Application of herba ecliptae extract in preparation of whitening product

Abstract

The invention discloses an application of a herba ecliptae extract in preparation of a whitening product. The skin-whitening product takes the herba perillae extract as a main functional component, and comprises a skin-care product, a washing care product and an aromatherapy product with a skin-whitening effect. The herba ecliptae extract comprises guaiacol, alpha-eudesmol, beta-eudesmol, D-camphor and beta-caryophyllene, and is obtained by performing steam distillation extraction on the overground part of herba ecliptae as a raw material. The invention discloses an effect of inhibiting melanin synthesis of the herba perillae acutae extract, can directly reduce the melanin content of human melanoma cells MNT1, and also can indirectly reduce the melanin content of the MNT1 cells by influencing human immortalized keratinocytes HaCaT. Under the same mass concentration, the effect of the whitening agent is superior to that of the whitening agents alpha-arbutin and kojic acid which are widely used at present. Therefore, the herba perillae extract can be used as a natural extract component to be applied to development of related whitening products such as cosmetic technology and aromatherapy.

Description

Technical Field

[0001] The present invention relates to the fields of cosmetics technology and aromatherapy, and specifically, to the application of perilla frutescens extract in the preparation of whitening products. Background Art

[0002] The color of human skin is largely determined by the type, quantity, and distribution pattern of melanin in the skin. Melanin is synthesized in the organelles of basal melanocytes - melanosomes, which are then transported into the surrounding keratinocytes and finally excreted out of the body as the keratinocytes shed. The function of melanocytes to synthesize melanin is regulated by a complex network of paracrine factors, and keratinocytes are an important part of this network. The secretion of factors is affected by external stimuli such as ultraviolet rays and air pollutants. At the same time, these stimuli can also trigger oxidative stress in cells, leading to the generation of a large number of free radicals, and the presence of excessive free radicals can also induce the production of melanin.

[0003] Existing whitening raw materials used in the cosmetics field include kojic acid, arbutin, phenylethyl resorcinol, niacinamide, vitamin C, etc. However, the application of the above-mentioned whitening agents has limitations. For example, vitamin C has weak stability and is easily oxidized, kojic acid has strong irritation, and arbutin can be converted into hydroquinone under certain conditions, presenting potential safety risks. Based on the traditional aesthetic concept of "beauty lies in whiteness", whitening cosmetics have become one of the most active fields in the domestic cosmetics market. Finding whitening raw materials with better whitening effects, which can make up for the defects of existing raw materials and help create differentiated products, is a research hotspot in the industry.

[0004] Based on the current consumers' preference for natural and sustainable ingredients, the application of plant extracts of natural origin in cosmetics has become a popular research direction in the industry. Most plant extracts have functions such as anti-inflammatory, antibacterial, and soothing effects, and are generally safer than chemically synthesized raw materials, having great application potential in the cosmetics field. Plant essential oils are obtained by methods such as steam distillation, pressing, or organic solvent extraction from parts of plants such as leaves, seeds, flowers, roots, etc., and are mixtures composed of volatile natural compounds. It not only has the characteristics of both safety and efficacy of plant extracts, but also can affect emotions, stress levels, etc. through volatile aroma substances, bringing additional benefits to the product and improving the consumer experience.

[0005] The plant species of the genus Salvia (Lamiaceae) are rich in species and widely distributed. There are about 1,000 species globally, and more than 80 species have been discovered in China. They have development value in aspects such as food, medicine, ornament, and essential oils, but the germplasms widely cultivated and applied in China are very limited. Currently, the essential oil of Salvia sclarea from the genus Salvia is commonly used in aromatherapy in China, while the extracts of Salvia japonica and Salvia officinalis are often used as raw materials for cosmetics. Salvia greggii is a perennial herb of the genus Salvia. It is regarded as a medicinal and edible plant in its native places, Mexico and the United States, and is used to treat digestive and oral diseases. It is usually cultivated as an ornamental plant in other regions of the world. The subtropical climate and altitude conditions in Yunnan are similar to its native place, which is very suitable for the growth of Salvia greggii. Although the above-ground part of Salvia greggii contains rich volatile fragrance substances, its potential in skin care has not been fully explored.

