A kind of perilla combined traditional Chinese medicine, effective component extraction method and application thereof

Abstract

The invention provides a perilla combination Chinese medicine, an effective component extraction method and application thereof, and relates to the technical field of skin whitening. The combination Chinese medicine comprises the following components in mass ratio: 40-50 parts of perilla leaves, 10-20 parts of cassia twigs, 8-12 parts of peony root, 8-12 parts of moutan bark, 8-15 parts of liquorice, and 6-10 parts of dried ginger. The invention adopts pure Chinese medicinal materials for proportioning, and extracts the total extract of the perilla prescription from the Chinese medicinal prescription by cellulase-assisted ultrasonic extraction method. It is found that the extract has rich active substances, strong DPPH free radical scavenging ability and the effect of inhibiting tyrosinase activity in vitro. The extract is applied in a melanin overexpression model of melanoma cells. Experimental results show that the extract can reduce the generation of intracellular melanin by inhibiting the intracellular tyrosinase activity, thereby achieving the effect of skin whitening.

Description

Technical Field

[0001] The invention relates to the technical field of skin whitening, and in particular to a perilla combined traditional Chinese medicine, a method for extracting effective components and applications thereof. Background Art

[0002] Skin is the largest organ in the human body and the first barrier separating the human body from the outside world. It is responsible for protecting the body, regulating body temperature, secretion, immunity and other functions. When people are exposed to the external environment for a long time, they will inevitably receive many external stimuli, such as ultraviolet rays in the atmosphere. When the skin is exposed to ultraviolet rays, the body will activate the activity of tyrosinase through a series of signal transduction pathways and protect the skin from ultraviolet damage by promoting the melanocytes in the basal layer of the skin to produce melanin. However, the abnormal accumulation of melanin will cause the formation of spots such as sun spots and freckles on the epidermis. In severe cases, it may evolve into diseases such as skin melanoma, which endangers human health.

[0003] With the improvement of living standards, people are paying more and more attention to the whitening effect of products to obtain a more satisfactory and comfortable use experience. Commonly used whitening ingredients in products include arbutin, kojic acid, ascorbic acid, etc., but these ingredients are often reported to be highly irritating and carcinogenic, and can cause adverse reactions such as contact dermatitis, making it difficult to expand market demand. Plant extract ingredients from natural sources are often favored by consumers due to their mild and natural characteristics. Therefore, finding and developing natural green whitening raw materials from plants has become a new trend in the development of the industry. Perilla is a Chinese medicine that is both a medicine and a food. It contains a variety of active substances, including phenolic acids, flavonoids, polyphenols, terpenes, polysaccharides and volatile oils, etc. It has multiple pharmacological activities such as anti-inflammatory, antioxidant, anti-depressant, blood pressure lowering, antibacterial, and regulating glucose and lipid metabolism. Taking perilla as the main medicinal flavor and combining it with other Chinese medicines that also have multiple functions, such as cinnamon twig, moutan bark, white peony root, etc., can form a Chinese medicine prescription with multiple functions. Therefore, the present invention proposes a perilla combined Chinese medicine, an effective component extraction method and its application to solve the problems existing in the prior art. Summary of the invention

[0004] In view of the above problems, the present invention provides a perilla combination Chinese medicine, an effective component extraction method and application thereof. The extract contains rich active substances, has good antioxidant capacity and safety, and has extremely high tyrosinase activity inhibition ability.

[0005] To achieve the purpose of the present invention, the present invention is implemented through the following technical scheme: a perilla combination Chinese medicine, comprising the following components in mass ratio: 40-50 parts of perilla leaves, 10-20 parts of cassia twigs, 8-12 parts of peony root, 8-12 parts of moutan bark, 8-15 parts of licorice, and 6-10 parts of dried ginger.

[0006] The further improvement is that it comprises the following ingredients in weight ratio: 50 parts of perilla leaves, 10 parts of cassia twigs, 10 parts of peony root, 10 parts of moutan bark, 10 parts of liquorice and 10 parts of dried ginger.

[0007] A method for extracting effective components of a perilla combination Chinese medicine comprises the following steps:

[0008] S1: Take dried perilla leaves, cinnamon twigs, peony root, moutan bark, liquorice and dried ginger, grind them into powder, use ethanol aqueous solution as extraction solvent, and add the powder to the extraction solvent at a solid-liquid ratio of 1g / 10mL;

[0009] S2: Add cellulase, set the extraction temperature to 53°C, the ultrasonic power to 40kHz, perform ultrasonic extraction for 40min, filter the liquid with a Buchner funnel, discard the residue, and obtain the ultrasonic extract of Perilla frutescens;

[0010] S3: Take the ultrasonic extract of Perilla frutescens prescription and place it on a rotary evaporator, reduce the pressure and concentrate it to an extract, place it in a freeze dryer, and freeze-dry it to powder to obtain the Perilla frutescens prescription extract.

