Composition with synergistic whitening effect and application thereof

Abstract

The invention provides a composition with a synergistic whitening effect. The composition comprises a rhodiola rosea extract and glabridin or is basically composed of the rhodiola rosea extract and the glabridin. The invention also provides another composition with a synergistic whitening effect. The composition contains salidroside and glabridin or is basically composed of salidroside and glabridin. The invention also relates to the use of said composition in the preparation of medicaments or cosmetics, and further relates to medicaments or cosmetics containing said composition.

Description

Technical Field

[0001] The present invention relates to a composition with synergistic whitening effect. The composition of the present invention comprises a rhodiola rosea extract and glabridin or is substantially composed of a rhodiola rosea extract and glabridin. The composition of the present invention comprises salidroside and glabridin or is substantially composed of salidroside and glabridin. The present invention also relates to the use of the composition in the preparation of medicines or cosmetics, and the present invention further relates to medicines or cosmetics containing the composition. Background Art

[0002] Melanin is a key component that determines the color of mammalian skin and hair, and is commonly found in structures such as skin, hair, and mucous membranes. Melanosomes are unique lysosome-related organelles in melanocytes and are the site of melanin synthesis, storage, and transportation. After being synthesized in melanocytes, melanin is transported from the basal layer to differentiated keratinocytes in the upper layer of the skin in the form of melanosomes, and tends to be distributed in keratinocytes in a protective manner against ultraviolet radiation (UVR), forming a supernuclear cap that co-localizes with the cytoskeletal motor protein dynein. Melanin plays an important role in protecting the skin from ultraviolet damage, but the melanin produced in the body is a stable substance that is not easily degraded, and moves to the surface of the skin with keratinocytes, leading to the formation of chloasma, freckles, age spots, and skin pigmentation.

[0003] Melanin is formed by a combination of enzyme catalysis and chemical reactions, a process called melanogenesis. The enzymes involved in melanogenesis are tyrosinase (TYR), tyrosinase-related proteins 1 and 2 (TRP1 and TRP2), but only tyrosinase is absolutely necessary for melanogenesis and plays a key role in the process. The initial stage is the oxidation of phenylalanine and tyrosine to dopaquinone under the catalysis of tyrosinase (TYR). This first step is the only rate-limiting step in melanin synthesis, and the rest of the reactions can proceed spontaneously at physiological pH. After tyrosinase forms dopaquinone, the compound is converted into dopa and dopachrome by auto-oxidation. Dopa is also a substrate for tyrosinase and is oxidized again to dopaquinone by the enzyme. Finally, eumelanin is formed by a series of oxidation reactions of dihydroxyindole (DHI), indole-2-carboxylic acid-5,6-quinone (ICAQ) and dihydroxyindole-2-carboxylic acid (DHICA), the reaction products of dopachrome. In the presence of cysteine ​​or glutathione, dopaquinone is converted to cysteine-based dopa or glutathione-based dopa, which is then oxidized to 5-hydroxy-1,4-benzothiazinyl alanine (HBTA), which then forms pheomelanin. Eumelanin and pheomelanin eventually synthesize melanin.

[0004] In recent years, with the increasing pursuit of the safety and effectiveness of natural ingredients, the use of plant extracts in skin whitening products has gained more and more attention. These naturally derived ingredients are generally regarded as a gentler, safer and healthier choice than synthetic whitening agents. They act on different stages of melanin synthesis through various mechanisms, mostly as tyrosinase inhibitors to inhibit melanin synthesis, thereby reducing skin pigmentation.

[0005] At present, there are many studies on the skin whitening effect of tyrosinase inhibitors. For example, Huang Jie et al. found that a composition (including glabridin, resveratrol, and polydatin) can significantly inhibit melanin production and tyrosinase activity, and the combined effect is significantly better than each single component (Huang Jie et al., Flavors and Fragrances Cosmetics, DOI: 10.20099 / j.issn.1000-4475.2024.0017). Patent CN105342916B discloses a plant extract composition, which is composed of ethanol extracts of acacia and peony, and has a more significant tyrosinase inhibitory effect with fewer components. However, there are still some problems in the current research, such as 1) the synergistic effect of the composition is not significant, 2) there are only in vitro biochemical experimental results, which are still far from the real physiological state, 3) the mechanism of synergistic enhancement of the composition is not clarified, and 4) the safety is low.

[0006] Therefore, there is an urgent need for a composition with strong synergistic whitening activity, which can effectively reduce the amount of a single component (such as glabridin, Rhodiola rosea extract) added in the composition due to the synergistic effect, thereby achieving the purpose of reducing the amount of a single component added in cosmetics while maintaining high whitening activity, thereby reducing the cost of raw materials. Summary of the invention

[0007] The present invention relates to a composition comprising a Rhodiola rosea extract and glabridin in a mass ratio of 5:3 to 20:1. The composition has a synergistic effect in whitening effect, so it can reduce the addition amount of a single component (such as Rhodiola rosea extract or glabridin) in cosmetics while maintaining high activity, thereby reducing the cost of raw materials.

[0008] A first aspect of the present invention relates to a composition comprising or essentially consisting of or consisting of a Rhodiola rosea extract and glabridin, wherein the mass ratio of the Rhodiola rosea extract to glabridin is 5:3 to 20:1.

[0009] The second aspect of the present invention relates to a composition comprising or essentially consisting of or consisting of a Rhodiola rosea extract and glabridin, wherein the mass ratio of the Rhodiola rosea extract to the glabridin is 5:3 to 20:1, wherein the Rhodiola rosea extract is prepared by ethanol extraction, macroporous resin column purification, concentration and drying.

[0010] A third aspect of the present invention relates to a composition comprising or consisting essentially of a Rhodiola rosea extract and glabridin or consisting of a Rhodiola rosea extract and glabridin, wherein the mass ratio of the Rhodiola rosea extract to the glabridin is 5:3 to 20:1, wherein the Rhodiola rosea extract is obtained by a method comprising the following steps:

[0011] (1) extracting the Rhodiola rosea raw material with 65%-85% ethanol by ultrasonic extraction at 20-80° C. for 30-60 minutes, and filtering the Rhodiola rosea raw material residue after the extraction to obtain the Rhodiola rosea alcohol extract;

[0012] (2) extracting the Rhodiola rosea raw material residue filtered out in step (1) again with ethanol under the same conditions as in step (1), and performing step (2) 0-3 times;

[0013] (3) if step (2) is performed 0 times, the rhodiola rosea alcohol extract of step (1) is directly concentrated; if step (2) is performed 1-3 times, the rhodiola rosea alcohol extracts of step (1) and step (2) are combined; and the concentrated solution is purified by a macroporous adsorption resin, wherein the macroporous adsorption resin is first eluted with water to remove impurities, and then the macroporous adsorption resin is eluted with 70-80% ethanol to obtain an eluate;

[0014] (4) The eluate obtained in step (3) is concentrated and then dried to obtain a Rhodiola rosea extract.

[0015] A fourth aspect of the present invention relates to a composition comprising or consisting essentially of or consisting of salidroside and glabridin, wherein the weight ratio of salidroside to glabridin is 1:6 to 5000:1, preferably 5:1 to 800:1.

[0016] The fifth aspect of the present invention relates to use of the composition according to any one of the first to fourth aspects of the present invention in the preparation of medicines / cosmetics having whitening effects.

[0017] The sixth aspect of the present invention relates to a cosmetic or a medicine comprising the composition according to any one of the first to fourth aspects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1These are the apparent chromaticity test results of the blank control group, negative control group, positive control group, and five sample groups A, B, C, D and E in Example 3 of the present invention.

