A traditional Chinese medicine compound phenolic extract and its application in preparing tyrosinase inhibitors

Abstract

The present invention relates to the field of medical technology, and specifically discloses a Chinese medicine compound phenol extract, which can extract multiple components from the materials of Wuwei Xiaodu Yin Fang by using ultrasound-assisted enzymatic hydrolysis and combining with a macroporous adsorption resin separation and purification method, and can maximize the extraction rate of polyphenols by determining the optimal extraction conditions of enzyme mass fraction, material-liquid ratio, enzymatic hydrolysis temperature and ultrasonic time. The present invention has been determined through experiments that the selected extracted polyphenol extract has a strong ability to inhibit tyrosinase, can be used as a potential high-efficiency tyrosinase inhibitor, and can be effectively applied to the cosmetics and pharmaceutical industries.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and more specifically, relates to a traditional Chinese medicine compound phenol extract and application thereof in preparing a tyrosinase inhibitor. Background Art

[0002] Melanin is the main pigment of human skin, hair and eye pigmentation, produced by melanocytes. Melanin production and skin pigmentation are the most important photoprotective factors for the human body against damage from solar ultraviolet radiation and skin photocarcinogenesis. However, excessive synthesis and accumulation of these pigments can lead to many skin diseases, including melasma, freckles, periorbital pigmentation, skin cancer risk, etc.

[0003] Tyrosinase is a biological enzyme containing multiple copper ions in living organisms, and plays an important role in the production of melanin and enzymatic browning. Tyrosinase inhibitors can be used as whitening agents and are widely used in the cosmetics and pharmaceutical industries. Natural anti-tyrosinase inhibitors isolated from plants have low toxicity and good bioavailability. They are particularly suitable for applications in the fields of food, cosmetics and medicine, and have received increasing attention in recent years. Therefore, screening and developing natural tyrosinase inhibitors is an important way to treat excessive melanin deposition and subsequent lesions. Many plant extracts have tyrosinase inhibitory activity, and natural plants have become an important source for finding new anti-tyrosinase compounds.

[0004] Polyphenol compounds are the main active ingredients of Wuwei Xiaodu Drink, which have significant antioxidant and anti-inflammatory effects. At present, the existing tyrosinase inhibitors are not effective in treating excessive melanin deposition and subsequent lesions. Based on this, the present application was developed. Summary of the invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a traditional Chinese medicine compound phenolic extract, which has a good inhibitory effect on tyrosinase and can be used to prepare a tyrosinase inhibitor.

[0006] The present invention also provides the use of the above-mentioned traditional Chinese medicine compound phenol extract in the preparation of tyrosinase inhibitors.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing a Chinese medicine compound phenolic extract comprises the following steps:

[0009] Step 1) Ultrasound-assisted enzyme method for crude extraction of polyphenolic compounds:

[0010] Take honeysuckle, wild chrysanthemum, Viola yedoensis, dandelion, and Gynura ovata, mix them evenly, add different amounts of cellulase, add distilled water according to different material-liquid ratios, adjust the pH to 4.5-5.5, and perform ultrasonic enzymolysis;

[0011] After ultrasonic enzymatic hydrolysis, the solid and liquid are separated, and the filter residue is extracted with ethanol, and the two filtrates are combined and concentrated under reduced pressure to obtain a concentrated solution;

[0012] Step 2) Purification of polyphenol extract by macroporous adsorption resin:

[0013] The concentrated solution is added to a macroporous adsorption resin column, and washed with pure water, 30% ethanol solution, 60% ethanol solution, and 90% ethanol solution in sequence until the eluent is colorless to obtain the corresponding target segment I component, target segment II component, target segment III component, and target segment IV component eluent. The eluent of each target segment component is concentrated under reduced pressure until it becomes viscous, and freeze-dried to obtain the product.

[0014] Specifically, in step 1), the amount of cellulase added can be 0.2-0.4% of the total mass of honeysuckle, wild chrysanthemum, Viola yedoensis, dandelion and Gynura ovata.

[0015] Specifically, in step 1), 28-32g of honeysuckle, 10-12g of wild chrysanthemum, 10-12g of Viola yedoensis, 10-12g of dandelion, and 10-12g of Gastrodia elata are taken. Further preferably, the Chinese medicine Wuwei Xiaodu Yin is used, that is, 30g of honeysuckle, 12g of wild chrysanthemum, 12g of Viola yedoensis, 12g of dandelion, and 12g of Gastrodia elata are taken and mixed evenly.