[0011] A further improvement is that in said S1, the volume percentage of ethanol in the ethanol aqueous solution is 50%-60%.

[0012] A further improvement is that in said S2, the content of the added cellulase is 1-3%.

[0013] The extract obtained by the above-mentioned method for extracting the effective ingredients of the perilla combination Chinese medicine is used in the preparation of whitening products. The whitening products are selected from external medicines and cosmetics, and the whitening products use the extract as the only active ingredient.

[0014] A further improvement is that the content of the extract in the cosmetic is 0.5%-5%.

[0015] A further improvement is that the content of the extract in the cosmetic or external medicine is 3%-10%.

[0016] A further improvement is that the cosmetic is one of toner, essence, lotion, face cream and mask.

[0017] A further improvement is that the cosmetic is one of loose powder, pressed powder, liquid foundation, foundation cream, BB cream, isolation cream, sunscreen, lipstick, lip balm, rouge, blush and eye shadow.

[0018] The beneficial effects of the present invention are:

[0019] 1. The present invention adopts pure Chinese medicinal materials for proportioning, and utilizes cellulase-assisted ultrasonic extraction method to extract the total extract of Perilla prescription from the Chinese medicinal prescription. The detection finds that the extract is rich in active substances, has strong DPPH free radical scavenging ability and inhibits tyrosinase activity in vitro. It is applied in a melanoma cell melanin overexpression model. The experimental results show that the extract can reduce the production of intracellular melanin by inhibiting intracellular tyrosinase activity, thereby achieving the effect of skin whitening.

[0020] 2. The present invention can adjust the effects of multiple medicinal flavors through the ratio of multiple medicinal flavors, enhance the effect and efficacy of the prescription. Comparative experiments show that the Chinese medicinal prescription of perilla combination is superior to perilla single herb in terms of in vitro antioxidant activity, intracellular tyrosinase inhibition, melanin synthesis inhibition, etc., and has a better whitening effect.

[0021] 3. The perilla extract of the present invention has the characteristics of being natural and mild, and can be used as a functional raw material component with whitening effect, and applied in cosmetics, external medicines and cosmetics, which is safer and more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of the detection of the effective component content of the perilla prescription extract and the perilla extract obtained by ultrasonic extraction and hot water extraction of the present invention, wherein A is the anthocyanin content; B is the total flavonoid content; C is the rosmarinic acid content; D is the caffeic acid content; *P≤0.05, **P≤0.01, ***P≤0.001 indicate significant differences compared with the perilla prescription extract and the perilla extract;

[0023] Figure 2 The DPPH free radical scavenging rate result diagram of the perilla prescription extract and the perilla extract of the present invention, wherein the yellow bar graph is the vitamin C result; the pink bar graph is the perilla extract result; the blue bar graph is the perilla prescription extract result; *P≤0.05, **P≤0.01, ***P≤0.001 indicate significant differences compared with the perilla prescription extract and the perilla extract;

[0024] Figure 3 The graph is a result of the in vitro tyrosinase inhibition rate of the perilla prescription extract and the perilla extract of the present invention, wherein the yellow bar graph is the result of arbutin; the pink bar graph is the result of the perilla extract; the blue bar graph is the result of the perilla prescription extract; *P≤0.05, **P≤0.01, ***P≤0.001 indicate significant differences between different test substances;

[0025] Figure 4The figure is the effect of the extract of the perilla prescription and the perilla extract of the present invention on the cell viability of B16-H10 cells, wherein A is the result of the extract of the perilla prescription; B is the result of the perilla extract; *P≤0.05, **P≤0.01, ***P≤0.001 indicate that the different concentrations of the drug administration groups are significantly different from the blank;

[0026] Figure 5 The results of the effect of the extract of the perilla prescription of the present invention on the melanin content and tyrosinase activity of B16-H10 cells are shown in Figure 1, wherein A is a melanin production phenotype diagram of the extract of the perilla prescription acting on B16-H10 cells; B is a histogram of the melanin content of B16-H10 cells under different treatments; C is a histogram of the tyrosinase activity of B16-H10 cells under different treatments; ###P≤0.001 indicates a significant difference compared with the blank group, *P≤0.05, **P≤0.01, ***P≤0.001 indicate a significant difference compared with the model group;