[0019] Figure 2 These are the apparent brightness L* value test results of the blank control group, negative control group, positive control group, and five sample groups A, B, C, D, and E in Example 3 of the present invention, where ## indicates P<0.01 compared with the blank control group; ** indicates P<0.01 compared with the negative control.

[0020] Figure 3 These are the melanin content test results for the blank control group, negative control group, positive control group, and five sample groups A, B, C, D and E in Example 3 of the present invention.

[0021] Figure 4 These are the melanin particle distribution detection results of the blank control group, negative control group, positive control group, and five sample groups A, B, C, D and E in Example 3 of the present invention.

[0022] Figure 5 Detailed diagram of the binding mode of human tyrosinase with salidroside (A) and glabridin (B) obtained according to the molecular dynamics simulation in Example 4 of the present invention.

[0023] Figure 6 The results show the inhibitory effect of salidroside and glabridin on melanin content after compounding according to Example 5 of the present application. Figure 6 The left figure shows the inhibition rate of melanin by 0.000125% glabridin alone, 0.05% salidroside alone, and a combination of 0.000125% glabridin and 0.05% salidroside (including actual values ​​and estimated values). Figure 6 The right graph shows the inhibition rate of melanin by 0.0000625% glabridin alone, 0.05% salidroside alone, and a combination of 0.0000625% glabridin and 0.05% salidroside (including actual values ​​and estimated values). DETAILED DESCRIPTION OF THE INVENTION

[0025] definition

[0026] As used herein, the terms "s", "min" and "h" stand for "seconds", "minutes" and "hours", respectively.

[0027] As used herein, the term "comprising", which is synonymous with "including", "containing" or "characterized by", is inclusive or open-ended and does not exclude additional, undescribed elements or method steps. However, in the text, each time "comprising" is mentioned, it is intended to cover the alternative embodiments of "consisting essentially of" and "consisting of", wherein "consisting of" excludes any elements or steps not specified, and "consisting essentially of" allows the inclusion of other elements or steps not described that do not materially affect the essential or basic characteristics and novel characteristics of the composition or method in question.

[0028] Specifically, as used herein, the term "consisting essentially of..." allows the inclusion of other components that are not described and do not substantially affect the necessary or basic characteristics and novel characteristics of the composition in question, such as adjuvants, diluents or excipients acceptable in the cosmetic field, and other cosmetic ingredients that do not have a whitening effect, but does not allow the inclusion of other substances with a whitening effect other than Rhodiola rosea extract, salidroside and glabridin.

[0029] The term "about" as used herein refers to ±10%, more preferably ±5%, and most preferably ±2% of the value modified by the term, so that a person skilled in the art can clearly determine the scope of the term "about" based on the modified value.

[0030] Composition comprising rhodiola rosea extract and glabridin

[0031] A first aspect of the present invention relates to a composition comprising or essentially consisting of or consisting of a Rhodiola rosea extract and glabridin, wherein the mass ratio of the Rhodiola rosea extract to glabridin is 5:3 to 20:1.

[0032] The second aspect of the present invention relates to a composition comprising or essentially consisting of or consisting of a Rhodiola rosea extract and glabridin, wherein the mass ratio of the Rhodiola rosea extract to the glabridin is 5:3 to 20:1, wherein the Rhodiola rosea extract is prepared by ethanol extraction, macroporous resin column purification, concentration and drying.

[0033] A third aspect of the present invention relates to a composition comprising or consisting essentially of a Rhodiola rosea extract and glabridin or consisting of a Rhodiola rosea extract and glabridin, wherein the mass ratio of the Rhodiola rosea extract to the glabridin is 5:3 to 20:1, wherein the Rhodiola rosea extract is obtained by a method comprising the following steps:

[0034] (1) extracting the Rhodiola rosea raw material with 65%-85% ethanol by ultrasonic extraction at 20-80° C. for 30-60 minutes, and filtering the Rhodiola rosea raw material residue after the extraction to obtain the Rhodiola rosea alcohol extract;

[0035] (2) extracting the Rhodiola rosea raw material residue filtered out in step (1) again with ethanol under the same conditions as in step (1), and performing step (2) 0-3 times;

[0036] (3) if step (2) is performed 0 times, the rhodiola rosea alcohol extract of step (1) is directly concentrated; if step (2) is performed 1-3 times, the rhodiola rosea alcohol extracts of step (1) and step (2) are combined; and the concentrated solution is purified by a macroporous adsorption resin, wherein the macroporous adsorption resin is first eluted with water to remove impurities, and then the macroporous adsorption resin is eluted with 70-80% ethanol to obtain an eluate;

[0037] (4) The eluate obtained in step (3) is concentrated and then dried to obtain a Rhodiola rosea extract.

[0038] In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 5:3 to 20:1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 5:3 to 5:2. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 5:2 to 5:1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 5:1 to 6:1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 6:1 to 7:1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 7:1 to 8:1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 8:1 to 9:1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 9:1 to 10:1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 10:1 to 11:1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 11:1 to 12:1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 12: 1 to 13: 1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 13: 1 to 14: 1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 14: 1 to 15: 1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 15: 1 to 16: 1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 16: 1 to 17: 1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 17: 1 to 18: 1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 18: 1 to 19: 1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 19: 1 to 20: 1. In some embodiments, the mass ratio of Rhodiola rosea extract and glabridin is 10: 1.

[0039] In some embodiments, the Rhodiola rosea raw material is Rhodiola rosea root, Rhodiola rosea stem or other parts of Rhodiola rosea. In some embodiments, the Rhodiola rosea raw material is Rhodiola rosea root.

[0040] In some embodiments, the Rhodiola rosea is selected from one or more of the following: Rhodiola rosea (Rhodiolacrenulata (Hook.f. & Thomson) H.Ohba), Rhodiola calliantha (H.Ohba) H.Ohba), Rhodiola cretinii (Raym.-Hamet) H.Ohba, Rhodiola heterodonta (Hook.f. & Thomson) Boriss.), Rhodiola kirilowii (Regel) Maxim., Rhodiola linearifolia A.Bor., Rhodiola akirilowii (Regel) Maxim., Rhodiola rosea L., Rhodiola sachalinensis Boriss., Rhodiola serrata H.Ohba) and Rhodiola dumulosa (Franch.) SHFu. In some embodiments, the Rhodiola is Rhodiola crenulata (Hook.f. & Thomson) H.Ohba.

[0041] In some embodiments, the Rhodiola rosea extract is obtained by ethanol extraction, wherein the concentration of the extraction solvent ethanol is 50%-100%. In some embodiments, the concentration of the extraction solvent ethanol is 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95% or 95%-100%. In some embodiments, the concentration of the extraction solvent ethanol is 65%-85%.

[0042] In some embodiments, the Rhodiola rosea extract is obtained by ethanol extraction, and the amount of ethanol used for extraction is 2-50 times the amount of the Rhodiola rosea raw material. In some embodiments, the amount of ethanol used for extraction is 2-3 times, 3-4 times, 4-5 times, 5-6 times, 6-7 times, 7-8 times, 8-9 times, 9-10 times, 10-11 times, 11-12 times, 12-13 times, 13-14 times, 14-15 times, 15-16 times, 16-17 times, 17-18 times, 18-19 times, 19-20 times, 20-21 times, 21-22 times, 22-23 times, 23-24 times, 24-25 times the amount of the Rhodiola rosea raw material. , 25-26 times, 26-27 times, 27-28 times, 28-29 times, 29-30 times, 30-31 times, 31-32 times, 32-33 times, 33-34 times, 34-35 times, 35-36 times, 36-37 times, 37-38 times, 38-39 times, 39-40 times, 40-41 times, 41-42 times, 42-43 times, 43-44 times, 44-45 times, 45-46 times, 46-47 times, 47-48 times, 48-49 times or 49-50 times. In some embodiments, the amount of the extraction solvent ethanol used for extraction is 6-10 times the amount of the Rhodiola rosea raw material.