[0016] Specifically, in step 1), distilled water can be added at a material-liquid ratio of 1 g: 55-65 ml. In step 1), the ultrasonic enzymatic hydrolysis temperature is 55-65° C., and the ultrasonic enzymatic hydrolysis time is 25-35 min.

[0017] Further preferably, in step 1), the filter residue is extracted with 25-35% ethanol at room temperature, and the extraction time is 25-35 minutes.

[0018] The invention provides a traditional Chinese medicine compound phenol extract prepared by the preparation method.

[0019] The present invention also provides the use of the above-mentioned traditional Chinese medicine compound phenol extract in the preparation of tyrosinase inhibitors. The present invention measures the inhibitory activity of the traditional Chinese medicine compound phenol extract on tyrosinase, including: 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 segment component on the tyrosinase activity of B16 cells; and determining the optimal ratio and content limit with strong tyrosinase inhibitory activity according to its tyrosinase inhibitory activity.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention adopts ultrasound-assisted enzymatic hydrolysis for crude extraction, combined with a macroporous adsorption resin separation and purification method, to extract extracts of multiple components from the materials of the Wuwei Disinfection Drink Recipe, and by determining the optimal extraction conditions of enzyme mass fraction, material-liquid ratio, enzymatic hydrolysis time and ultrasonic time, the extraction rate of polyphenols can be improved to the greatest extent, and the selected extracted polyphenol extract has a strong ability to inhibit tyrosinase, and can be used as a potential high-efficiency tyrosinase inhibitor, and effectively applied to the cosmetics and pharmaceutical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 HPLC chromatogram of target I-IV segment polyphenol extract;

[0023] Figure 2 The effect of polyphenol extracts from target II on B16 cell viability;

[0024] Figure 3 The effect of target III polyphenol extract on B16 cell viability;

[0025] Figure 4 The effect of polyphenol extracts from target II on the content of cellular melanin;

[0026] Figure 5 The effect of target III polyphenol extract on cellular melanin content;

[0027] Figure 6 The effect of polyphenol extracts from target II on tyrosinase activity;

[0028] Figure 7 The effect of segment III polyphenol extract on tyrosinase activity was investigated. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0030] In the following examples, the raw materials used are all common commercial products that can be directly purchased or can be prepared by referring to conventional methods in the art. If there are processes or experimental operations not mentioned in detail, conventional techniques in the art can be used.

[0031] Unless otherwise specified in the examples, ethanol refers to the volume percentage concentration. Room temperature refers to 25±5°C.

[0032] Example 1

[0033] A method for preparing a Chinese medicine compound phenolic extract comprises the following steps:

[0034] Step 1: Ultrasonic-assisted enzyme method to crudely extract polyphenol compounds:

[0035] Take 30g of honeysuckle, 12g of wild chrysanthemum, 12g of Viola yedoensis, 12g of dandelion, and 12g of Gynura ovata, mix them evenly, add different amounts of cellulase, add distilled water according to different material-liquid ratios, adjust the pH to 5.0, and perform ultrasonic enzymolysis;

[0036] After ultrasonic enzymatic hydrolysis, the mixture was taken out and filtered, and the filter residue was extracted with 30% ethanol at room temperature for 30 minutes. The two filtrates were combined and concentrated to 80 ml under reduced pressure at 50°C, and the volume was adjusted to 100 mL with distilled water to obtain a concentrated solution.

[0037] Step 2: Orthogonal experiment to optimize the parameters of ultrasound-assisted enzymatic crude polyphenol extraction:

[0038] The effects of cellulase mass fraction, solid-liquid ratio, ultrasonic enzymatic hydrolysis temperature and ultrasonic enzymatic hydrolysis time on the extraction of polyphenols from Wuwei Xiaodu Yin were investigated. 4 ) Orthogonal test was used to optimize the process, and the total polyphenol content was used as the evaluation index. The total polyphenol content was determined by the Folin phenol method. As shown in the following table, it is a table of 4 factor levels.