[0027] Figure 6 The figures are result diagrams showing the effect of the perilla extract of the present invention on the melanin content and tyrosinase activity of B16-H10 cells, wherein A is a melanin production phenotype diagram of the perilla extract acting on B16-H10 cells; B is a bar graph of the melanin content of B16-H10 cells under different treatments; C is a bar graph of the tyrosinase activity of B16-H10 cells under different treatments; ###P≤0.001 indicates a significant difference compared with the blank group, *P≤0.05, **P≤0.01, ***P≤0.001 indicate a significant difference compared with the model group. DETAILED DESCRIPTION

[0028] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0029] Embodiment 1

[0030] This embodiment proposes the preparation of the perilla prescription extract, and the perilla prescription extract and the perilla extract are extracted by the following method, comprising the following steps:

[0031] S1, extraction: take dried perilla leaves and cinnamon twigs, peony root, moutan bark, licorice, and dried ginger (or take only dried perilla leaves), grind and crush, use 60% ethanol as the extraction solvent, add the powder to the extract at a solid-liquid ratio of 1g / 10mL, add 1% cellulase, the extraction temperature is 53°C, the ultrasonic power is 40kHz, ultrasonic extraction is performed for 40min, filter with a Buchner funnel, discard the filter residue, and obtain the ultrasonic extract of perilla prescription;

[0032] S2. Concentration: Take the ultrasonic extract of Perilla frutescens prescription and place it on a rotary evaporator, concentrate it under reduced pressure to an extract, place it in a freeze dryer, and freeze-dry it to powder to obtain the Perilla frutescens prescription extract.

[0033] Embodiment 2

[0034] Extracts of perilla prescription and content of active ingredients in perilla extracts

[0035] The effective component contents of the perilla extract and the perilla extract prepared in Example 1 were respectively detected, including anthocyanins, total flavonoids, rosmarinic acid, and caffeic acid. At the same time, the perilla extract and the perilla extract were extracted by hot water extraction to compare the difference in effective component content between the ultrasonic extraction method and the hot water extraction method. The specific experimental steps are as follows:

[0036] 1. Hot water extraction method:

[0037] Take dried perilla leaves and cinnamon twigs, peony root, moutan bark, licorice, and dried ginger (or only take dried perilla leaves alone), grind them into powder, use pure water as the extraction solvent, add the powder to the pure water at a solid-liquid ratio of 1g / 10mL, heat to boiling over high heat, keep it boiling for 30 minutes over low heat, filter with a Buchner funnel, discard the filter residue, concentrate under reduced pressure to an extract, and freeze-dry to powder to obtain the perilla prescription and perilla extract.

[0038] 2. Detection of anthocyanin content in perilla extract and perilla extract

[0039] Weigh 0.1g of perilla extract and perilla extract powder respectively into a conical flask, add 10mL of 0.1mol / L hydrochloric acid methanol solution respectively, extract by ultrasonic for 40min, make up the lost weight with 0.1mol / L hydrochloric acid methanol solution and take out, centrifuge at 4000r / min for 10min, take out the supernatant and dilute 10 times. Analyze on a spectrophotometer. Use 0.1mol / L hydrochloric acid methanol solution as a blank control. Determine the optical density value at wavelengths of 530, 620, and 650nm. Calculate the anthocyanin content according to the Greey formula, as follows:

[0040] Anthocyanin optical density value ODλ=(OD530-OD620)-0.1(OD650-OD620)

[0041] Anthocyanin content (nmol / g) = [(ODλ / ε) × (V / m)] × 1000000

[0042] Where: ε is the molar extinction coefficient of anthocyanin 4.62×106; V is the total volume of the extract (mL); m is the sample mass (g).

[0043] 3. Detection of total flavonoids content in perilla prescription extract and perilla extract

[0044] Accurately weigh 0.1 g of perilla prescription extract and perilla extract powder respectively, place in a stoppered conical flask, add 10 mL of 70% ethanol respectively, extract by ultrasonication for 45 min, cool, weigh again, make up the lost weight with 70% ethanol, take out, centrifuge at 4000 r / min for 10 min, and aspirate 1.0 mL of supernatant respectively, and wait for color development (color development is the same as below).

[0045] Preparation of standard curve of total flavonoids: take about 8.5mg of rutin reference substance, weigh accurately, place in a 25mL brown volumetric flask, add an appropriate amount of 70% ethanol, place in warm water and shake gently to dissolve, cool, add 70% ethanol to the scale, shake well, and the reference solution is obtained; accurately draw 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL, 3.0mL of reference solution, place in a 25mL volumetric flask respectively, add 70% ethanol to 3.0mL; then add 1mL of 5% sodium nitrite solution, shake well, and let stand for 6min; then add 1mL of 10% aluminum nitrate test solution, shake well, and let stand for 6min; add 10mL of sodium hydroxide test solution, finally use 70% ethanol to the scale, shake well, and let stand for 15min. According to the UV-visible spectrophotometry method, the absorbance is measured at a wavelength of 500nm, and the standard curve is drawn with the mass of rutin as the horizontal axis and the absorbance as the vertical axis.