[0043] In some embodiments, the temperature of ultrasonic extraction is 20-80° C. In some embodiments, the temperature of ultrasonic extraction is 20-25° C., 25-30° C., 30-35° C., 35-40° C., 40-45° C., 45-50° C., 50-55° C., 55-60° C., 60-65° C., 65-70° C., 70-75° C., or 75-80° C.

[0044] In some embodiments, the time of ultrasonic extraction is 10-180 minutes. In some embodiments, the time of ultrasonic extraction is 10-20 minutes, 20-30 minutes, 30-40 minutes, 40-50 minutes, 50-60 minutes, 60-70 minutes, 70-80 minutes, 80-90 minutes, 90-100 minutes, 100-110 minutes, 110-120 minutes, 120-130 minutes, 130-140 minutes, 140-150 minutes, 150-160 minutes, 160-170 minutes or 170-180 minutes. In some embodiments, the time of ultrasonic extraction is 30-60 minutes.

[0045] In some embodiments, step (2) according to the third aspect of the present application is performed 0-3 times. In some embodiments, step (2) according to the third aspect of the present application is performed 0 times, 1 time, 2 times or 3 times.

[0046] In some embodiments, the macroporous adsorption resin is selected from macroporous adsorption resin AB8, D101, DB-301 and DEAE-52. In some embodiments, the macroporous adsorption resin is macroporous adsorption resin AB8. In some embodiments, the macroporous adsorption resin is D101.

[0047] In some embodiments, the purification by macroporous adsorption resin includes the steps of first eluting the macroporous adsorption resin with water to remove impurities, and then eluting the macroporous adsorption resin with 70-80% ethanol to obtain an eluent. In some embodiments, the amount of water eluent used is 1-5 times the column volume. In some embodiments, the amount of water eluent used is 1-1.5 times the column volume, 1.5-2 times the column volume, 2-2.5 times the column volume, 2.5-3 times the column volume, 3-3.5 times the column volume, 3.5-4 times the column volume, 4-4.5 times the column volume or 4.5-5 times the column volume. In some embodiments, the amount of water eluent used is 1.5-3 times the column volume.

[0048] In some embodiments, the purification by macroporous adsorption resin includes the steps of first eluting the macroporous adsorption resin with water to remove impurities, and then eluting the macroporous adsorption resin with 70-80% ethanol to obtain an eluent. In some embodiments, the amount of ethanol eluent used is 1-10 times the column volume. In some embodiments, the amount of ethanol eluent used is 1-2 times the column volume, 2-3 times the column volume, 3-4 times the column volume, 4-5 times the column volume, 5-6 times the column volume, 6-7 times the column volume, 7-8 times the column volume, 8-9 times the column volume or 9-10 times the column volume. In some embodiments, the amount of ethanol eluent used is 2-5 times the column volume.

[0049] In some embodiments, the composition does not include other substances with whitening effect except Rhodiola rosea extract, salidroside and glabridin. In some embodiments, the composition does not include other substances with whitening effect except Rhodiola rosea extract and glabridin. In some embodiments, the composition does not include niacinamide. In some embodiments, the composition does not include arbutin. In some embodiments, the composition does not include vitamin C. In some embodiments, the composition does not include glutathione. In some embodiments, the composition does not include kojic acid. In some embodiments, the composition does not include azelaic acid. In some embodiments, the composition does not include tranexamic acid. In some embodiments, the composition does not include Tenaka extract.

[0050] In some embodiments, the Rhodiola rosea extract is not an extract obtained after fermentation of Rhodiola rosea.

[0051] In some embodiments, the composition includes other components not described that do not substantially affect the essential or basic characteristics and novel features of the composition in question, such as cosmetically acceptable excipients, diluents or vehicles, and other cosmetic ingredients that do not have a whitening effect.

[0052] In some embodiments, the concentration of the Rhodiola rosea extract is 100 μg / mL to 2500 μg / mL. In some embodiments, the concentration of the Rhodiola rosea extract is 100 μg / mL to 120 μg / mL, 120 μg / mL to 140 μg / mL, 140 μg / mL to 160 μg / mL, 160 μg / mL to 180 μg / mL, 180 μg / mL to 200 μg / mL, 200 μg / mL to 220 μg / mL, 220 μg / mL to 240 μg / mL, 240 μg / mL to 260 μg / mL, 260 μg / mL to 280 μg / mL, 280 μg / mL to 300 μg / mL, 300 μg / mL to 320 μg / mL, 320 μg / mL to 340 μg / mL, 3 40μg / mL to 360μg / mL, 360μg / mL to 380μg / mL, 380μg / mL to 400μg / mL, 400μg / mL to 420μg / mL, 420μg / mL to 440μg / mL, 440μg / mL to 460μg / mL, 460μg / mL to 480μg / mL, 480μg / mL to 500μg / mL, 500μg / mL to 520μg / mL, 520μg / mL to 540μg / mL, 540μg / mL to 560μg / mL, 560μg / mL to 580μg / mL, 580μg / mL to 600μg / mL, 600μg / mL / mL to 620μg / mL, 620μg / mL to 640μg / mL, 640μg / mL to 660μg / mL, 660μg / mL to 680μg / mL, 680μg / mL to 700μg / mL, 700μg / mL to 720μg / mL, 720μg / mL to 740μg / mL, 740μg / mL to 760μg / mL, 760μg / mL to 780μg / mL, 780μg / mL to 800μg / mL, 800μg / mL to 820μg / mL, 820μg / mL to 840μg / mL, 840μg / mL to 860μg / mL, 860μg / mL to 880μg / mL, 880μg / mL to 900μg / mL, 900μg / mL to 920μg / mL, 920μg / mL to 940μg / mL, 940μg / mL to 960μg / mL, 960μg / mL to 980μg / mL, 980μg / mL to 1000μg / mL, 1000μg / mL to 1020μg / mL, 1020μg / mL to 1040μg / mL, 1040μg / mL to 1060μg / mL, 1060μg / mL to 1080μg / mL, 1080μg / mL to 1200μg / mL, 1100μg / mL to 1120μg / mL,1120μg / mL to 1140μg / mL, 1140μg / mL to 1160μg / mL, 1160μg / mL to 1180μg / mL, 1180μg / mL to 1200μg / mL, 1200μg / mL to 1220μg / mL, 1220μg / mL to 1240μg / mL, 1240μg / mL to 1260μg / mL, 1260μg / mL to 1280μg / mL, 1280μg / mL to 1300μg / mL, 1300μg / mL to 1320μg / mL, 1320μg / mL to 1340μg / mL, 1340μg / mL to 1360μg / mL, 1360μg / mL to 1380μg / mL, 1380μg / mL to 1400μg / mL, 1400μg / mL to 1420μg / mL, 1420μg / mL to 1440μg / mL, 1440μg / mL to 1460μg / mL, 1460μg / mL to 1480μg / mL, 1480μg / mL to 1500μg / mL, 1500μg / mL to 1520μg / mL, 1520μg / mL to 1540μg / mL, 1540μg / mL to 1560μg / mL, 1560μg / mL to 1580μg / mL, 1580μg / mL to 1600μg / mL, 1600μg / mL to 1620μg / mL, 1620μg / mL to 1640μg / mL, 1640μg / mL to 1660μg / mL, 1660μg / mL to 1680μg / mL, 1680μg / mL to 1700μg / mL, 1700μg / mL to 1720μg / mL, 1720μg / mL to 1740μg / mL, 1740μg / mL to 1760μg / mL, 1760μg / mL to 1780μg / mL, 1780μg / mL to 1800μg / mL, 1800μg / mL to 1820μg / mL, 1820μg / mL to 1840μg / mL, 1840μg / mL to 1860μg / mL, 1860μg / mL to 1880μg / mL, 1880μg / mL to 1900μg / mL, 1900μg / mL to 1920μg / mL, 1920μg / mL to 1940μg / mL, 1940μg / mL to 1960μg / mL, 1960μg / mL to 1980μg / mL, 1980μg / mL to 2000μg / mL, 2000μg / mL to 2020μg / mL, 2020μg / mL to 2040μg / mL, 2040μg / mL to 2060μg / mL, 2060μg / mL to 2080μg / mL, 2080μg / mL to 2200μg / mL, 2100μg / mL to 2120μg / mL,2120μg / mL to 2140μg / mL, 2140μg / mL to 2160μg / mL, 2160μg / mL to 2180μg / mL, 2180μg / mL to 2200μg / mL, 2200μg / mL to 2220μg / mL, 2220μg / mL to 2240μg / mL, 2240μg / mL to 2260μg / mL, 2260μg / mL to 2280μg / mL, 2280μg / mL to 2300μg / mL, 2300μg / mL to 2320μg / mL, 2320μg / mL to 2340μg / mL, 2340μg / mL to 2360μg / mL, 2360μg / mL to 2380μg / mL, 2380μg / mL to 2400μg / mL, 2400μg / mL to 2420μg / mL, 2420μg / mL to 2440μg / mL, 2440μg / mL to 2460μg / mL, 2460μg / mL to 2480μg / mL or 2480μg / mL to 2500μg / mL. In some embodiments, the concentration of the Rhodiola rosea extract is 140μg / mL to 1500μg / mL. In some embodiments, the concentration of the Rhodiola rosea extract is 140μg / mL to 500μg / mL. 、