[0006] In summary, the exploration of new raw materials and their new uses can not only promote the further understanding of natural plant extracts, but also provide scientific basis and support for the development of the cosmetics industry, the field of aromatherapy, and the local economy. Summary of the Invention

[0007] To solve the above technical problems, the purpose of the present invention is to provide an application of Salvia greggii extract in the preparation of whitening products. Provide skin care products, washing and care products, and aromatherapy products containing Salvia greggii extract. Using Salvia greggii extract as the main active ingredient can effectively reduce the melanin content in the epidermis, providing new ideas and effective ways for the use of natural ingredients in whitening means for cosmetics and aromatherapy products.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides an application of Salvia greggii extract in the preparation of whitening products. The whitening products use Salvia greggii extract as the main efficacy ingredient, and the Salvia greggii extract is the essential oil extract of the above-ground part of Salvia greggii.

[0010] As some specific embodiments of the present invention, in the whitening products, the content of Salvia greggii extract is 0.2% - 5% by mass fraction.

[0011] As some specific embodiments of the present invention, by mass percentage, the Salvia greggii extract contains the following components: guaiol 21.0% - 26.0%, α-eudesmol 11.5% - 14.5%, β-eudesmol 9.0% - 12.0%, D-camphor 7.5% - 9.5%, and β-caryophyllene 5.5% - 7.5%.

[0012] In some specific embodiments of the present invention, by mass percentage, the extract of Satureja montana L. contains the following components: guaiol 23.6155% ± 1.3187%, α-eudesmol 12.9751% ± 0.1697%, β-eudesmol 10.4303% ± 0.0574%, D-camphor 8.3704% ± 0.1706%, and β-caryophyllene 6.4713% ± 0.0117%.

[0013] In some specific embodiments of the present invention, the extract of Satureja montana L. further includes the following components:

[0014] α-pinene, α-thujene, camphene, β-pinene, sabinene, terpinene, D-limonene, eucalyptol, γ-terpinene, o-cymene, terpinolene, 3-octanol, α-cubebene, (2R,5R)-2-methyl-5-propan-2-ylbicyclo[3.1.0]hexan-2-ol, 2-methyl-5-isopropyl-bicyclo[3.1.0]hexan-3-ol, cyclocitral, 2-amino-3-methylbenzyl alcohol, α-copaene, borneal, β-bourbonene, 1-methyl-4-(1-methylethenyl)cyclohexanol, (-)-isocaryophyllene, (-)-bornyl acetate, caryophyllene, β-humulene, (-)-α-bergamotene, 4-terpineol, α-caryophyllene, trans-veratrol, γ-muurolene, borneol, α-terpineol, ferulene, α-muurolene, δ-cadinene, β-sesquiphellandrene, α-curcumene, α-ionone, 1-(5,6,7,8-tetrahydro-2,8,8-trimethyl-4H-cyclohepta[b]furan-5-yl)ethanone, α-dehydrocalamenene, (3R,3aR,3bR,4S,7R,7aR)-4-isopropyl-3,7-dimethyloctahydro-1H-cyclopenta[1,3]cyclopropa[1,2]benzene-3-ol, caryophyllin, spiroxatone, 2-isopropyl-5-methyl-9-methylenebicyclo[4.4.0]dec-1-ene, bicyclogermacrene, γ-gurjunene, γ-selinene, valeranone, (+)-rosifoliol, eucalypta-4(15),7-dien-1β-ol, γ-eudesmol, eugenol, acorenene, aristolene, valenciaene, α-gurjunene, isoguaiol, 1,2,4-triethylbenzene, T-eudesmol, hexamethylbenzene, 10,10-dimethyl-2,6-methylenebicyclo[7.2.0]undecan-5β-ol, α-cubaben-11-ol.

[0015] In some specific embodiments of the present invention, the preparation method of the extract of Satureja montana L. includes: taking the raw material of Satureja montana L. for steam distillation, collecting the upper oily liquid, and dehydrating and drying to obtain a yellow essential oil, which is the extract of Satureja montana L.