[0039] Table 1, Four factor level table

[0040]

[0041] Step 3: Purification of polyphenol extract by macroporous adsorption resin:

[0042] 101 macroporous adsorption resin was soaked in 95% ethanol solution for 24h, and loaded into the column after being fully swollen. The resin was rinsed with distilled water until the effluent was clear and no longer contained ethanol. The above concentrate was added to the macroporous adsorption resin column at a frequency of 10ml / time, and washed with pure water, 30% ethanol solution, 60% ethanol solution, and 90% ethanol solution at a rate of 2BV / h until the eluent was colorless, and the corresponding eluents of the target segment I component, target segment II component, target segment III component, and target segment IV component were obtained. The eluents of each target segment component were concentrated under reduced pressure at 50°C until they were viscous, and freeze-dried to dry powder to obtain the target I-IV segment polyphenol extracts.

[0043] Step 4: Component analysis of each target segment

[0044] Chromatographic conditions: C18 (4.6mm*250mm, 5μm) chromatographic column, flow rate of 0.8-1.0mL / min; column temperature of 25-35°C; mobile phase of 0.2% acetic acid (phosphoric acid, acetic acid) water (A)-acetonitrile (B), with gradient elution conditions of 0-29min, 4-35% B; injection volume of 10μL.

[0045] 1.1. Preparation of mixed reference solution

[0046] Preparation of mixed reference substance solution: Weigh an appropriate amount of each reference substance, accurately weigh it, and place it in a 100 mL brown volumetric flask. Use methanol as solvent to prepare a mixed reference substance solution 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 40.75 mg / L for monocaffeoyltartaric acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, fraxin, chicoric acid, luteolin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and montanol, respectively;

[0047] 1.2. Ingredient identification and content determination

[0048] The components of each target eluate were identified by referring to the retention time and spectral absorption curve of the reference substance. The content of each polyphenol compound in the extract was calculated by the external standard method;

[0049] Step 5: Determination of the inhibitory activity of polyphenol extract on tyrosinase

[0050] 2.1. Determination of the effect of B16 cell activity

[0051] B16 cells (mouse melanoma cells) growing in logarithmic phase were taken and cultured at 5×10 4 The cells were inoculated at a concentration of 100 μL / mL in a 96-well plate with 100 μL of DMEM culture medium per well. 2 The cells were cultured in a 5% CO environment for 24 h; the supernatant was discarded, and 100 μL of DMEM culture medium containing different concentrations of polyphenol extracts (10, 50, 100, 200, 400, 600 μg / mL) was added to each well. The control group was the same volume of DMEM culture medium without polyphenol extracts. The blank group was not inoculated with cells, and only 100 μL of DMEM culture medium was added. Each group was set to 6 replicates and incubated at 37°C and 5% CO 2 Culture in the environment for 24 hours; discard the cell supernatant, add 100 μL of Cell Counting Kit-8 cell counting reagent (CCK8) containing 10% culture medium, measure the absorbance at 450nm with a microplate reader after 2 hours, and calculate the 24-hour cell viability. The cell viability of the cells at 48 hours and 72 hours was determined in the same way.

[0052] The calculation formula of cell viability is as follows:

[0053]

[0054] In the formula, A i is the sample absorbance; A 0 is the absorbance of the blank group; A c is the absorbance of the control group.

[0055] 2.2 Effect of polyphenol extract on melanin content in B16 cells

[0056] The melanin content of B16 cells was determined by NaOH dissolution method. Reference is as follows:

[0057] B16 cells growing in logarithmic phase were inoculated into 6-well plates (5×10 4 pcs / well, 2 mL) and incubated at 37°C, 5% CO 2 The cells were cultured in an environment for 24 h; the DMEM culture medium was discarded, and 1 mL of DMEM culture medium with different concentrations of polyphenol extracts (10, 50, 100, and 200 μg / mL) was added. The control group was the same volume of DMEM culture medium without polyphenol extracts, and the blank group was only added with the same volume of DMEM culture medium. The positive control was an equal volume of 50 μg / mL arbutin. The cells were cultured in an environment at 37°C and 5% CO. 2 After continuing to culture in the environment for 24h, 48h, and 72h, rinse with PBS buffer three times, add 2mL of 0.2mol / L NaOH solution, bathe in 80℃ water for 1h to completely dissolve the cell clumps, and then measure the absorbance at 492nm.

[0058] The calculation formula of melanin relative content is as follows:

[0059]

[0060] In the formula, A i is the sample absorbance; A 0 is the absorbance of the blank group; A c is the absorbance of the control group.