[0046] The absorbance of the test solution was measured at the maximum absorption wavelength, and the content of total flavonoids was calculated using the standard curve.

[0047] 4. Detection of rosmarinic acid and caffeic acid content in perilla extract and perilla extract

[0048] The rosmarinic acid and caffeic acid contents were detected by high performance liquid chromatography, and the steps are as follows:

[0049] Chromatographic conditions: C18 chromatographic column, methanol: 0.2% phosphoric acid aqueous solution (25:75) as mobile phase, detection wavelength 330 nm, flow rate 1 mL / min.

[0050] Preparation of test solution: Accurately weigh 0.01 g of the extract of Perilla frutescens prescription and Perilla frutescens extract, respectively, add 1.5 mL of 80% methanol, extract by ultrasonic at 40 °C for 40 min, take the supernatant and centrifuge at 12000 rpn for 10 min, and filter to obtain the solution.

[0051] Preparation of reference solution: Take appropriate amount of rosmarinic acid and caffeic acid reference substances, weigh accurately, and add 80% methanol to prepare a rosmarinic acid reference solution containing 0.16 mg per 1 mL and a caffeic acid reference solution containing 0.26 mg per 1 mL, respectively.

[0052] Determination of rosmarinic acid and caffeic acid content: According to the high performance liquid chromatography method, accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0053] The results showed that both the perilla prescription and the perilla extract contained abundant active ingredients ( Figure 1 ). Compared with thermal extraction, ultrasonic extraction can extract higher contents of rosmarinic acid and caffeic acid, but less anthocyanins and total flavonoids. Since rosmarinic acid has been proven to be a strong antioxidant and caffeic acid has been added to cosmetics, ultrasonic extraction was selected as the extraction method for later research, which is more conducive to the application of later products. In addition, the contents of anthocyanins, total flavonoids, rosmarinic acid and caffeic acid in the extract of perilla prescription are not as high as those in perilla extract. This may be because these four components are all contained in large quantities in perilla, and the perilla prescription, which is composed of multiple medicinal flavors, contains a wider range of active ingredients and needs further testing.

[0054] Embodiment 3

[0055] Antioxidant activity of extracts from Perilla frutescens in vitro

[0056] The extract of the perilla prescription and the perilla extract prepared in Example 1 were tested by a DPPH free radical scavenging rate experiment. Vitamin C was used as a positive control. The specific experimental steps are as follows:

[0057] The extract of perilla prescription and perilla extract were reconstituted with pure water into aqueous solutions of different concentrations, and 25, 50, 100, 200, 500, and 1000 μg / mL were selected as experimental concentrations, with vitamin C as the positive control. The DPPH free radical scavenging ability detection kit was used, and the control tube solution, the test tube solution, and the blank tube solution were prepared according to the kit instructions. They were placed indoors in the dark for 30 minutes, and then centrifuged to take the supernatant and detect the absorbance at 517 nm. The DPPH free radical scavenging rate of each sample was calculated according to the scavenging rate calculation formula, and the formula is as follows:

[0058] DPPH free radical scavenging rate (%) = [(1-(A determination - A control) ÷ A blank) × 100]%;

[0059] Where Aassay is the absorbance value of the assay tube, Acontrol is the absorbance value of the control tube, and Ablank is the absorbance value of the blank tube.

[0060] The results showed that both the extract of Perilla frutescens prescription and the extract of Perilla frutescens had good concentration dependence ( Figure 2). The free radical scavenging rate of 200μg / mL of Perilla frutescens extract was 88.8%; when the concentration reached 1000μg / mL, the free radical scavenging rate could reach 95% of vitamin C, and its antioxidant capacity was comparable to that of the strong antioxidant vitamin C. The free radical scavenging rate of Perilla frutescens extract was higher than that of Perilla frutescens extract, indicating that the composition of the formula can improve the antioxidant capacity of the extract, and it has better efficacy than Perilla frutescens alone. The experimental results show that Perilla frutescens extract has good in vitro antioxidant activity and is less dose-dependent.