[0016] As some specific embodiments of the present invention, the raw material of Salvia officinalis L. is selected from the aerial parts of at least one of the plants of the genus Salvia in the family Lamiaceae, namely Salvia officinalis L. and its variants and cultivars, and the aerial parts include stems and leaves.

[0017] As some specific embodiments of the present invention, the temperature of the steam distillation is 210 - 230 °C, and the time is 4 - 6 h.

[0018] As some specific embodiments of the present invention, the method of dehydration and drying is the anhydrous sodium sulfate drying method, the drying temperature is 25 - 30 °C, preferably at room temperature, and the time is 1 - 2 h.

[0019] As some specific embodiments of the present invention, the whitening product includes at least one of skin care products, washing and care products, and aromatherapy products having a whitening effect.

[0020] As some specific embodiments of the present invention, the skin care product is selected from any one of the following forms: aqueous solution, emulsion, spray, cream, essence, and facial mask.

[0021] As some specific embodiments of the present invention, the washing and care product is selected from any one of the following forms: hand sanitizer, body wash, and facial cleanser.

[0022] As some specific embodiments of the present invention, the aromatherapy product is selected from any one of the following forms: massage oil, aromatherapy essential oil, and aromatherapy candle.

[0023] The aromatherapy refers to aromatherapy, which utilizes the aromatic smell or active ingredients of Salvia officinalis L. essential oil. Through methods such as massage or aromatherapy, it is absorbed by the human body through the olfactory organ or skin to help people relieve stress and relax both physically and mentally.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention provides a new use of Salvia officinalis L. extract in whitening. The results of cell experiments show that Salvia officinalis L. essential oil can reduce the melanin content of MNT1 cells. At the same mass concentration, its effect exceeds that of the currently widely used whitening raw materials α - arbutin and kojic acid. In addition, the technical solution described in the present invention finds that Salvia officinalis L. essential oil can indirectly reduce the melanin content of MNT1 cells by affecting HaCaT cells, and its effect is also better than that of α - arbutin and kojic acid. Moreover, Salvia officinalis L. essential oil also has certain antioxidant properties and can play a whitening role through multiple pathways such as acting on melanocytes and keratinocytes to exert antioxidant ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] By reading the detailed description of the non - restrictive embodiments with reference to the following drawings, other features, objects, and advantages of the present invention will become more apparent:

[0027] Figure 1 It is the experimental result diagram of the scavenging ability of perilla frutescens essential oil on DPPH free radicals in Example 2;

[0028] Figure 2 It is the experimental result diagram of the scavenging ability of perilla frutescens essential oil on ABTS free radicals in Example 2;

[0029] Figure 3 It is the experimental result diagram of the toxicity of different concentrations of perilla frutescens essential oil on HaCaT cells in Example 3;

[0030] Figure 4 It is the experimental result diagram of the toxicity of different concentrations of perilla frutescens essential oil on MNT1 cells in Example 3;

[0031] Figure 5 It is the experimental result diagram of the influence of different test substances and concentrations on the melanin content of MNT1 cells in Example 4;

[0032] Figure 6 It is the experimental result diagram of the influence of the supernatant of HaCaT cells treated with different test substances on the melanin content of MNT1 cells in Example 5. Detailed implementation mode

[0033] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0034] Unless otherwise specified, the raw materials, reagents, and instruments used in the following embodiments can all be obtained from conventional commercial channels or can be obtained by existing known methods.

[0035] Example 1 Extraction and component analysis of perilla frutescens essential oil

[0036] 1. Experimental method

[0037] (1) Essential oil extraction

[0038] Weigh 400 g of the fresh above-ground part of perilla frutescens, cut it into pieces and place it in a 5 L round-bottom flask. Add 2.5 L of water, then turn on the condensation reflux device and heat it at 220 °C in an electric hot pot until the volume of the oil layer in the separatory funnel no longer increases. Stop heating. After the separatory funnel cools down, rotate the piston to release the lower-layer water and collect the upper-layer oily liquid. After drying, yellow essential oil is obtained. The essential oil is collected and sealed in a glass container essential oil bottle. After weighing, it is stored in the refrigerator at 4 °C in the dark for later use.

