultrasonic enzymolysis temperature is 55-65 ℃ and the ultrasonic enzymolysis time is 25-35min.

It is further preferred that in step 1), the filter residue is extracted with 25-35% ethanol at room temperature for 25-35min.

The application provides the traditional Chinese medicine compound phenolic extract prepared by the preparation method.

The application also provides application of the traditional Chinese medicine compound phenolic extract in preparing tyrosinase inhibitors. The application determines the inhibition activity of the extracted traditional Chinese medicine compound phenolic extract on tyrosinase, and comprises the following steps: measuring the activity of B16 cells; detecting the effect of the polyphenol extract of each target segment component on the melanin content of B16 cells; detecting the effect of the polyphenol extract of each target fragment component on the tyrosinase activity of B16 cells; and determining the optimal proportion and content limit with strong tyrosinase inhibitory activity according to the tyrosinase inhibitory activity.

Compared with the prior art, the application has the following beneficial effects:

the application adopts the ultrasonic-assisted enzymolysis method for crude extraction and combines the macroporous adsorption resin separation and purification method, can extract the extracts of multiple sections of components in the materials of the five-flavor disinfection drink, can maximally improve the extraction rate of polyphenol by determining the optimal extraction conditions of the enzyme mass fraction, the feed-liquid ratio, the enzymolysis time and the ultrasonic time, has strong tyrosinase inhibition capability by selecting the extracted polyphenol extract, can be used as a potential high-efficiency tyrosinase inhibitor, and is effectively applied to the cosmetic and pharmaceutical industries.

Drawings

FIG. 1 is an HPLC chromatogram of a target I-IV stage polyphenol extract;

FIG. 2 is a graph showing the effect of a target stage II polyphenol extract on B16 cell viability;

FIG. 3 is a graph showing the effect of a target III-stage polyphenol extract on B16 cell viability;

FIG. 4 is a graph showing the effect of a target fraction II polyphenol extract on cellular melanin content;

FIG. 5 is a graph showing the effect of a target III-stage polyphenol extract on cellular melanin content;

FIG. 6 is an effect of a target stage II polyphenol extract on tyrosinase activity;

FIG. 7 shows the effect of the target III-stage polyphenol extract on tyrosinase activity.

Detailed Description

The following description of the technical solutions in the embodiments of the present application will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

In the examples below, the starting materials used are either commercially available products which are commercially available as such or can be prepared by methods conventional in the art. If not mentioned in detail, the process or experimental operation is carried out by conventional techniques in the art.

In the examples, unless otherwise specified, ethanol refers to the volume percent concentration. Room temperature refers to 25±5 ℃.

Example 1

step 1, carrying out ultrasonic-assisted enzymatic crude extraction on polyphenol compounds:

taking 30g of honeysuckle, 12g of wild chrysanthemum, 12g of viola yedoensis, 12g of dandelion and 12g of semiaquilegia root, uniformly mixing, adding different amounts of cellulase, adding distilled water according to different feed liquid ratios, regulating the pH value to be 5.0, and carrying out ultrasonic enzymolysis;

taking out after ultrasonic enzymolysis, carrying out suction filtration, extracting filter residues with 30% ethanol at room temperature for 30min, combining 2 times of filtrates, concentrating under reduced pressure to 80 mL at 50 ℃, and fixing the volume to 100mL with distilled water to obtain concentrated solution.

Step 2, optimizing extraction parameters of the crude polyphenol extracted by an ultrasonic-assisted enzymatic method through an orthogonal test:

the influence of 4 factors of cellulose quality fraction, feed-liquid ratio, ultrasonic enzymolysis temperature and ultrasonic enzymolysis time on the extraction amount of polyphenol in the five-flavor disinfection drink is respectively examined, and L9 (3 ⁴ ) The orthogonal test is carried out for process optimization, the total polyphenol content is used as an evaluation index, and Fu Lin Fenfa is adopted for the total polyphenol content measurement. As shown in the following table, a table of 4 factor levels.