[0061] 2.3 Effects of polyphenol extracts on tyrosinase activity in B16 cells

[0062] Follow the procedure in 2.2 until the cells are rinsed three times with PBS buffer, then add 50 μL of cell lysis solution (1% Triton X-100), freeze at low temperature for 1 hour, take out and thaw at room temperature; incubate at 37°C and add 10 μL of 1% L-DOPA solution (levodopa), react for 1 hour, and measure the absorbance at 492 nm.

[0063] The relative activity calculation formula of tyrosinase is as follows:

[0064]

[0065] In the formula, A i is the sample absorbance; A 0 is the absorbance of the blank group; A c is the absorbance of the control group.

[0066] All data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test using statistical software SPSS 16.0. Data are expressed as mean ± SD, and p < 0.05 was considered to be significantly different.

[0067] The orthogonal experimental design and result analysis tables are shown in Tables 2 and 3 below.

[0068] Table 2. Orthogonal experimental design

[0069]

[0070]

[0071] Note: 1, 2, 3 in the table are the levels of each factor.

[0072] Table 3, ANOVA table

[0073]

[0074] It can be seen from the above table: by investigating the extraction effect of enzyme mass fraction, solid-liquid ratio, ultrasonic enzymatic hydrolysis temperature and ultrasonic enzymatic hydrolysis time on polyphenol compounds in Wuwei Xiaodu Drink, a single-factor experiment and a four-factor three-level orthogonal experiment were designed. According to the extraction rate of polyphenol compounds under different extraction conditions, the optimal extraction conditions were intuitively determined to be A2B3C3D2, that is, the cellulase mass fraction was 0.3%, the solid-liquid ratio was 1:60, the ultrasonic enzymatic hydrolysis temperature was 60℃ and the ultrasonic enzymatic hydrolysis time was 30min, which were the optimal extraction conditions for polyphenols; and through the results of variance analysis, it was found that the degree of influence of the four factors of enzyme mass fraction (A), solid-liquid ratio (B), ultrasonic enzymatic hydrolysis time (C) and ultrasonic enzymatic hydrolysis time (D) on the polyphenol extraction rate was A>B>C>D.

[0075] Example 2

[0076] The components of the eluate of each target segment of Wuwei Xiaoduyin obtained in step 3 of the above Example 1 were analyzed, and the results are shown in Figure 1 .

[0077] like Figure 1 The HPLC chromatograms of the target segments I-IV eluates are shown in FIG. 1 , and the components of the eluates of each target segment are identified by referring to the retention time and spectral absorption curve of the reference substance. The results are shown in FIG. Figure 1; In the figure, No. 1-11 are monocaffeoyltartaric acid, cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid, quercetin, chicoric acid, luteolin, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and montmorillonine; taking peak No. 3 as the reference chromatographic peak, the relative retention times of polyphenol compounds No. 1-11 are 0.555, 0.707, 1.000, 1.072, 1.216, 1.459, 2.091, 2.168, 2.256, 2.404, and 3.082.

[0078] The dry powder yield and qualitative polyphenol content determination results of the target segment I-IV eluate refer to the target segment I-IV polyphenol proportion analysis table, as shown in Tables 4 and 5 below.

[0079] Table 4, Content of polyphenolic compounds in target segments I-IV

[0080]

[0081] Table 5. Analysis of the proportion of polyphenolic compounds in target segments I-IV

[0082]

[0083] The results in the table above show that the peak area of ​​the identified polyphenols in the target I-IV segments at 330nm accounts for more than 76% of the total peak area. The target segment I-IV extract has the highest yield in segment I, but the main component in segment I is chlorogenic acid, and the content is also low. It is inferred that I is mostly sugars and proteins and other substances without UV absorption. There are many types of polyphenol compounds in the extract of target segment II, and the content of chlorogenic acid is high. There are many types of polyphenol compounds in the target segment III, and the content is generally high, especially isochlorogenic acid A and monanthin. The target segment IV has the lowest yield, and the content of polyphenol compounds is very low.

[0084] In summary, polyphenol compounds are mainly concentrated in segments II and III, so the activity evaluation of polyphenol extracts from segments II and III was focused.