[0039] The above experiments were repeated in parallel three times to calculate the average extraction rate of savory essential oil obtained by steam distillation. The calculation formula is as follows:

[0040] Essential oil extraction rate = (mass of obtained essential oil / mass of the above-ground part of savory weighed) × 100%

[0041] (2) Analysis of essential oil components

[0042] The components of the essential oil were analyzed by gas chromatography-mass spectrometry (GC-MS) technology, and the detection parameters are as follows:

[0043] Gas chromatography conditions: The chromatographic column was a DB-WAX capillary column (30 m × 250 μm × 0.25 μm); the carrier gas was high-purity helium (99.999%), and the flow rate was 1 mL / min; 10 μL of the sample before injection was diluted with 500 μL of ethanol and 500 μL of n-hexane; the injection port temperature was 260 °C, split injection, the injection volume was 1.0 μL, and the split ratio was 30:1. The temperature programming: The initial temperature was 50 °C, held for 3 min; then heated to 120 °C at a rate of 4 °C / min and held for 10 min; then heated to 220 °C at a rate of 5 °C / min and held for 2 min; the solvent delay was 3.5 min.

[0044] Mass spectrometry conditions: Electron ionization source (EI), electron energy 70 eV, GC-MS interface temperature 280 °C; ion source temperature 230 °C; scanning mode was full scan; mass scanning range was 35 - 450 amu.

[0045] Each separated peak was retrieved by a computer using a standard mass spectrometry library (Agilent NIST14 library), and the spectra were manually analyzed. The relative percentage content of each component was obtained by the peak area normalization method. The above experiments were repeated in parallel three times to calculate the average relative percentage content of each component.

[0046] 2. Experimental results

[0047] The results are shown in Tables 1 - 2. Table 1 shows the extraction rate of savory essential oil obtained, and Table 2 shows the component information.

[0048] Table 1 Extraction rate of savory essential oil

[0049] Plant weight / g Essential oil weight / g Extraction rate / % Replicate 1 395.6 0.940 0.238 Replicate 2 412.2 1.012 0.246 Replicate 3 405.0 0.975 0.241 Average 0.241 Standard deviation 0.003

[0050] Table 2 Main components of savory essential oil

[0051]

[0052]

[0053]

[0054]

[0055] As can be seen from Table 1, the extraction rate of essential oil obtained from the aerial part of *Salvia officinalis* is 0.241±0.003%. As can be seen from Table 2, a total of 67 components were identified through detection of the essential oil of *Salvia officinalis*, and the 5 components with relatively high contents are as follows: guaiol (23.6155%±1.3187%), α-eudesmol (12.9751%±0.1697%), β-eudesmol (10.4303%±0.0574%), D-camphor (8.3704%±0.1706%), and β-caryophyllene (6.4713%±0.0117%). The main components of the essential oil of *Salvia sclarea*, which is commonly used in aromatherapy, are linalyl acetate and linalool. CN110302128A discloses a whitening, anti-allergic, soothing and sleep-aiding facial mask rich in natural product extraction components and a preparation method thereof. The sage extract, which is one of the natural product extraction components, is an ethanol extract, and the main component is salvianolic acid. By comparing with the component analysis results, it can be seen that the main components of the essential oil of *Salvia officinalis* are very different from the components of the extracts of other *Salvia* plants.

[0056] Example 2 Experiment on the scavenging ability of *Salvia officinalis* essential oil against DPPH and ABTS free radicals

[0057] 1. Experimental method

[0058] (1) Determination of DPPH free radical scavenging ability

[0059] Weigh 0.01 mmol of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) into a 100 mL volumetric flask, make up the volume with anhydrous ethanol and shake well to prepare a 0.1 mmol / L DPPH ethanol solution, and let it stand in the dark for 30 min for later use. Add 100 μL of the previously prepared ethanol solutions of *Salvia officinalis* essential oil with different concentrations of 0.1, 1, 10, 100, 1000, 10000, 100000, and 200000 μg / mL to each well of a 96-well plate. Additionally, set 100 μL of anhydrous ethanol as the solvent control group. Then add 100 μL of the DPPH ethanol solution to each well, mix well and let it stand in the dark for 30 min. Set three replicates for each treatment concentration. Use a microplate reader to measure the absorbance value (OD value) at a wavelength of 517 nm. According to the formula, compare and calculate the OD values of the solvent control group A1 and the essential oil group A2 to obtain the DPPH free radical scavenging rate. The calculation formula is as follows:

[0060] DPPH free radical scavenging rate = (A1 - A2) / A1 × 100%

[0061] (2) Determination of ABTS free radical scavenging ability

[0062] Weigh 1.4 mmol of ABTS into a beaker, add ultrapure water to 200 mL and shake well to prepare a 7 mmol / L ABTS solution; weigh 0.49 mmol of potassium persulfate into a beaker, add ultrapure water to 200 mL and shake well to prepare a 2.45 mmol / L potassium persulfate solution; mix the 7 mmol / L ABTS solution and the 2.45 mmol / L potassium persulfate solution in a volume ratio of 1:1, shake well, and let it stand in the dark for 12 - 16 h to obtain the ABTS working mother liquor. Dilute the working mother liquor with absolute ethanol so that the OD value is 0.70 ± 0.02 at a wavelength of 734 nm to obtain the ABTS working solution. Add 10 μL of the previously prepared ethanol solutions of savory essential oil with different concentrations of 0.1, 1, 10, 100, 1000, 10000, 100000, 200000 μg / mL to each well of a 96-well plate, and set 10 μL of absolute ethanol as the solvent control group. Then add 200 μL of the ABTS working solution to each well, mix well, and let it stand in the dark for 30 min. Set three replicates for each treatment concentration. Measure the OD value at a wavelength of 734 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Compare and calculate the OD values of the solvent control group A1 and the essential oil group A2 according to the formula to obtain the ABTS radical scavenging rate. The calculation formula is as follows:

[0063] ABTS radical scavenging rate = (A1 - A2) / A1 × 100%

[0064] 2. Experimental results

[0065] It can be understood that when the skin is stimulated by external factors such as ultraviolet rays and air pollutants, cells will produce oxidative stress and a large number of free radicals will be generated. The existence of too many free radicals will induce the formation of melanin. On the other hand, the biosynthesis of melanin requires a series of oxidation reactions. Therefore, raw materials with antioxidant activity can play a whitening role by inhibiting free radicals and reducing melanin and the intermediate substances in its synthesis process. For the antioxidant activity of raw materials, physical and chemical detection can be carried out by using the methods of DPPH and ABTS radical scavenging.

[0066] Table 3 Scavenging ability of savory essential oil on DPPH radicals

[0067]

[0068] Table 4 Scavenging ability of savory essential oil on ABTS radicals

[0069]

[0070] The experimental results are shown in Tables 3 - 4 and Appendix Figures 1 - 2 Among them, Tables 3 and Appendix Figure 1 show the effects of savory essential oil at different concentrations on the DPPH radical scavenging rate. Tables 4 and AppendixFigure 2 Effect of Thymus vulgaris L. essential oil at different concentrations on ABTS radical scavenging rate. One-way ANOVA was used for statistical analysis, and Dunnett's post hoc multiple comparison test was used to test significance. Compared with the solvent control group, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

[0071] As shown in Table 3 and Appendix Figure 1 It can be seen that Thymus vulgaris L. essential oil with a concentration higher than 1 mg / mL has significant DPPH radical scavenging ability, and the IC50 value is 2.645 ± 0.290 mg / mL; as shown in Table 4 and Appendix Figure 2 It can be seen that Thymus vulgaris L. essential oil with a concentration higher than 1 mg / mL has significant ABTS radical scavenging ability, and the IC50 value is 6.148 ± 0.656 mg / mL, indicating that Thymus vulgaris L. essential oil has certain antioxidant activity.