[0061] Embodiment 4

[0062] Inhibitory Effect of Extracts from Perilla frutescens on Tyrosinase Activity in Vitro

[0063] The shiso extract and shiso extract prepared in Example 1 were tested by tyrosinase inhibition test. Arbutin was used as a positive control. The specific experimental steps are as follows:

[0064] Perilla extract and perilla extract were reconstituted with pure water into aqueous solutions of different concentrations, and 5, 50, and 100 μg / mL were selected as experimental concentrations, with arbutin as a positive control. Tyrosinase activity detection kit (Solebo) was used to prepare different solutions according to the instructions of the kit. Tyrosinase solution: 25kU tyrosinase powder was added to 12.5mL of pure water to prepare 2000U / mL. A blank group and a measurement group were set up. Blank group: 50μL pure water + 50μL tyrosinase solution; measurement group: 50μL of the solution to be tested + 50μL tyrosinase solution.

[0065] Add 900 μL of reagent 1 to the above test solution, start timing immediately, and measure the absorbance at 475 nm at 10 s (total time); continue the reaction at room temperature for 180 s, and measure the absorbance at 475 nm at 190 s (total time); test each group of test solutions 3 times. The tyrosinase inhibition rate is calculated according to the following formula:

[0066] Inhibition ratio (%) = 1-((CD) / (AB)) × 100%

[0067] Wherein, A is the absorbance value at 475 nm of the blank group at 10 s (total time); B is the absorbance value at 475 nm of the blank group at 190 s (total time); C is the absorbance value at 475 nm of the measurement group at 10 s (total time); D is the absorbance value at 475 nm of the measurement group at 190 s (total time).

[0068] The results showed that the extracts of Perilla frutescens prescription and Perilla frutescens extract had good in vitro tyrosinase inhibition ability in a concentration-dependent manner ( Figure 3). The tyrosinase inhibition rate of the extract of Perilla frutescens and Perilla frutescens extract was higher than that of the positive drug arbutin. When the concentration was 100 μg / mL, the tyrosinase inhibition rate of the extract of Perilla frutescens could reach 84.8%, which was 5 times that of arbutin at the same concentration, showing a very high inhibition ability. In addition, the extract of Perilla frutescens was higher than that of Perilla frutescens extract at all concentrations, indicating that the effect of Perilla frutescens was better than that of Perilla frutescens alone and had a stronger whitening effect.

[0069] Embodiment 5

[0070] Effect of Perilla frutescens extract on proliferation of melanoma cells (B16F10 cells)

[0071] The effect of the extract of Perilla frutescens prescription on the proliferation of melanoma cells (B16F10 cells) was detected by CCK-8 cell proliferation toxicity test. The specific experimental steps are as follows:

[0072] 100 μL of B16F10 cell suspension was prepared in a 96-well plate, with each well containing approximately 5,000 cells; the culture plate was pre-cultured in an incubator at 37°C, 5% CO2 for 24 hours; 10 μL of the test substance (Perilla extract and Perilla extract) of different concentrations was added to the culture plate, and the plate was incubated in an incubator for 24 hours; the culture dish was removed, the old culture medium was removed, 100 mL of PBS was added to each well, and the plate was washed twice, and 90 μL of complete culture medium and 10 μL of CCK-8 solution were added to each well; the plate was incubated in an incubator for 2 hours; and the absorbance at 450 nm was measured using an enzyme marker.

[0073] The results showed that the cell viability gradually decreased with the increase of the concentration of the extract of Perilla frutescens and Perilla frutescens extract in the range of 5 μg / mL to 1200 μg / mL ( Figure 4 ). When the concentration of perilla extract was 80 μg / mL, the cell viability was > 89%, indicating that this concentration had no inhibitory effect on cell proliferation. When the concentration was 150 μg / mL, the cell viability was 82.2%, indicating that this concentration may have a certain inhibitory effect on cell viability, so the perilla extract below 100 μg / mL was selected as the subsequent dosing concentration; when the perilla extract was 300 μg / mL, the cell viability was 83.3%, indicating that this concentration may have a certain inhibitory effect on cell proliferation, so the perilla extract below 200 μg / mL was selected as the subsequent dosing concentration.

[0074] Embodiment 6

[0075] Effects of extracts from Perilla frutescens on melanin synthesis in melanoma cells

[0076] The melanin overexpression model of B16H10 cells was used to evaluate the effects of Perilla prescription extract and Perilla extract on melanin synthesis. The experiment was divided into normal group, model group, control group and different concentration drug administration groups.

[0077] Normal group: cells were cultured normally; model group: 10 μM α-MSH was added; control group: 100 μg / mL of Perilla extract was added; low-concentration administration group: 10 μM α-MSH and 10 μg / mL of Perilla extract were added; medium-concentration administration group: 10 μM α-MSH and 30 μg / mL of Perilla extract were added; high-concentration administration group: 10 μM α-MSH and 100 μg / mL of Perilla extract were added.