[0053] In some embodiments, the concentration of glabridin is 10 μg / mL to 150 μg / mL. In some embodiments, the concentration of glabridin is 50 μg / mL to 100 μg / mL. In some embodiments, the concentration of glabridin is 50 μg / mL to 80 μg / mL. In some embodiments, the concentration of glabridin is 10 μg / mL to 15 μg / mL, 15 μg / mL to 20 μg / mL, 20 μg / mL to 25 μg / mL, 25 μg / mL to 30 μg / mL, 30 μg / mL to 35 μg / mL, 35 μg / mL to 40 μg / mL, 40 μg / mL to 45 μg / mL, 45 μg / mL to 50 μg / mL, 50 μg / mL to 55 μg / mL, 55 μg / mL to 60 μg / mL, 60 μg / mL to 65 μg / mL, 65 μg / mL to 70 μg / mL, 70 μg / mL to 75 μg / mL, 75 μg / mL to 80 μg / mL, 80 μg / mL to 85 μg / mL, 85 μg / mL to 90 μg / mL, 90 μg / mL to 95 μg / mL, 90 μg / mL to 100 μg / mL, 10 ... 0μg / mL to 85μg / mL, 85μg / mL to 90μg / mL, 90μg / mL to 95μg / mL, 95μg / mL to 100μg / mL, 100μg / mL to 105μg / mL, 105μg / mL to 110μg / mL, 110μg / mL to 115μg / mL, 115μg / mL to 120μg / mL, 120μg / mL to 125μg / mL, 125μg / mL to 130μg / mL, 130μg / mL to 135μg / mL, 135μg / mL to 140μg / mL, 140μg / mL to 145μg / mL, 145μg / mL to 150μg / mL.

[0054] Composition containing salidroside and glabridin

[0055] A fourth aspect of the present invention relates to a composition comprising or consisting essentially of or consisting of salidroside and glabridin, wherein the weight ratio of salidroside to glabridin is 1:6 to 5000:1, preferably 5:1 to 800:1.

[0056] In some embodiments, the weight ratio of salidroside to glabridin is 1:6 to 1:3, 1:3 to 1:1, 1:1 to 3:1, 3:1 to 5:1, 5:1 to 8:1, 8:1 to 10:1, 10:1 to 15:1, 15:1 to 30:1, 30:1 to 50:1, 50:1 to 100:1, 100:1 to 150:1, 150:1 to 200:1, 200:1 to 250:1, 250:1 to 300:1. :1 to 300:1, 300:1 to 350:1, 350:1 to 400:1, 400:1 to 450:1, 450:1 to 500:1, 500:1 to 550:1, 550:1 to 600:1, 600:1 to 650:1, 650:1 to 700:1, 700:1 to 750:1, 750:1 to 800:1, 800:1 to 850:1, 850:1 to 900:1, 900:1 to 950:1, 950:1 to 1000:1, 1000:1 to 1200:1, 1200:1 to 1400:1, 1400:1 to 1600:1, 1600:1 to 1800:1, 1800:1 to 2000:1, 2000:1 to 2200:1, 2200:1 to 2400:1, 2400:1 to 2600:1, 2600:1 to 2800:1, 2 800: 1 to 3000: 1, 3000: 1 to 3200: 1, 3200: 1 to 3400: 1, 3400: 1 to 3600: 1, 3600: 1 to 3800: 1, 3800: 1 to 4000: 1, 4000: 1 to 4200: 1, 4200: 1 to 4400: 1, 4400: 1 to 4600: 1, 4600: 1 to 4800: 1 or 4800: 1 to 5000: 1. In some embodiments, the weight ratio of salidroside to glabridin is 400: 1. In some embodiments, the weight ratio of salidroside to glabridin is 10: 1.

[0057] In some embodiments, the composition does not include other substances with whitening effect except Rhodiola rosea extract, salidroside and glabridin. In some embodiments, the composition does not include other substances with whitening effect except salidroside and glabridin. In some embodiments, the composition does not include niacinamide. In some embodiments, the composition does not include arbutin. In some embodiments, the composition does not include vitamin C. In some embodiments, the composition does not include glutathione. In some embodiments, the composition does not include kojic acid. In some embodiments, the composition does not include azelaic acid. In some embodiments, the composition does not include tranexamic acid. In some embodiments, the composition does not include Tenaka extract.

[0058] In some embodiments, the composition includes other components not described that do not substantially affect the essential or basic characteristics and novel features of the composition in question, such as cosmetically acceptable excipients, diluents or vehicles, and other cosmetic ingredients that do not have a whitening effect.

[0059] In some embodiments, the concentration of salidroside is 0.01% w / w to 1% w / w. In some embodiments, the concentration of salidroside is 0.01% w / w to 0.02% w / w, 0.02% w / w to 0.025% w / w, 0.025% w / w to 0.03% w / w, 0.03% w / w to 0.04% w / w, 0.04% w / w to 0.05% w / w, 0.05% w / w to 0.06% w / w, 0.06% w / w to 0.07% w / w, 0.07% w / w to 0.08% w / w, 0.08% w / w. In some embodiments, the concentration of salidroside is 0.05% w / w. In some embodiments, the concentration of salidroside is 0.025% w / w.

[0060] In some embodiments, the concentration of glabridin is 0.00001% w / w-0.1% w / w. In some embodiments, the concentration of glabridin is 0.00001% w / w-0.00005% w / w, 0.00005% w / w-0.0000625% w / w, 0.0000625% w / w-0.0001% w / w, 0.0001% w / w-0.000125% w / w, 0.000125% w / w In some embodiments, the concentration of glabridin is 0.000125% w / w. In some embodiments, the concentration of glabridin is 0.0000625% w / w. In some embodiments, the concentration of glabridin is 0.00025% w / w.

[0061] Use of the composition of the present invention for preparing medicine or cosmetics

[0062] The fifth aspect of the present invention relates to use of the composition according to any one of the first to fourth aspects of the present invention in the preparation of medicines / cosmetics having whitening effects.

[0063] In some embodiments, the whitening effect is an inhibitory effect on tyrosinase.