[0072] Example 3 Toxicity experiment of Thymus vulgaris L. essential oil on HaCaT and MNT1 cells

[0073] 1. Experimental method

[0074] The CCK-8 method was used to determine the safe concentration range of Thymus vulgaris L. essential oil. Take human immortalized keratinocytes HaCaT and human melanoma cells MNT1 in the logarithmic growth phase with good status. After digestion, collection and viable cell counting, the control group and the essential oil group were inoculated into 96-well plates at a cell density of 2×10 5 cells / mL, 100 μL / well. The blank group was added with 100 μL of complete medium (the composition of the complete medium is shown in Table 5), and each group was set with three replicates. After culturing in a 5% CO2, 37 °C incubator for 24 h, the original medium was removed. For the essential oil groups of HaCaT cells, 100 μL of complete medium containing 1, 10, 100, 150, 200, 250, 300, 350 μg / mL essential oil was added to each well respectively. For the essential oil groups of MNT1 cells, 100 μL of complete medium containing 1, 10, 25, 50, 75, 100, 150, 200, 300 μg / mL essential oil was added to each well respectively. The control group and the blank group were added with 100 μL of complete medium. After continuing to culture for 24 h, the original medium was removed, and 100 μL of fresh complete medium containing 10% CCK-8 was added to each well. After incubating at 37 °C for 2 h, the OD value at a wavelength of 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. According to the formula, the OD values of the blank group A0, the control group A1 and the essential oil group A2 were compared and calculated to obtain the relative cell viability and determine the safe concentration of the essential oil. The calculation formula is as follows:

[0075] Relative cell viability = (A2 - A0) / (A1 - A0) × 100%.

[0076] Table 5 Composition of Complete Medium

[0077] Component Brand Item number Volume fraction DMEM medium (high glucose) Gibco 11965092 88％ Fetal bovine serum Gibco 10099-141 10％ Penicillin - streptomycin mixed solution (100×) Yuanye Bio-Technology R20016 - 100ml 1％ MEM non-essential amino acid solution (100×) Yeasen Biotech 60707ES60 1％

[0078] 2. Experimental Results

[0079] The experimental results are shown in Appendix Figure 3 and Appendix Figure 4 . Among them, Appendix Figure 3 shows the effect of different concentrations of Thymus vulgaris essential oil on the relative viability of HaCaT cells, and Appendix Figure 4 shows the effect of different concentrations of Thymus vulgaris essential oil on the relative viability of MNT1 cells. One-way ANOVA was used for statistical analysis, and Dunnett's post hoc multiple comparison test was used to test for significance. Compared with the control group, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

[0080] It can be seen from Appendix Figures 3 - 4 that when the concentration of Thymus vulgaris essential oil is 1 - 100 μg / mL, it has no toxic effect on HaCaT and MNT1 cells; when the concentration of Thymus vulgaris essential oil is greater than 200 μg / mL, there is a significant difference in the relative viability of MNT1 cells compared with the control group. Therefore, the concentration below 100 μg / mL was finally selected for subsequent experiments, and three working concentrations of 10, 25, and 50 μg / mL were set. It is understandable that when the test substance has no cytotoxicity, if it has a certain inhibitory effect on melanin synthesis, it indicates that the test substance has a certain whitening effect at the cellular level.

[0081] Example 4 Direct Inhibitory Effect of Thymus vulgaris Essential Oil on Melanin Content in MNT1 Cells

[0082] 1. Experimental Method

[0083] The NaOH lysis method was used to detect the melanin content in MNT1 cells. Take MNT1 cells in the logarithmic growth phase with good status, digest, collect and count the live cells. Then, the solvent control group, positive control group and essential oil group were seeded at a cell density of 6×10 4Inoculate at a density of

[0084] 2. Experimental Results

[0085] It is understandable that the amount of melanin directly affects the skin color of the human body. Therefore, the melanin content can be evaluated using melanoma cells to determine the whitening effect of the test substance. The experimental results are shown in the appendix Figure 5 , showing the effects of different concentrations of savory essential oil, α-arbutin, and kojic acid on the melanin content of MNT1 cells. One-way ANOVA was used for statistical analysis, and Dunnett's post hoc multiple comparison test was used to test for significance. Compared with the control group, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

[0086] As can be seen from the appendix Figure 5 , the effects of savory essential oil, α-arbutin, and kojic acid on melanin content were concentration-dependent. Compared with the solvent control group, the melanin content decreased significantly after treatment with low and medium concentrations (10, 25 μg / mL) of savory essential oil. In contrast, the positive controls α-arbutin and kojic acid showed significant effects only at high concentration (50 μg / mL), and the inhibitory effect of high-concentration savory essential oil on melanin content was better than that of the two positive control substances. In summary, compared with the two positive control substances, savory essential oil had a more significant effect on the melanin content of MNT1 cells and had the effect of inhibiting melanin synthesis in cells.