[0085] 3. Effect of polyphenol extracts on tyrosinase activity

[0086] 3.1 Effects of polyphenol extracts on B16 cell activity

[0087] Figure 2 The effects of target II polyphenol extract on B16 cell viability are given. Figure 2As shown, the polyphenol extracts of different concentrations of target segment II affect the cell activity of B16 cells. The polyphenol extracts of target segment II in the range of 10-200 μg / mL have no significant inhibitory effect on the activity of B16 cells within 72 hours, and the concentration range of 10-100 μg / mL is dose-dependent with the viability of B16 cells. The polyphenol extracts of target segment II with a concentration greater than 400 μg / mL will significantly inhibit the activity of B16 cells. After treatment with 400 μg / mL for 72 hours, the viability of B16 cells decreased to 92.15%; after treatment with 600 μg / mL for 24 hours, the viability of B16 cells decreased to 89.26%. The above results show that the polyphenol extracts in the range of 10-200 μg / mL have no significant effect on the cell activity of B16 cells.

[0088] Figure 3 The effects of target III polyphenol extract on B16 cell viability are given. Figure 3 As shown in the figure, the target segment III polyphenol extracts of different concentrations affect the cell activity of B16 cells. The target segment III polyphenol extracts in the range of 10-200 μg / mL have no significant inhibitory effect on the activity of B16 cells within 72 hours, and the concentration range of 10-100 μg / mL is dose-dependent with the activity of B16 cells. When the concentration is greater than 400 μg / mL, the target segment III polyphenol extract will significantly inhibit the activity of B16 cells. After 72 hours of treatment with 400 μg / mL, the activity of B16 cells is reduced to 92.49%; after 24 hours of treatment with 600 μg / mL, the activity of B16 cells is reduced to 90.5%.

[0089] The above results indicate that the target II and III segment polyphenol extracts within the range of 10 to 200 μg / mL had no significant effect on the cell activity of B16 cells.

[0090] 3.2 Effect of polyphenol extract on melanin content in B16 cells

[0091] Figure 4 , 5 The effects of target II and III polyphenol extracts on cell melanin content are given respectively. Figure 4 and Figure 5As shown in the figure, the effect of target II and III polyphenol extracts on the melanin content of B16 cells, different concentrations of polyphenol extracts (10, 50, 100, 200 μg / mL) have significant effects on B16 cell melanin; 10 μg / mL of target II and III polyphenol extracts have no obvious inhibitory effect on B16 cell melanin synthesis; with the increase of polyphenol extract concentration, the inhibitory effect gradually increases, 200 μg / mL of polyphenol extract has a significant effect on 72h B16 cells, the inhibition rate of target II segment on melanin production is 34.22%, the inhibition rate of target III segment on melanin production is 39.56%, and the inhibition rate of positive control arbutin on melanin production is 48.79%. It can be seen that the target II and III extracts have a significant inhibitory effect on melanin, and the inhibitory effect of the target III extract on melanin production is better than that of the target II extract.

[0092] 3.3 Effects of polyphenol extracts on tyrosinase activity in B16 cells

[0093] Figure 6 , 7 The effects of target II and III polyphenol extracts on tyrosinase activity are given respectively. Figure 6 and Figure 7 As shown in the results, compared with the blank group, the target II and III polyphenol extracts had a significant inhibitory effect on B16 cell tyrosinase; 10 μg / mL of the target II polyphenol extract reduced the 24h B16 cell tyrosinase activity to 91.49%, and 10 μg / mL of the target III polyphenol extract reduced the 24h B16 cell tyrosinase activity to 90.89%. At low concentrations, the inhibitory effects of the target II and III polyphenol extracts on B16 cell tyrosinase were not significantly different; with the increase of the concentration of polyphenol extracts and the treatment With the increase of treatment time, the tyrosinase activity of B16 cells gradually decreased, and the tyrosinase activity was dose-dependent; 200 μg / mL of target II and III polyphenol extracts reduced the tyrosinase activity of B16 cells to 58.36% and 51.69% respectively at 72 h; the inhibitory effect of target III extract on tyrosinase activity was stronger than that of target II extract. It can be seen that the target II and III extracts of Wuwei Xiaodu Drink are potential tyrosinase activity inhibitors and can be used as potential tyrosinase inhibitors in the cosmetics and pharmaceutical industries.