[0064] In some embodiments, the whitening effect is a reduction effect on the secretion of melanin.

[0065] Medicines and cosmetics

[0066] The sixth aspect of the present invention relates to a cosmetic or a medicine comprising the composition according to any one of the first to fourth aspects of the present invention.

[0067] In some embodiments, the composition comprises 0.01% to 20% by weight of the cosmetic or medicament.

[0068] In some embodiments, the composition accounts for 0.01%-0.02%, 0.02%-0.03%, 0.03%-0.04%, 0.04%-0.05%, 0.05%-0.06%, 0.06%-0.07%, 0.07%-0.08%, 0.08%-0.09%, 0.09%-0.1%, 0.1 %-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 10%-11%, 11%-12%, 12%-13%, 13%-14%, 14%-15%, 15%-16%, 16%-17%, 17%-18%, 18%-19%, or 19%-20%. In some embodiments, the composition accounts for about 0.11% of the weight of the drug or cosmetic by weight.

[0069] In some embodiments, the medicine or cosmetics are used for skin care. In some embodiments, the medicine and cosmetics are used for skin whitening. In some embodiments, the medicine and cosmetics include the composition according to any one of the first to fourth aspects of the present invention and one or more pharmaceutically or cosmetically acceptable carriers, diluents or excipients. In some embodiments, the medicine and cosmetics are creams, emulsions, pastes, ointments, facial masks, gels, lotions or essences. In some embodiments, the medicine and cosmetics of the present invention may also contain one or more other ingredients, such as plant extracts, nutritional additives, surfactants, flavors and fragrances, pigments, preservatives, antioxidants, moisturizers, ultraviolet absorbers, astringents, penetration aids, pH regulators, etc. Those skilled in the art can make a selection according to their common sense and specific needs.

[0070] In some embodiments, the medicine or cosmetic does not contain other substances with whitening effect except Rhodiola rosea extract, salidroside and glabridin. In some embodiments, the medicine or cosmetic does not contain niacinamide. In some embodiments, the medicine or cosmetic does not contain arbutin. In some embodiments, the medicine or cosmetic does not contain vitamin C. In some embodiments, the medicine or cosmetic does not contain glutathione. In some embodiments, the medicine or cosmetic does not contain kojic acid. In some embodiments, the medicine or cosmetic does not contain azelaic acid. In some embodiments, the medicine or cosmetic does not contain tranexamic acid. In some embodiments, the medicine or cosmetic does not contain Tenaka extract.

[0071] In some embodiments, the medicine or cosmetic comprises the composition according to any one of the first to fourth aspects of the present invention and one or more cosmetically acceptable carriers, diluents or excipients. In some embodiments, the one or more cosmetically acceptable carriers, diluents or excipients include thickeners, humectants, emulsifiers, surfactants, solubilizers, water or a combination of one or more thereof.

[0072] In some embodiments, the medicine or cosmetic comprises the composition according to any one of the first to fourth aspects of the present invention, a thickener, a moisturizer, an emulsifier, a surfactant, a solubilizer and water.

[0073] In some embodiments, the thickener is polyacrylate crosspolymer-6 (SEPIMAX ZEN). In some embodiments, the humectant includes 1,3-propylene glycol, 2-methyl-2,4-pentanediol (isomeric hexanediol) or a combination of one or more thereof. In certain embodiments, the surfactant includes PEG-26-butanol polyether-26, PEG-40 hydrogenated castor oil, SOLUBILISANT LRI or a combination of one or more thereof.

[0074] In some embodiments, the drug or cosmetic further comprises polyacrylate crosspolymer-6, 1,3-propylene glycol, 2-methyl-2,4-pentanediol, SOLUBILISANT LRI and water.

[0075] In some embodiments, the medicine or cosmetic comprises 0.01%-20% by weight of the composition according to any one of the first to fourth aspects of the present invention, 0.1-5% by weight of polyacrylate cross-linked polymer-6, 0.1%-10% by weight of 1,3-propylene glycol, 0.1%-10% by weight of 2-methyl-2,4-pentanediol, 0.01%-1% by weight of SOLUBILISANT LRI and 50%-99% by weight of water.

[0076] In some embodiments, the medicine or cosmetic comprises 0.11% by weight of the composition according to any one of the first to fourth aspects of the present invention, 0.4% by weight of polyacrylate cross-polymer-6, 1% by weight of 1,3-propylene glycol, 1% by weight of 2-methyl-2,4-pentanediol, 0.05% by weight of SOLUBILISANT LRI and 97.44% by weight of water.

[0077] In some embodiments, the medicine or cosmetic comprises the composition according to any one of the first to third aspects of the present invention, wherein the content of the Rhodiola rosea extract accounts for 0.01%-1% by weight of the medicine or cosmetic, and the content of glabridin accounts for 0.001%-0.1% by weight of the medicine or cosmetic. In some embodiments, the cosmetic comprises the composition according to any one of the first to third aspects of the present invention, wherein the content of the Rhodiola rosea extract accounts for 0.1% by weight of the medicine or cosmetic, and the content of glabridin accounts for 0.01% by weight of the medicine or cosmetic.

[0078] In some embodiments, the medicine or cosmetic comprises the composition according to the fourth aspect of the present invention, wherein the content of salidroside is 0.01%-1% by weight of the medicine or cosmetic, and the content of glabridin is 0.001%-0.1% by weight of the medicine or cosmetic. In some embodiments, the medicine or cosmetic comprises the composition according to the fourth aspect of the present invention, wherein the content of salidroside is 0.1% by weight of the medicine or cosmetic, and the content of glabridin is 0.01% by weight of the medicine or cosmetic.

[0079] In some embodiments, the medicine or cosmetic further comprises one or more other cosmetic active ingredients, such as but not limited to moisturizers, keratin repair agents, oil control agents, oil repellent agents, hydrating agents, anti-allergic agents, anti-aging and anti-wrinkle agents, etc. DETAILED DESCRIPTION

[0080] The present invention can be implemented through the following embodiments, but the present invention is not limited thereto.

[0081] The instruments and equipment used in the embodiments of the present invention are all conventional instruments and equipment in the field and can be replaced by instruments and equipment that meet the corresponding standards.

[0082] The Chinese herbal medicine raw materials used in the embodiments of the present invention are all commercially available unless otherwise specified, and the reagents used in the embodiments of the present invention are all commercially available analytically pure chemical reagents unless otherwise specified.

[0083] The raw materials and instruments and equipment mentioned in the present invention are all commonly used raw materials and instruments and equipment in the field, which are only examples and are not intended to limit the scope of protection of the present invention. Those skilled in the art can select equivalent raw materials and related instruments and equipment based on the disclosure of the present invention.

[0084] Experimental materials, equipment and statistical analysis methods

[0085] Experimental reagents and equipment: Rhodiola crenulata root (commercially purchased from the root of Rhodiola crenulata (Hook.f. & Thomson) H.Ohba, a plant of the Crassulaceae family, produced in Sichuan at an altitude of more than 4200 m), glabridin (purchased from Gansu Panzhi Pharmaceutical Co., Ltd.), tyrosinase (Sigma, T3824-25KU), L-DOPA (Sigma, D9628), NaH 2 PO 4 (Greagent, G81292A), Na 2 HPO 4(Greagent, G81291A), hydrochloric acid (CP, 36.0-38.0%, Shanghai test), kojic acid (RG, Adamas).