[0078] B16H10 cells in the logarithmic growth phase were inoculated in 6-well culture plates, divided into groups according to the above experiments, and 10 μM α-MSH and different concentrations of the test substances were added respectively, and cultured in an incubator for 72 hours. Subsequently, the old culture medium was discarded, PBS was washed twice, and trypsin was added to collect the cell precipitate. PBS buffer containing 1% Triton X-100 was added to each tube, and the cells were lysed in an ice bath, centrifuged at 10000r / min, 4℃ for 15min, and the supernatant was used to detect tyrosinase activity; black cell precipitates were taken, 1mL ethanol: ether solution (V / V=1:1) was added to dissolve the opaque particles therein, centrifuged at 3000r / min for 5min, the supernatant was discarded, and 1mol / L NaOH solution containing 10% DMSO was added to the precipitate, and the precipitate was incubated at 80℃ for 1h. After cooling, centrifuged at 3000r / min for 5min, and the supernatant was taken to measure the absorbance of each group at 405nm.

[0079] Depend on Figure 5 The results showed that the melanin content of the model group administered with α-MSH was significantly increased compared with the blank group (P>0.01), indicating that the melanin overexpression model of B16-F10 cells was successfully constructed. The melanin content of the control group administered with 100μg / mL perilla extract alone was equivalent to that of the blank group. The melanin content of the group administered with α-MSH and 100μg / mL perilla extract at the same time was significantly decreased compared with the model group (P>0.05), indicating that the perilla extract at these two concentrations has the effect of significantly reducing the synthesis of intracellular melanin and has a whitening effect.

[0080] Embodiment 7

[0081] Effects of Perilla frutescens extract on tyrosinase activity in melanoma cells

[0082] The cell supernatant in Example 6 was taken to determine the intracellular tyrosinase activity. The protein concentration in each group was determined by the BCA method. The supernatant was placed in a 96-well plate, 150 μL of 1 mg / mL L-DOPA was added to each well, and cells without the test substance were used as blank control wells. The cells were incubated at 37°C in the dark for 3 h, and the absorbance at 470 nm was measured. The results were normalized and plotted.

[0083] Tyrosinase activity (%) = A absorbance / protein concentration × 100%

[0084] Depend on Figure 5 The results showed that the tyrosinase activity of the model group administered with α-MSH was significantly increased compared with the blank group (P>0.001), indicating that the melanin overexpression model of B16-F10 cells was successfully constructed. The tyrosinase activity of the control group administered with 100 μg / mL perilla extract alone was equivalent to that of the blank group. The tyrosinase activity of the groups administered with α-MSH and 10 and 100 μg / mL perilla extract at the same time was significantly decreased compared with the model group (P>0.05), indicating that the perilla extract at these two concentrations has a significant inhibitory effect on intracellular tyrosinase activity, and also indicates that the perilla extract reduces melanin production by inhibiting intracellular tyrosinase activity and has a whitening effect. In addition, compared with the perilla extract, the perilla extract showed a more effective whitening effect, indicating that the preparation of multiple medicinal flavors can enhance the therapeutic effect of the drug.

[0085] Based on the above research results, the extract of the Chinese herbal prescription of Perilla combination is highly safe, has good antioxidant capacity and excellent in vitro tyrosinase activity inhibition ability, and can reduce melanin synthesis in melanoma cells by inhibiting tyrosinase activity. It can be used in daily skin care, whitening and antioxidant cosmetics, cosmetics and topical medicines, and can be used as a new type of cosmetic raw material that is natural, mild and effective.

[0086] Embodiment 8

[0087] Perilla extract is used in aqueous solution

[0088] Cosmetics used in aqueous solutions are made from the following raw material ratios:

[0089] Table 1 Ratio of raw materials for aqueous cosmetics

[0090] raw material Proportion (mass %) Dipotassium glycyrrhizinate 0.3 β-Glucan 2 Butanediol 5 0.5 Phenoxyethanol 0.3 Allantoin 0.2 Ethanol 8 Perilla extract 1 Polyoxyethylene oleyl alcohol 0.1 Distilled water Add to 100

[0091] When making an aqueous solution, the production steps are as follows: heat each material and dissolve it in water.