Table 1, four factor level table

Step 3, purifying the polyphenol extract by macroporous adsorption resin:

the 101 macroporous adsorption resin is soaked in 95% ethanol solution for 24 hours, the column is filled after full swelling, and distilled water is used for washing the resin until effluent liquid is clear and no ethanol is contained. Adding the concentrated solution into a macroporous adsorption resin column at the frequency of 10 ml/time, washing with pure water, 30% ethanol solution, 60% ethanol solution and 90% ethanol solution at the rate of 2BV/h until the eluent is colorless to obtain corresponding eluent of a target I section component, a target II section component, a target III section component and a target IV section component, concentrating the eluent of each target section component under reduced pressure at 50 ℃ to be sticky, and freeze-drying to obtain a target I-IV section polyphenol extract.

Step 4, analyzing the components of each target segment

Chromatographic conditions: c18 (4.6 mm x 250mm,5 μm) chromatographic column at a flow rate of 0.8-1.0mL/min; the column temperature is 25-35 ℃; the mobile phase is 0.2% acetic acid (phosphoric acid, acetic acid) water (A) -acetonitrile (B), and the gradient elution condition is that 0-29min is adopted, and 4-35% B is adopted; the sample volume was 10. Mu.L.

1.1 preparation of a Mixed control solution

Preparing a mixed reference substance solution: respectively weighing a proper amount of each reference substance, precisely weighing, placing in a 100mL brown volumetric flask, and taking methanol as a solvent to prepare mixed reference substance solutions of single caffeoyl tartaric acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, aesculin, chicoric acid, luteolin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C and linarin with mass concentrations of 26.80, 12.31, 143.99, 28.50, 17.12, 25.82, 12.74, 18.0, 33.6, 46.7 and 40.75mg/L respectively;

1.2 component identification and content determination

And (5) carrying out component identification on the eluent of each target segment according to the retention time and the spectrum absorption curve of the reference substance. Calculating the content of each polyphenol compound in the extract by an external standard method;

step 5, determination of tyrosinase inhibitory activity of polyphenol extract

2.1, B16 cell Activity Effect assay

The log phase grown B16 cells (mouse melanoma cells) were taken according to a 5X 10 protocol ⁴ The concentration of each/mL was inoculated into a 96-well plate, and 100. Mu.L of DMEM medium was used per well. At a temperature of 37℃and CO ₂ Culturing for 24h in 5% environment; the supernatant was discarded, 100. Mu.L of DMEM medium containing different concentrations of polyphenol extract (10, 50, 100, 200, 400, 600. Mu.g/mL) was added to each well, the control group was the same volume of DMEM medium without polyphenol extract, the blank group was not inoculated with cells, only 100. Mu.L of DMEM medium was added, 6 replicates were set for each group, and the concentration of CO was 5% at 37 ℃C ₂ Culturing in the environment for 24h; the cell supernatant was discarded, 100. Mu.L of Cell Counting Kit-8 cell counting reagent (CCK 8) containing 10% of the culture solution was added, and after 2 hours, the absorbance at 450nm was measured by an ELISA reader, and 24 hours of cell viability was calculated. Cell viability was determined for 48h and 72h in the same manner.

Wherein, the calculation formula of the cell viability is as follows:

wherein A is _i Absorbance for the sample; a is that ₀ Absorbance for the blank group; a is that _c Absorbance for control group。

2.2 Effect of polyphenol extract on B16 cell melanin content

The melanin content of B16 cells was determined using NaOH lysis. The references are as follows:

b16 cells grown in log phase were seeded in 6-well plates (5×10) ⁴ Individual/well, 2 mL), at 37 ℃, 5% co ₂ Culturing in the environment for 24h; discarding DMEM culture solution, adding 1mL of DMEM culture solution containing polyphenol extracts (10, 50, 100, 200 μg/mL) at different concentrations, adding the same volume of DMEM culture solution without polyphenol extract as control group, adding equal volume of DMEM culture solution as blank group, adding arbutin as positive control with equal volume of 50 μg/mL, and adding water solution at 37deg.C and 5% CO ₂ After further culturing in the environment for 24 hours, 48 hours and 72 hours, the cells were washed 3 times with PBS buffer, 2mL of NaOH solution with a concentration of 0.2mol/L was added, the cells were completely dissolved in a water bath at 80℃for 1 hour, and the absorbance at 492nm was measured.

The relative melanin content is calculated as follows:

wherein A is _i Absorbance for the sample; a is that ₀ Absorbance for the blank group; a is that _c Absorbance was used as control.