[0094] In summary, the ultrasound-assisted enzymatic method was used to crudely extract the polyphenol extract of Wuwei Xiaodu Drink, and the crude extract was eluted on a macroporous adsorption resin column with pure water, 30% ethanol solution, 60% ethanol solution, and 90% ethanol solution in sequence to obtain purified target segment I eluate, target segment II eluate, target segment III eluate, and target segment IV eluate. Among them, the segment II and segment III eluates contained many types of polyphenol compounds with high content. By further investigating the effects of II and III polyphenol extracts on tyrosinase activity, it was found that within the range of 10-200 μg / mL, II and III polyphenol extracts had no obvious toxicity to B16 cell viability, and III polyphenol extracts had better inhibitory activity on B16 cell melanin and tyrosinase than II polyphenol extracts. Therefore, it was concluded that the target II and III extracts of Wuwei Xiaodu Drink are potential melanin synthesis inhibitors and tyrosinase inhibitors; secondly, the target III extract is composed of cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid, quercetin, chicoric acid, olibanum, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C and montmorillonine. Honeysuckle, wild chrysanthemum, dandelion, Viola yedoensis and Gynura ovata from 5 main producing areas were randomly divided into 10 groups. Ultrasound-assisted enzymatic extraction combined with macroporous adsorption resin column separation and purification were used to obtain 10 batches of target segment III polyphenol extracts. Combined with the results of B16 cell tyrosinase inhibitory activity assay, the average content of the target segment III polyphenol compounds was set as 70-130% as the content limit of the tyrosinase inhibitor. Among the polyphenol extracts used as tyrosinase inhibitors, the content of cryptochlorogenic acid ranged from 0.014±0.03%, the content of chlorogenic acid ranged from 1.070±0.3%, the content of neochlorogenic acid ranged from 0.011±0.03%, the content of quercetin ranged from 0.574±0.15%, the content of chicoric acid ranged from 0.019±0.01%, the content of olibanum glucoside ranged from 0.707±0.21%, the content of isochlorogenic acid B ranged from 0.552±0.16%, the content of isochlorogenic acid A ranged from 12.399±3.72%, the content of isochlorogenic acid C ranged from 3.986±1.19%, and the content of monanthus glycoside ranged from 9.007±2.70%. The target segment III polyphenol extract within this content range has the best tyrosinase inhibitory activity and can be used as a potential tyrosinase inhibitor in the cosmetics and pharmaceutical industries.

[0095] Finally, a few points should be explained: Although the present invention has been described in detail above with general descriptions and specific embodiments, on the basis of the present invention, the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the aforementioned embodiments, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a Chinese medicine compound phenolic extract, characterized in that: The following steps are involved: Step 1) Ultrasound-assisted enzyme method for crude extraction of polyphenolic compounds: Take honeysuckle, wild chrysanthemum, Viola yedoensis, dandelion, and Gynura ovata, mix them evenly, add cellulase and distilled water, adjust the pH to 4.5-5.5, and perform ultrasonic enzymolysis; After ultrasonic enzymatic hydrolysis, the solid and liquid are separated, and the filter residue is extracted with ethanol, and the two filtrates are combined and concentrated under reduced pressure to obtain a concentrated solution; Step 2) Purification of polyphenol extract by macroporous adsorption resin: The concentrated solution is added to a macroporous adsorption resin column, and washed with pure water, 30% ethanol solution, 60% ethanol solution, and 90% ethanol solution in sequence until the eluent is colorless, to obtain the corresponding target segment I component, target segment II component, target segment III component, and target segment IV component eluent, and the eluent of each target segment component is concentrated under reduced pressure until viscous, and freeze-dried to obtain; The traditional Chinese medicine compound phenolic extract comprises target segment II components and / or target segment III components; In step 1), the amount of cellulase added is 0.2-0.4% of the total mass of honeysuckle, wild chrysanthemum, Viola yedoensis, dandelion and Gynura ovata; In step 1), take 28-32g of honeysuckle, 10-12g of wild chrysanthemum, 10-12g of Viola yedoensis, 10-12g of dandelion, and 10-12g of Gynura ovata; In step 1), distilled water is added at a material-liquid ratio of 1g:55-65ml; In step 1), the ultrasonic enzymatic hydrolysis temperature is 55-65°C, and the ultrasonic enzymatic hydrolysis time is 25-35 min; In step 1), the filter residue is extracted with 25-35% ethanol for 25-35 minutes.

2. The Chinese herbal compound phenolic extract prepared by the preparation method according to claim 1.

3. Use of the Chinese herbal compound phenolic extract according to claim 2 in the preparation of tyrosinase inhibitors.

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