[0087] Example 5 Indirect inhibitory effect of savory essential oil on melanin content of MNT1 cells by affecting HaCaT cells

[0088] 1. Experimental method

[0089] Collect the supernatant of HaCaT cells treated or untreated with savory essential oil as the conditioned medium for the growth of MNT1 cells, and detect the melanin content in MNT1 cells by the NaOH lysis method. Take HaCaT cells in the logarithmic growth phase with good status, digest, collect and count the viable cells. Then, the solvent control group, positive control group and essential oil group were inoculated into 6-well plates at a cell density of 4×10 5 cells / mL, 1 mL / well. After culturing in a 5% CO2, 37 °C incubator for 24 h, remove the original medium. In the positive control group, each group was added with 1.5 mL of complete medium containing 10, 25, 50 μg / mL of kojic acid or α-arbutin. In the essential oil group, each group was added with 1.5 mL of complete medium containing 10, 25, 50 μg / mL of essential oil. The solvent control group was added with 1.5 mL of complete medium. After continuing to culture for 48 h, remove the original medium, wash twice with phosphate buffer (PBS), add 1.5 mL of fresh medium to each group, continue to culture for 24 h, centrifuge to collect the cell supernatant, and add an equal volume of fresh medium to obtain the conditioned medium.

[0090] Take MNT1 cells in the logarithmic growth phase with good status, digest, collect and count the viable cells. Then, the blank control group, solvent control group, positive control group and essential oil group were inoculated into 12-well plates at a cell density of 6×10 4 cells / mL, 1 mL / well. Each group was set with six replicates, three of which were used to detect the melanin content and the other three were used to detect the total protein content of the cells. After culturing in a 5% CO2, 37 °C incubator for 24 h, remove the original medium. The solvent control group was added with 1.5 mL of conditioned medium obtained without positive control or essential oil treatment. In the positive control group, each group was added with 1.5 mL of conditioned medium obtained by treatment with 10, 25, 50 μg / mL of kojic acid or α-arbutin. In the essential oil group, each group was added with 1.5 mL of conditioned medium obtained by treatment with 10, 25, 50 μg / mL of essential oil. The blank control group was added with 1.5 mL of complete medium. The subsequent methods for determining the total protein content and melanin content were the same as those in Example 4.

[0091] 2. Experimental results

[0092] The experimental results are shown in the appendix Figure 6, Effects of supernatants of HaCaT cells treated with different concentrations of savory essential oil, α-arbutin, and kojic acid on the melanin content of MNT1 cells. One-way ANOVA was used for statistical analysis, and Dunnett's post hoc multiple comparison test was used to test for significance. Compared with the control group, *: p < 0.05; **: p < 0.01; ***: p < 0.001.

[0093] As shown by Figure 6 It can be seen that compared with the blank control group, the melanin content in the solvent control group increased significantly, indicating that the supernatant of HaCaT cells has the effect of promoting melanin synthesis. Compared with the solvent control group, the cell supernatants treated with low and medium concentrations (10, 25 μg / mL) of savory essential oil could significantly reduce the melanin content, suggesting that the essential oil can indirectly affect the synthesis of melanin in MNT1 cells by influencing the paracrine effect of HaCaT cells. The cell supernatants treated with the two positive control substances had no significant effect on the cell melanin level. In summary, within the tested concentration range, savory essential oil has the effect of indirectly inhibiting melanin synthesis by influencing HaCaT cells, while the two positive control substances cannot exert their whitening effects through this pathway.

[0094] In summary, the technical solution described in the present invention has proven through multiple experimental studies that savory essential oil has a whitening effect, and the effect is better than that of the positive control substances α-arbutin and kojic acid. Savory essential oil can exert its whitening effect through pathways such as antioxidant activity, directly reducing the melanin content of melanocytes, and indirectly reducing the cell melanin content by influencing the paracrine of keratinocytes.

[0095] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various modifications or alterations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. Use of a savory extract in the preparation of a skin-whitening product, characterized in that, The whitening product uses perilla frutescens extract as the main active ingredient.