2.3 Effect of polyphenol extract on tyrosinase activity in B16 cells

After 3 washes with PBS buffer according to 2.2, 50. Mu.L of 1% Triton X-100 as cell lysate was added, frozen at low temperature for 1h, and thawed at room temperature after removal; incubation was carried out at 37℃and 10. Mu.L of 1% strength L-DOPA solution (L-DOPA) was added and reacted for 1h, and the absorbance at 492nm was measured.

Wherein, the calculation formula of the tyrosinase relative activity is as follows:

wherein A is _i Absorbance for the sample; a is that ₀ Is blank groupAbsorbance; a is that _c Absorbance was used as control.

All data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett multiple comparison test using statistical software SPSS 16.0. Data are expressed as mean.+ -. SD, p <0.05 is considered to be significantly different.

Orthogonal experimental design and results analysis tables are shown in tables 2 and 3 below.

TABLE 2 orthogonal experimental design

Note that: the tables 1,2,3 represent the levels of each factor.

TABLE 3 analysis of variance table

From the above table, it can be seen that: by examining the extraction effect of enzyme mass fraction, feed liquid ratio, ultrasonic enzymolysis temperature and ultrasonic enzymolysis time on polyphenol compounds in the five-flavor disinfection drink, designing a single-factor experiment and a four-factor three-level orthogonal experiment, intuitively determining that the optimal extraction condition is A2B3C3D2 according to the extraction rate of the polyphenol compounds under different extraction conditions, namely that the mass fraction of cellulase is 0.3 percent and the feed liquid ratio is 1: 60. the ultrasonic enzymolysis temperature is 60 ℃, and the ultrasonic enzymolysis time is 30min, which is the optimal extraction condition of polyphenol; and the analysis of variance results show that the influence degree of four factors of enzyme mass fraction (A), feed-liquid ratio (B), ultrasonic enzymolysis time (C) and ultrasonic enzymolysis time (D) on the polyphenol extraction rate is A > B > C > D.

Example 2

The eluent components of each target segment of the five-flavor disinfection drink obtained in the step 3 of the above example 1 were analyzed, and the results are shown in fig. 1.

The HPLC chromatograms of the target section I-IV eluents shown in the figure 1 are used for carrying out component identification on the target section eluents by referring to the retention time and the spectral absorption curve of the reference substance, and the result is shown in the figure 1; in the figure, no. 1-11 are monocaffeoyltartaric acid, cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid, aesculin, chicoric acid, luteolin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C and linarin in sequence; the relative retention times of the polyphenols 1-11 were 0.555, 0.707, 1.000, 1.072, 1.216, 1.459, 2.091, 2.168, 2.256, 2.404, 3.082 with respect to peak 3 as reference chromatographic peak.

The dry powder yields and qualitative polyphenol content determinations of the target I-IV eluents are referenced to target I-IV polyphenol fraction analysis tables, as shown in tables 4 and 5 below.

TABLE 4 content of polyphenols in target paragraphs I-IV

TABLE 5 target segment I-IV polyphenols duty cycle analysis

The results of the table above show: the percentage of the area of the qualitative polyphenol peak in the target section I-IV at 330nm is more than 76%, the highest yield of the extract of the target section I-IV is section I, but the main component in section I is chlorogenic acid, the content is also lower, and most of substances without ultraviolet absorption such as saccharides and proteins in section I are deduced. The extract of the target section II has various polyphenol compounds and high chlorogenic acid content. The target III section contains various polyphenols, and has high content, especially isochlorogenic acid A and linarin. The yield of the target section IV is the lowest, and the content of the polyphenol compounds is very low.

In conclusion, the polyphenols are mainly concentrated in the II and III sections, so that the activity evaluation of the polyphenol extracts of the II and III sections is focused.