[0086] Bio-Architect -WS, Genofei), cell expansion system (Liangfang, Genofei), cell culture incubator (HERAcell 150i, Thermo), multifunctional microplate reader (Varioskan LUX, ThermoFisher), constant temperature water bath (BK310C, Yamato), fluorescence upright microscope (ECLIPSE Ni-U, Nikon), UV irradiator (UV-621111801, Genofei), colorimeter (CR-10, KONICA MINOLTA), SLR camera (SONY), -Mela melanin model (RB0402, Genefi), melanin model growth medium (BR020203, Genefi), MTT (M5665, Sigma), Masson-Fontana kit (G2032, Solarbio).

[0087] Precision 7920 tower workstation (DELL), GROMACS molecular dynamics simulation software, Ambertools20 software.

[0088] DMEM medium (without phenol red), FBS, double antibody, B16F10 cells, enhanced CCK-8 kit.

[0089] Statistical methods: The experimental results are the mean ± standard deviation of multiple parallel experimental data. Paired t-test was used to test whether the differences between groups were statistically significant. The statistical software was GraphPad 5.0.

[0090] Example

[0091] Example 1. Preparation of Rhodiola rosea root extract

[0092] Add 65%-85% ethanol in a weight of 6-10 times the weight of the raw medicinal material of Rhodiola rosea root, and ultrasonically extract for 30-60 minutes at a temperature of 20-80°C. After filtering, the filtered medicinal material residue is extracted for a second time, and the extraction conditions are consistent with the first time, and the filtrates filtered twice are combined to obtain an alcohol extract. After the alcohol extract is vacuum concentrated to a solid content of 10%-15%, the ethanol is removed to obtain an alcohol extract concentrate. The alcohol extract concentrate is loaded onto a macroporous resin column AB8 or D101, first eluted with 1.5-3 times the column volume of water to remove impurities, and then eluted with 2-5 times the column volume of 70-80% ethanol to obtain an eluent. The eluent is concentrated and dried to prepare a final dried Rhodiola rosea extract.

[0093] Example 2. In vitro tyrosinase inhibition assay

[0094] (1) Solution preparation

[0095] 80 mM phosphate buffer (PB): 1) Add 9.60 g NaH 2 PO 4 Dissolve in 1L deionized water; 2) 11.36g Na 2 HPO 4 Dissolve in 1L of deionized water; 3) Add 2) to 1) until the pH is 6.8.

[0096] 1M HCl: Add 1 mL of concentrated HCl to 11 mL of deionized water.

[0097] 6.0 mM L-DOPA solution: 7.1 mg of L-DOPA was dissolved in 100 μL of 1 M HCl and the solution was made up to 6 mL by adding 80 mM PB.

[0098] 250U / mL tyrosinase: Dissolve 25KU tyrosinase solid in 1mL of 80mM PB and dispense into 40μL tubes. Add PB to 4mL before use.

[0099] Kojic acid solution (positive control): Weigh 4.5 mg of kojic acid and dissolve it in 1 mL of deionized water (32 mM). Dilute it with deionized water to 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 mM for later use.

[0100] (2) Experimental procedures

[0101] Add reagents according to Table 1 below and react, and detect the absorbance at 475 nm using an enzyme reader.

[0102] Table 1

[0103]

[0104] 1) The calculation formula of the tyrosinase inhibition rate of the sample to be tested is as follows:

[0105] Tyrosinase inhibition rate % = [1-(A s -A sb ) / (A c -A cb )]×100%

[0106] 2) IC of the sample to be tested for tyrosinase inhibition 50 The values ​​(50% inhibition concentration) were estimated by GraphPad prism software.

[0107] 3) Synergistic effect index: The synergistic effect index (CI) is used to determine whether the inhibitory effect of the composition containing component a and component b on tyrosinase is synergistic or antagonistic. The synergistic effect index is calculated based on the report in the literature (Siridechakorn, Ittipon et al. "Synergistic impact of arbutin and kaempferol-7-O-α-L-rhamnopyranoside from Nephelium lappaceum L. on whitening efficacy and stability of cosmetic formulations." Scientific reports vol. 13, 122004.12Dec.2023), and the specific calculation formula is as follows:

[0108]

[0109] In this calculation formula, IC50 amix and IC50 bmix IC50 is the mass concentration when the inhibition rate of the combined action of component a and component b is 50%, in μg / mL; a and IC50 b It is the mass concentration of component a and component b at which the inhibition rate is 50%, expressed in μg / mL.

[0110] When CI<1, the combined effect of component a and component b is synergistic, and the smaller the CI value, the more significant the synergistic effect; when CI=1, the combined effect of component a and component b is additive; when CI>1, the combined effect of component 1 and component 2 is antagonistic.

[0111] (3) IC of a composition comprising a Rhodiola rosea root extract and glabridin on tyrosine kinase inhibition 50 and determination of the combined effect index

[0112] The corresponding samples were dissolved in anhydrous ethanol according to the ratios shown in Table 2 to prepare samples of Rhodiola rosea root extract, samples of glabridin and samples of a composition comprising Rhodiola rosea root extract and glabridin. The IC values ​​of each group of samples for tyrosinase inhibition were calculated as described above. 50 The values ​​and CI values ​​are shown in Table 2 below.

[0113] Table 2

[0114]

[0115]

[0116] Note: IC50 amix and IC50 bmix They represent the mass concentrations of Rhodiola rosea root extract and glabridin when the inhibition rate of the combined action is 50%, in μg / mL.

[0117] As shown in Table 2, when the mass ratio of the two components of the Rhodiola rosea root extract and glabridin is 5:3, 5:2, 5:1, 10:1, 15:1 and 20:1, the combined action index CI of the composition containing these two components in the inhibition of tyrosinase is less than 1, showing a synergistic effect. Since the smaller the CI value, the stronger the synergistic effect, and when the mass ratio of the Rhodiola rosea root extract and glabridin is 10:1, the CI value is the smallest, which is 0.018, so 10:1 is the most preferred mass ratio of the Rhodiola rosea root extract and glabridin.

[0118] The data in Table 2 also show that when the mass ratio of the root extract of Rhodiola rosea and glabridin is less than 1:1 of the above-mentioned synergistic ratio range, the CI value is 1.0363, showing a weak antagonistic effect. And when the mass ratio of the root extract of Rhodiola rosea and glabridin is greater than 30:1 of the above-mentioned synergistic ratio range, the CI value is 1.1174, showing an antagonistic effect. These all indicate that the combination of the root extract of Rhodiola rosea and glabridin in any proportion does not have a synergistic effect on the inhibition of tyrosinase. The inventors of the present application have found through research that the combination of Rhodiola rosea extract and glabridin in a specific ratio (i.e., 5:3 to 20:1) has a strong synergistic effect on the inhibition of tyrosinase, which is beyond the expectations of those skilled in the art.

[0119] Example 3. Using 3D Melanin Model -Mela's study on the whitening effect of the composition

[0120] (1) Experimental principles and significance of test indicators

[0121] In vitro 3D melanin model -Mela has a similar tissue structure and function to real human skin. Its function is mainly reflected in the blackening response ability of the 3D melanin model under external factors (such as ultraviolet rays) and the whitening response ability under the action of whitening raw material formula. Therefore, we based on UVB-induced 3D melanin model -Mela, evaluates the whitening effect of the samples from four aspects: apparent chromaticity, apparent brightness L* value, melanin content and melanin particle distribution.

[0122] Apparent chromaticity: The overall color of the melanin model, obtained by taking a photo with a color gradient color circle card. The whiter the overall color, the better the whitening effect of the sample.

[0123] Apparent brightness L* value: The L* value measured by the colorimetric system (Lab colorimetric system) colorimeter is used to characterize brightness. Its change indicates the change in skin black and white. The larger the value, the more white the color is, indicating that the whitening effect of the sample is better.

[0124] Melanin content: The amount of melanin produced by melanocytes in the melanin model. Melanin is the most important determinant of skin color. By measuring the melanin content in the melanin model, the whitening effect of the sample can be reflected. The lower the melanin content, the better the whitening effect of the sample.