[0092] Embodiment 9

[0093] Perilla extract is used in lotions

[0094] Cosmetics used in lotions and creams are made from the following raw materials:

[0095] Table 2 Ratio of raw materials for lotions and creams

[0096]

[0097] When making the emulsion, the production steps are as follows:

[0098] Mix A and heat it to 95°C to prepare the oil phase; heat B to 95°C to prepare the water phase; slowly add the oil phase A to the water phase B, homogenize and emulsify for 5 minutes; stir continuously, cool to room temperature and discharge.

[0099] Embodiment 10

[0100] Perilla extract is used in patch-type facial masks

[0101] Cosmetics used in patch-type facial masks are made from the following raw material ratios:

[0102] Table 3 Raw material ratios of cosmetics for patch-type facial masks

[0103]

[0104]

[0105] When making a facial mask, the production steps are as follows:

[0106] Mix phase A in advance and dissolve phase B in advance. Mix the two phases and fully dissolve them. After all substances are dissolved, soak the non-woven fabric patch with the mask liquid and package it in a single-piece aseptic manner.

[0107] Embodiment 11

[0108] Perilla extract is used in creams and ointments

[0109] Cosmetics used in creams are made from the following raw materials:

[0110] Table 4 Raw material ratios of creams and cosmetics

[0111]

[0112] When making the cream, the production steps are as follows:

[0113] Mix the oil phase A and heat to 90°C to melt and mix; mix the water phase B evenly; slowly add the oil phase A to the water phase B, stir for 30 minutes, cool to 40°C and then pack.

[0114] Embodiment 12

[0115] Perilla extract is used in essence liquid

[0116] Cosmetics used in serums are made from the following raw materials:

[0117] Table 5 Raw material ratio of essence cosmetics

[0118]

[0119]

[0120] When making the essence, the production steps are as follows:

[0121] Mix the oil phase A component and stir it evenly until it is completely dissolved, heat it to 80°C, keep it warm for 20 minutes, wait until the temperature drops to 45°C, add the mixed water phase B component, stir evenly, wait until the temperature drops to 36°C, and filter it with a 400-mesh filter cloth.

[0122] Embodiment 13

[0123] Perilla extract is used in lipsticks

[0124] The cosmetics used in lipstick are made from the following raw materials:

[0125] Table 6 Lipstick cosmetic raw material ratio table

[0126] raw material Proportion (mass %) Beeswax 28.4 Niacinamide 0.6 Rapeseed Oil 28 beeswax 25 Nano zinc oxide 4 Peppermint essential oil 5 Perilla extract 5 Ethylparaben 4

[0127] When making lipstick, the production steps are as follows:

[0128] Weigh the above raw materials according to the formula, mix and heat, stir evenly, and pour into a mold.

[0129] Embodiment 14

[0130] Perilla extract is used in blush creams

[0131] Cosmetics used in blush creams are made from the following raw material ratios:

[0132] Table 7 Raw material ratio of blush cream cosmetics

[0133] raw material Proportion (mass %) Kaolin 8 Titanium dioxide 4 Ozokerite 5 Lanolin 2.5 Polyethylene glycol 4 Propylene glycol 3 Butanediol 4 Mica 2.5 Distilled water Add to 100 Perilla extract 2

[0134] Weigh the above raw materials according to the formula, mix and stir evenly, and then pour into a mold to shape.

[0135] The Chinese medicines selected in this invention are all from clinical experience, and the functions of the effective components are briefly introduced as follows:

[0136] Perilla: It can relieve the symptoms of cold. It contains rich active ingredients and has antioxidant, anti-inflammatory and antibacterial effects. It can improve skin diseases such as dermatitis and acne, and improve skin condition.

[0137] Cinnamon twig: relieves muscles and dispels wind, harmonizes Ying and Wei. Cinnamon twig with added flavor can treat skin diseases such as erythema, eczema, urticaria, and pruritus.

[0138] Peony: nourishes blood and softens the liver, relieves depression and removes blood stasis, has anti-inflammatory, soothing, liver-nourishing and liver-protecting effects, and can improve and treat facial chloasma.

[0139] Cortex Moutan: Clears away heat and cools blood, promotes blood circulation and removes blood stasis. It has antibacterial, anti-inflammatory and antioxidant effects, and can treat skin diseases such as skin pigmentation, itching, eczema, etc.

[0140] Licorice: nourishes the spleen and replenishes Qi, clears away heat and detoxifies. It can regulate the skin's immune function and enhance the skin's disease resistance.

[0141] Dried ginger: warms the middle and dispels cold, restores yang and unclogs the meridians. It can promote blood circulation, regulate skin immunity, etc.

[0142] By combining multiple medicinal ingredients, the effects of multiple medicinal ingredients can be harmonized and the efficacy of the prescription can be enhanced.