3. Influence of polyphenol extract on tyrosinase activity

3.1 Effect of polyphenol extract on B16 cell Activity

FIG. 2 shows the effect of the target stage II polyphenol extract on B16 cell viability. As shown in FIG. 2, the effect of the polyphenol extracts of different concentrations of the target II on the cell activity of B16 cells is shown, the polyphenol extract of the target II in the range of 10-200 mug/mL has no obvious inhibition effect on the activity of B16 cells in 72h, and the concentration range of 10-100 mug/mL is dose-dependent with the activity of B16 cells. The target II-stage polyphenol extract with the concentration of more than 400 mug/mL can obviously inhibit the activity of B16 cells, the activity of the B16 cells is reduced to 92.15 percent after 72 hours of treatment with 400 mug/mL; the viability of B16 cells was reduced to 89.26% by treatment with 600. Mu.g/mL for 24 h. The above results indicate that polyphenol extracts in the range of 10-200 μg/mL have no significant effect on the cellular activity of B16 cells.

FIG. 3 shows the effect of the target III-stage polyphenol extract on B16 cell viability. As shown in FIG. 3, the effect of the polyphenol extracts of different concentrations of the target III on the cell activity of B16 cells is shown, the polyphenol extract of the target III in the range of 10-200 mug/mL has no obvious inhibition effect on the activity of B16 cells in 72h, and the concentration range of 10-100 mug/mL is dose-dependent with the activity of B16 cells. When the target III-stage polyphenol extract with the concentration of more than 400 mug/mL can obviously inhibit the activity of B16 cells, the activity of the B16 cells is reduced to 92.49 percent after 72 hours of treatment with 400 mug/mL; the viability of B16 cells was reduced to 90.5% by treatment with 600. Mu.g/mL for 24 h.

The results show that the target II and III-stage polyphenol extracts in the range of 10-200 mug/mL have no obvious influence on the cell activity of B16 cells.

3.2 Effect of polyphenol extract on B16 cell melanin content

FIGS. 4 and 5 show the effect of the target II and III polyphenol extracts on the melanin content of cells, respectively. As shown in fig. 4 and 5, the effect of the target II and III polyphenol extracts on the melanin content of B16 cells, and the effect of different concentrations of polyphenol extracts (10, 50, 100, 200 μg/mL) on the melanin content of B16 cells was significant; the extraction of the target II and III section polyphenols of 10 mug/mL has no obvious inhibiting effect on the melanin synthesis of B16 cells; with the increase of the concentration of the polyphenol extract, the inhibition effect is gradually enhanced, the action of the polyphenol extract with the concentration of 200 mug/mL on B16 cells for 72 hours is remarkable, the inhibition rate of the target II section on melanin generation amount is 34.22%, the inhibition rate of the target III section on melanin generation amount is 39.56%, and the inhibition rate of the positive control arbutin on melanin generation amount is 48.79%. Therefore, the extracts of the target II and III sections have obvious inhibiting effect on melanin, and the inhibiting effect of the extract of the target III section on melanin generation amount is better than that of the extract of the target II section.

3.3 Effect of polyphenol extract on B16 cell tyrosinase Activity

FIGS. 6 and 7 show the effect of the target II and III polyphenol extracts on tyrosinase activity, respectively. As shown in fig. 6 and 7, the target II, III polyphenol extracts have significant inhibition effect on B16 cell tyrosinase compared to the blank group; the target II-stage polyphenol extract with the concentration of 10 mug/mL reduces the tyrosinase activity of B16 cells for 24 hours to 91.49 percent, the target III-stage polyphenol extract with the concentration of 10 mug/mL reduces the tyrosinase activity of B16 cells for 24 hours to 90.89 percent, and the inhibition effect of the target II-stage polyphenol extract and the III-stage polyphenol extract on the tyrosinase of B16 cells is not obvious at low concentration; along with the increase of the concentration of the polyphenol extract and the increase of the treatment time, the tyrosinase activity of B16 cells is gradually reduced, and the tyrosinase activity and the dosage are in a dependent relationship; 200 mug/mL of the target II-stage III-stage polyphenol extract respectively reduces the tyrosinase activity of B16 cells to 58.36 percent and 51.69 percent in 72 hours; the target III section extract has stronger inhibition effect on tyrosinase activity than the target II section extract, and the target II and III section extracts of the schisandra chinensis disinfection drink are potential tyrosinase activity inhibitors, can be used as potential tyrosinase inhibitors, and are applied to the industries of cosmetics and medicines.