[0125] Melanin particle distribution: The Masson-Fontana staining 3D melanin model can be used to observe the distribution of melanin particles in the tissue, reflecting the effect of the sample on melanin production and transport.

[0126] (2) Sample preparation

[0127] Five cosmetic samples A, B, C, D and E were prepared according to the ratios in Table 3 below.

[0128] Table 3

[0129]

[0130]

[0131] The main functional substances and states of these five cosmetic samples are summarized in Table 4:

[0132] Table 4

[0133] Sample No. Main active ingredients state A Rhodiola rosea root extract 0.1% Slightly yellow gel B Salidroside 0.1% Transparent gel C Glabridin 0.01% Transparent gel D Rhodiola rosea root extract 0.1% + glabridin 0.01% Slightly yellow gel E Salidroside 0.1% + Glabridin 0.01% Transparent gel

[0134] (3) Experimental methods

[0135] Grouping and loading

[0136] Will -Mela melanin model was randomly divided into blank control group, negative control group, sample group and positive control group. The positive control group, negative control group and sample group were irradiated with UVB for 6 consecutive days starting from the 5th day of gas-liquid exposure, with an irradiation dose of 50mJ / cm 2 / times. After each irradiation, the culture medium was replaced, and the liquid volume was 1.5mL / well. The blank control group was not irradiated with UVB, and only the culture medium was replaced every day, and the liquid volume was 1.5mL / well.

[0137] The samples were surface-administered at 7 days and 9 days of gas-liquid incubation, twice with a 48-hour interval and a volume of 10 μL. Kojic acid was used as a positive control with a concentration of 0.05% (w / v).

[0138] Whitening efficacy index detection

[0139] 1) Apparent chromaticity: Place the colorimetric card in the studio, fix the SLR camera so that the model is directly below the camera, use tweezers to place a single model in the colorimetric card circle, adjust the camera parameters and take a photo.

[0140] 2) Determination of apparent brightness L* value: Use clean tweezers to place the model on photo paper, ensuring that the stratum corneum faces upwards; after the colorimeter is self-calibrated, press the detection hole of the colorimeter vertically on the surface of the model and take three readings. The average value is taken as the final L* value reading of the single model.

[0141] 3) Melanin content detection: Place the model after the L* value is determined in a 1.5mL centrifuge tube, add 1mL PBS buffer, vortex shaker for 3min, centrifuge at 2000r / min for 10min, and discard the supernatant; add 200μL distilled water, 500μL anhydrous ethanol and 500μL ether to the 1.5mL centrifuge tube in turn, mix well, stand at room temperature for 20min, centrifuge at 3000r / min for 5min, and discard the supernatant; add 500μL 1mol / LNaOH aqueous solution containing 10% DMSO, heat in 80℃ water bath for 40min, pipette 200μL into 96-well plate, and read OD value at 405nm wavelength of microplate reader. Melanin content is positively correlated with OD405, and OD405 is used to characterize the melanin content of the sample.

[0142] 4) Distribution of melanin particles: After the model was cut along the edge with a surgical blade, it was placed in a 1.5 mL centrifuge tube containing 4% paraformaldehyde solution and fixed for 2 hours. After dehydration with ethanol and transparency with xylene, it was embedded in paraffin and sliced ​​(thickness 5-8 μm). After xylene dewaxing and gradient ethanol hydration, it was stained according to the instructions of the Masson-Fontana melanin staining kit. After sealing, it was placed under an upright microscope with a 40x objective lens to take pictures and observe the distribution of melanin particles.

[0143] (4) Statistical analysis of experimental data

[0144]

[0145] All data were expressed as mean ± standard deviation. The groups were compared using t-test. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0146] (5) Experimental conclusion

[0147] 1) Apparent colorimetric test results

[0148] The apparent colorimetric test results are as follows Figure 1 As shown, compared with the negative control group, the apparent chromaticity of sample groups A, B, C, D, and E all became white to varying degrees, among which D (Rhodiola rosea root extract + glabridin) and E (Salidroside + glabridin) had significantly higher whitening degrees than groups A, B, and C.

[0149] 2) Apparent brightness L* value test results

[0150] The apparent brightness L* value test results are shown in Table 5 and Figure 2 The results showed that compared with the negative control group, samples A, B, C, D, and E could significantly increase the model's apparent brightness L* value (P<0.01), with the increase rates being 4.2%±0.1%, 2.6%±0.1%, 4.5%±0.1%, 9.2%±0.3%, and 11.3%±0.1%, respectively. The apparent brightness L* values ​​of groups D and E were significantly higher than those of groups A, B, and C.

[0151] Table 5

[0152]

[0153]

[0154] Note: ## indicates P<0.01 compared with the blank control group; ** indicates P<0.01 compared with the negative control group.

[0155] 3) Melanin content test results

[0156] The results of melanin content detection are shown in Table 6 and Figure 3As shown. The results showed that compared with the negative control group, samples A, B, C, D, and E could significantly inhibit the synthesis of model melanin content (P<0.01). The actual value of the increase rate in group D was significantly higher than the estimated value (A+C), and the actual value of the increase rate in group E was significantly higher than the estimated value. This shows that group D (Rhodiola rosea root extract + glabridin) and group E (salidroside + glabridin) have a very significant synergistic effect in inhibiting melanin synthesis.

[0157] Explanation of the meaning of the estimated value: Estimated melanin content inhibition rate of the combination of Rhodiola rosea extract and glabridin. The estimated value of the synergistic melanin content inhibition rate of the combination of Rhodiola rosea extract and glabridin = melanin content inhibition rate of Rhodiola rosea extract + melanin content inhibition rate of glabridin. The actual value is the melanin content inhibition rate detected in the specific experiment.

[0158] Table 6

[0159]

[0160] Note: ## indicates P<0.01 compared with the blank control group; ** indicates P<0.01 compared with the negative control group.

[0161] 4) Melanin particle distribution test results

[0162] Melanin particle distribution test results Figure 4 The results showed that compared with the negative control group, samples A, B, C, D, and E could significantly reduce the distribution of melanin particles in the model. The distribution of melanin particles in groups D and E was significantly lower than that in groups A, B, and C.

[0163] This whitening efficacy evaluation system is based on the UVB-induced 3D melanin model -Mela, judging from the data of apparent chromaticity, apparent brightness (L* value), melanin content and melanin particle distribution, the actual whitening effect produced by group D (0.1% Rhodiola rosea root extract and 0.01% glabridin) and group E (0.1% salidroside and 0.01% glabridin) was significantly higher than the theoretical whitening effect produced by the combination, and had a very significant synergistic effect.

[0164] Example 4. Molecular dynamics simulation: human tyrosinase

[0165] The main active ingredient of Rhodiola rosea root extract is salidroside. From the composition of the extract's active ingredients and literature research, salidroside is the main compound component of the extract that has a whitening effect. Therefore, salidroside is used as the main object for subsequent synergistic enhancement mechanism research.

[0166] (1) Experimental procedures

[0167] All-atom molecular dynamics simulations of human tyrosinase-salidroside complex and human tyrosinase-glabridin complex were performed using GROMACS2023.1.

[0168] 1) The structures of salidroside and glabridin were optimized at the B3LYP / def2SVP level using Gaussian 16. Subsequently, electrostatic potentials were calculated at the same level. Charge assignment was then performed using Multiwfn 3.8dev according to the RESP2 program.

[0169] 2) The net charge of salidroside and glabridin is 0. The parameters of these ligands were determined using the antechamber module of AMBER22.

[0170] 3) The structure of human tyrosinase used for simulation was predicted by AlphaFold2. AutodockVina 1.2.3 was used to remove salidroside and glabridin and dock them into the human tyrosinase structure predicted by AlphaFold2.