2. The application according to claim 1, wherein, In the whitening product, the content of perilla frutescens extract is 0.2% - 5% by mass fraction.

3. The application according to claim 1, characterized in that, By mass percentage, the perilla frutescens extract contains the following components: guaiol 21.0% - 26.0%, α-eudesmol 11.5% - 14.5%, β-eudesmol 9.0% - 12.0%, D-camphor 7.5% - 9.5% and β-caryophyllene 5.5% - 7.5%.

4. The application according to claim 3, wherein By mass percentage, the perilla frutescens extract contains the following components: guaiol 23.6155% ± 1.3187%, α-eudesmol 12.9751% ± 0.1697%, β-eudesmol 10.4303% ± 0.0574%, D-camphor 8.3704% ± 0.1706% and β-caryophyllene 6.4713% ± 0.0117%.

5. The application according to claim 3 or 4, characterized in that, The perilla frutescens extract also includes the following components: α-pinene, α-thujene, camphene, β-pinene, sabinene, terpinene, D-limonene, eucalyptol, γ-terpinene, o-cymene, terpinolene, 3-octanol, α-cubebene, (2R,5R)-2-methyl-5-propan-2-ylbicyclo 3.1.0]hexan-2-ol, 2-methyl-5-isopropyl-bicyclo[3.1.0]hexan-3-ol, cyclocitral, 2-amino-3-methylbenzyl alcohol, α-copaene, bornylidenacetone, β-bourbonene, 1-methyl-4-(1-methylethenyl)cyclohexanol, (-)-isocaryophyllene, (-)-bornyl acetate, caryophyllene, β-humulene, (-)-α-bergamotene, 4-terpineol, α-caryophyllene, trans-veratrol, γ-ylangene, borneol, α-terpineol, costunolide, α-ylangene, δ-cadinene, β-sesquiphellandrene, α-curcumene, α-ionone, 1-(5,6,7,8-tetrahydro-2,8,8-trimethyl-4H-cyclohepta[b]furan-5-yl)ethanone, α-dihydrocalamenene, (3R,3aR,3bR,4S,7R,7aR)-4-isopropyl-3,7-dimethyloctahydro-1H-cyclopenta[1,3]cyclopropa[1,2]benzene-3-ol, caryophyllin, spiroalangicol, 2-isopropyl-5-methyl-9-methylene-bicyclo[4.4.0]dec-1-ene, bicyclogermacrene, γ-gurjunene, γ-selinene, valeranone, (+)-rosifoliol, eudesm-4(15),7-dien-1β-ol, γ-eudesmol, eugenol, acoradiene, aristolene, valenciaene, α-gurjunene, isoguaiol, 1,2,4-triethylbenzene, T-ylangol, hexamethylbenzene, 10,10-dimethyl-2,6-methylene-bicyclo[7.2.0]undecan-5β-ol, α-cubaben-11-ol.

6. The application according to claim 1, wherein The preparation method of the perilla frutescens extract includes: taking perilla frutescens raw materials for steam distillation, collecting the upper oily liquid, and dehydrating and drying to obtain yellow essential oil, which is the perilla frutescens extract.

7. The application according to claim 6, wherein The sage raw material is selected from the aerial parts of plants of the genus Salvia in the Lamiaceae family, including sage and its varieties and cultivars, and the aerial parts include stems and leaves.

8. The application according to claim 6, characterized in that, The temperature of the steam distillation is 210-230 °C, and the time is 4-6 h; The method of dehydration and drying is anhydrous sodium sulfate drying method, the drying temperature is 25-30 °C, and the time is 1-2 h.

9. The application according to claim 1, characterized in that, The whitening product includes at least one of skin care products, washing and care products, and aromatherapy products with whitening effects.

10. The application according to claim 9, wherein The skin care product is selected from any one of the following forms: aqueous solution, emulsion, spray, cream, essence, mask; And / or, the washing and care product is selected from any one of the following forms: hand sanitizer, body wash, facial cleanser; And / or, the aromatherapy product is selected from any one of the following forms: massage oil, aromatherapy essential oil, aromatherapy candle.

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