In summary, the ultrasonic-assisted enzymatic method is used for crude extraction of the schisandra disinfection drink polyphenol extract, and the crude extract is sequentially eluted by pure water, 30% ethanol solution, 60% ethanol solution and 90% ethanol solution on a macroporous adsorption resin column to obtain purified target I section eluate, target II section eluate, target III section eluate and target IV section eluate, wherein the II and III section eluate has more polyphenol compound types and high content. By further examining the influence of the II and III section polyphenol extracts on tyrosinase activity, the II and III section polyphenol extracts within the range of 10-200 mug/mL are found to have no obvious toxicity on the activity of B16 cells, and the III section polyphenol extract has better inhibiting activity on melanin and tyrosinase of the B16 cells than the II section polyphenol extract, so that the II and III section extracts of the target schistosome disinfection drink are potential melanin synthesis inhibitors and tyrosinase inhibitors; and the target III-stage extract comprises cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid, aesculin, chicoric acid, luteolin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C and linarin. 10 groups of honeysuckle, wild chrysanthemum, dandelion, viola yedoensis and semiaquilegia root from 5 main producing areas are randomly formed, 10 batches of target III-section polyphenol extracts are obtained by separating and purifying through ultrasonic-assisted enzyme extraction and macroporous adsorption resin column, and 70-130% of the average content of the target III-section polyphenol compounds is taken as the content limit of the tyrosinase inhibitor according to the B16 cell tyrosinase inhibitory activity determination result. The polyphenol extract used as the tyrosinase inhibitor has the content range of 0.014+/-0.03%, the content range of chlorogenic acid of 1.070+/-0.3%, the content range of neochlorogenic acid of 0.011+/-0.03%, the content range of aesculin of 0.574+/-0.15%, the content range of chicoric acid of 0.019+/-0.01%, the content range of luteolin of 0.707+/-0.21%, the content range of isochlorogenic acid B of 0.552+/-0.16%, the content range of isochlorogenic acid A of 12.399 +/-3.72%, the content range of isochlorogenic acid C of 3.986 +/-1.19% and the content range of linarin of 9.007+/-2.70%, and the target III-segment polyphenol extract in the content range has the best tyrosinase inhibition activity, and can be used as a potential tyrosinase inhibitor in cosmetics and pharmaceutical industry.

The last points to be described are: while the application has been described in detail in the foregoing general description and with reference to specific embodiments, the foregoing embodiments are merely illustrative of the technical aspects of the application and are not limiting thereof; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims

1. The preparation method of the compound phenol extract of the traditional Chinese medicine is characterized by comprising the following steps:

step 1) ultrasonic-assisted enzymatic crude extraction of polyphenol compounds:

mixing flos Lonicerae, flos Chrysanthemi Indici, herba Violae, herba Taraxaci, and radix Semiaquilegiae, adding cellulase and distilled water, adjusting pH to 4.5-5.5, and performing ultrasonic enzymolysis;

step 2) purifying the polyphenol extract by macroporous adsorption resin:

2. The method for preparing the compound phenol extract of traditional Chinese medicine according to claim 1, wherein in the step 1), the addition amount of cellulase is 0.2-0.4% of the sum of the mass of honeysuckle, wild chrysanthemum flower, viola philippica, dandelion and semiaquilegia root.

3. The method for preparing the compound phenol extract of traditional Chinese medicine according to claim 1, wherein in the step 1), 28-32g of honeysuckle, 10-12g of wild chrysanthemum flower, 10-12g of herba violae, 10-12g of dandelion and 10-12g of semiaquilegia root are taken.

4. The method for preparing the compound phenol extract of traditional Chinese medicine according to claim 1, wherein in the step 1), the ratio of the feed to the liquid is 1g: distilled water was added in 55-65 ml.

5. The method for preparing the compound phenol extract of traditional Chinese medicine according to claim 1, wherein in the step 1), the ultrasonic enzymolysis temperature is 55-65 ℃ and the ultrasonic enzymolysis time is 25-35min.

6. The method for preparing the compound phenol extract of traditional Chinese medicine according to claim 1, wherein in step 1), 25-35% ethanol is used for extracting filter residues for 25-35min.

7. The compound phenol extract of traditional Chinese medicine prepared by the preparation method of any one of claims 1 to 6.

8. The use of the compound phenolic extract of traditional Chinese medicine as claimed in claim 7 in preparing tyrosinase inhibitors.