[0171] 4) At pH 7.0, all titratable residues remain in a predominantly protonated state. The protein-ligand complex is solvated in a periodic box of TIP3P water, and the minimum distance between the protein and the box boundary is maintained at The simulation system was configured by introducing sodium and chloride ions into the water box so that the NaCl concentration reached 0.15 M.

[0172] 5) Using the tleap module of AMBER22, parameter topology and coordinate files were constructed. These files were then converted into GROMACS input files. All simulations used the GAFF2 standpoint to simulate the ligand and the ff14SB protein force field to simulate the protein.

[0173] 6) An extensive energy minimization process was performed on GROMACS with an integration time step of 2 fs and 50,000 steps. To maintain the stability of the system, constraints were imposed on the protein and ligand, all of which were The uniform force constant is . The bonds involving hydrogen atoms are constrained using the LINCS algorithm. The electrostatic interactions are calculated using the Particle Mesh Ewald method with a cutoff distance set to The van der Waals interactions were also calculated using a cutoff distance of The system temperature was then raised to 310 K. At this stage, the cycle box was equilibrated for 1 ns in a constant volume constant temperature system (NVT) with a v-scale thermostat. Subsequently, the system pressure was changed to 1 bar and equilibrated for 1 ns in a constant pressure constant temperature system (NPT) using a c-rescale barostat. The human tyrosinase-salidroside complex and the human tyrosinase-glabridin complex were simulated for 100 ns at 310 K and 1 bar, respectively.

[0174] 7) PyMOL 2.5.0 was used for visualization of protein-ligand complexes.

[0175] (2) Experimental results

[0176] Details of the binding of human tyrosinase to salidroside (A) and glabridin (B) Figure 5 As shown. From the results, it can be seen that salidroside binds to the substrate pocket of human tyrosinase and is a competitive inhibitor of tyrosinase. However, glabridin binds to the allosteric regulatory site far away from the catalytic site of human tyrosinase, which may affect the flexible conformation of the core domain and is a non-competitive inhibitor. Since salidroside and glabridin bind to different sites of human tyrosinase and have different modes of action, the two may play a synergistic role.

[0177] Example 5. Detection of melanin content in B16F10 cells

[0178] (1) Experimental procedures

[0179] Add 100 μL of 1×10 4 B16F10 cells / mL were cultured at 37°C and 5% CO 2 Incubate in the incubator for 24 hours. Add 100 μL of α-MSH (final concentration of 1 μM) to the sample group and the control group, and add an equal volume of solvent to the blank group; add 100 μL of the sample to be tested to the sample group, and add an equal volume of sample solvent to the blank group, and place in the incubator for 48 hours and 72 hours. Collect 100 μL of cell supernatant, transfer to a 96-well plate, and detect the absorbance at 405 nm.

[0180] (2) Experimental results

[0181] The inhibition rate of different ratios and concentrations of salidroside and glabridin on the melanin production of B16F10 cells induced by α-MSH is shown in Figure 6and Table 7. It can be seen from the experimental results that when salidroside and glabridin are combined in concentrations of 0.05% w / w + 0.00125% w / w, 0.025% w / w + 0.000125% w / w, 0.05% w / w + 0.0000625% w / w and 0.025% w / w + 0.0000625% w / w, the actual inhibition rate is greater than the estimated inhibition rate obtained by adding the inhibition rates of the two corresponding concentrations of the single components, showing a synergistic effect.

[0182] Table 7

[0183]

[0184] Note: %w / w is a concentration unit indicating mass fraction. For example, 0.05%w / w salidroside means a solution formed by 500μg of salidroside dissolved in 1g of solvent.

Claims

1. A composition comprising or essentially consisting of a Rhodiola rosea extract and glabridin, wherein the mass ratio of the Rhodiola rosea extract to glabridin is 5:3 to 20:

1.

2. The composition according to claim 1, wherein the Rhodiola rosea extract is prepared by ethanol extraction, macroporous resin column purification, concentration and drying.

3. The composition according to claim 1 or 2, wherein the Rhodiola rosea extract is obtained by a method comprising the following steps: (1) extracting the Rhodiola rosea raw material with 65%-85% ethanol by ultrasonic extraction at 20-80° C. for 30-60 minutes, and filtering the Rhodiola rosea raw material residue after the extraction to obtain the Rhodiola rosea alcohol extract; (2) extracting the Rhodiola rosea raw material residue filtered out in step (1) again with ethanol under the same conditions as in step (1), and performing step (2) 0-3 times; (3) if step (2) is performed 0 times, the rhodiola rosea alcohol extract of step (1) is directly concentrated; if step (2) is performed 1-3 times, the rhodiola rosea alcohol extracts of step (1) and step (2) are combined and the combined rhodiola rosea alcohol extract is concentrated; and the concentrated solution is purified by a macroporous adsorption resin, wherein the macroporous adsorption resin is first eluted with water to remove impurities, and then the macroporous adsorption resin is eluted with 70-80% ethanol to obtain an eluate; (4) The eluate obtained in step (3) is concentrated and then dried to obtain a Rhodiola rosea extract.

4. The composition according to claim 3, wherein the amount of ethanol in step (1) is 6-10 times the amount of the Rhodiola rosea raw material; the macroporous adsorption resin in step (3) is a macroporous resin column AB8 or D101; the amount of the water eluent used in step (3) is 1.5-3 times the column volume, and the amount of the alcohol eluent used is 2-5 times the column volume.

5. The composition according to any one of claims 1 to 4, wherein the Rhodiola rosea extract is a Rhodiola rosea root extract, preferably a Rhodiola crenulata (Hook. f. & Thomson) H. Ohba) root extract.

6. A composition comprising or consisting essentially of salidroside and glabridin, wherein the weight ratio of salidroside to glabridin is from 1:6 to 5000:1, preferably from 5:1 to 800:

1.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of medicines / cosmetics with whitening effects.

8. A medicine or cosmetic comprising the composition according to any one of claims 1 to 6, wherein the composition accounts for 0.01% to 20% by weight of the medicine or cosmetic.

9. The drug or cosmetic according to claim 8, further comprising one or more selected from the following ingredients: a thickener, a humectant, an emulsifier, a surfactant, a solubilizer, water and one or more other cosmetic active ingredients.

10. The medicine or cosmetic according to claim 8 or 9, comprising the composition according to any one of claims 1 to 6, polyacrylate crosspolymer-6, 1,3-propylene glycol, 2-methyl-2,4-pentanediol, SOLUBILISANT LRI and water.

11. The drug or cosmetic according to claim 10, comprising 0.01%-20%, preferably 0.11% by weight of a composition according to any one of claims 1 to 6; 0.1-5%, preferably 0.4% by weight of polyacrylate crosspolymer-6; 0.1%-10%, preferably 1% by weight of 1,3-propylene glycol; 0.1%-10%, preferably 1% by weight of 2-methyl-2,4-pentanediol; 0.01%-1%, preferably 0.05% by weight of SOLUBILISANT LRI and 50%-99%, preferably 97.44% by weight of water.

12. The medicine or cosmetic according to claims 8-11, comprising the composition according to any one of claims 1-5, wherein the content of Rhodiola rosea extract accounts for 0.01%-1% by weight, preferably 0.1% by weight of the medicine or cosmetic; the content of glabridin accounts for 0.001%-0.1% by weight, preferably 0.01% by weight of the medicine or cosmetic.

13. The medicine or cosmetic according to claims 8-11, comprising the composition according to claim 6, wherein the content of Rhodiola rosea extract is 0.01%-1% by weight, preferably 0.1% by weight, of the medicine or cosmetic; and the content of glabridin is 0.001%-0.1% by weight, preferably 0.01% by weight.

Images