[0143] The invention utilizes cellulase-assisted ultrasonic extraction to extract the total extract from the traditional Chinese medicine prescription. The reagents used in the method are green and safe, and the method is efficient and practical with high extraction efficiency.

[0144] The present invention proves that many active ingredients are contained in the extract by detecting the effective ingredients in the extract. By exploring the antioxidant capacity of the extract, the in vitro tyrosinase inhibition ability, and using the melanin overexpression model of melanoma cells (B16-H10 cells), it is proved that the perilla prescription extract can reduce the melanin synthesis in the cell by inhibiting the tyrosinase activity, thereby playing a whitening effect. Skin whitening is to have a high DPPH free radical scavenging rate and to inhibit the tyrosinase activity in vitro. Skin whitening is to reduce the intracellular melanin content. Skin whitening is to inhibit the intracellular tyrosinase activity, thereby playing a whitening effect.

[0145] The present invention utilizes cellulase-assisted ultrasonic extraction to extract the total extract of perilla prescription from Chinese medicine prescription, and detects and finds that the extract has rich active substances, such as rosmarinic acid, caffeic acid, anthocyanidin, total flavonoids, etc., has strong DPPH free radical scavenging ability and the effect of inhibiting tyrosinase activity in vitro, and is applied to the melanin overexpression model of melanoma cells. The experimental results show that the extract can reduce the generation of intracellular melanin by inhibiting intracellular tyrosinase activity, thereby achieving the effect of skin whitening. In addition, by comparing the whitening effect of perilla single-flavor extract, the results show that perilla prescription is superior to perilla single-flavor medicine in terms of in vitro antioxidant property, intracellular tyrosinase inhibition, melanin synthesis inhibition, etc., indicating that perilla prescription has a more excellent whitening effect. The perilla prescription extract is safe and reliable to be used in cosmetics, external medicines and cosmetics as a functional raw material component.

[0146] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A combination of Chinese medicines containing Perilla frutescens, characterized in that: The invention comprises the following ingredients in mass ratio: 40-50 parts of perilla leaves, 10-20 parts of cassia twigs, 8-12 parts of peony root, 8-12 parts of moutan bark, 8-15 parts of liquorice root and 6-10 parts of dried ginger.

2. A Perilla combination Chinese medicine according to claim 1, characterized in that: The invention comprises the following ingredients in weight ratio: 50 parts of perilla leaves, 10 parts of cassia twigs, 10 parts of peony root, 10 parts of moutan bark, 10 parts of liquorice and 10 parts of dried ginger.

3. A method for extracting effective components from a combination of Chinese herbal medicines containing Perilla frutescens, using the combination of Chinese herbal medicines containing Perilla frutescens as claimed in claim 1, characterized in that: The following steps are involved: S1: Take dried perilla leaves, cinnamon twigs, peony root, moutan bark, liquorice and dried ginger, grind them into powder, use ethanol aqueous solution as extraction solvent, and add the powder to the extraction solvent at a solid-liquid ratio of 1g / 10mL; S2: Add cellulase, set the extraction temperature to 53°C, the ultrasonic power to 40kHz, perform ultrasonic extraction for 40min, filter the liquid with a Buchner funnel, discard the filter residue, and obtain the ultrasonic extract; S3: Take the ultrasonic extraction solution and place it on a rotary evaporator, reduce the pressure and concentrate it to an extract, place it in a freeze dryer, and freeze-dry it to powder to obtain the perilla combination Chinese medicine extract.

4. The method for extracting effective components from a combination of Perilla Chinese medicines according to claim 3, characterized in that: In the S1, the volume percentage of ethanol in the ethanol aqueous solution is 50%-60%.

5. The method for extracting effective components from a combination of Perilla Chinese medicines according to claim 3, characterized in that: In the S2, the added cellulase content is 1-3%.

6. Use of the extract obtained by the method for extracting the effective components of a combination of Perilla Chinese medicines as described in any one of claims 3 to 5 in the preparation of whitening products, wherein the whitening products are selected from external medicines and cosmetics, and the whitening products use the extract as the only active component.

7. The use according to claim 6, characterized in that: In the cosmetics, the content of the extract is 0.5%-5%.

8. The use according to claim 6, characterized in that: In the cosmetics or external medicines, the content of the extract is 3%-10%.

9. The use according to claim 7, characterized in that: The cosmetic is one of toner, essence, lotion, face cream and facial mask.

10. The use according to claim 8, characterized in that: The cosmetic is one of loose powder, pressed powder, liquid foundation, foundation cream, BB cream, isolation cream, sunscreen, lipstick, lip balm, rouge, blush and eye shadow.

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