Whitening composition and application thereof

Abstract

The invention relates to the technical field of cosmetics, and discloses a whitening composition and application thereof, and the whitening composition comprises 3, 7-dihydroxy-2, 4-dimethoxyphenanthrene, 2, 6-dihydroxy-2, 4-dimethoxyphenanthrene, 2, 6-dihydroxy-2, 4-dimethoxyphenanthrene, 2, 3, 7-dihydroxy-2, 4-dimethoxyphenanthrene has a remarkable melanin inhibition activity effect and can inhibit the melanin content and tyrosinase activity, meanwhile, 3, 7-dihydroxy-2, 4-dimethoxyphenanthrene can reduce the active oxygen level of UV-A irradiated cells, and the whitening composition has a remarkable whitening effect, has a skin care effect and can be used for whitening skin. The effects of resisting oxidation, resisting wrinkles, repairing and the like are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care products / cosmetics, and particularly relates to a whitening composition and its application. Background Art

[0002] Melanin is a natural pigment mainly present in hair, skin and eyes, and is produced by melanocytes in the basal layer of the epidermis. Under physiological conditions, human skin can avoid skin damage caused by ultraviolet (UV) radiation through the synthesis and deposition of melanin. However, the excessive production, unnecessary accumulation and abnormal distribution of melanin can all lead to a wide range of pigmentary dermatoses, including melasma, vitiligo, and post-inflammatory hyperpigmentation, etc. More seriously, the excessive production of melanin can also lead to melanoma skin cancer. It is reported that more than 60% of melanoma cases are related to increased ultraviolet (UV) exposure. Excessive reactive oxygen species (ROS) generated by UVB radiation can disrupt the normal redox balance, thereby causing oxidative damage to cells and triggering melanocytes to synthesize melanin. Therefore, when developing effective skin protection products against ultraviolet radiation, in addition to considering inhibiting melanin production, antioxidant properties should also be taken into account.

[0003] Currently, more and more natural active substances have been used in the research of ultraviolet protection. Plant extracts, as natural ultraviolet filters for sunscreen, have attracted much attention due to their low skin irritation, photochemical stability, safety and reliability. For example, Ganoderma lucidum polysaccharide is a non-toxic natural antioxidant that inhibits UVB-induced melanin production by antagonizing mitochondrial damage and ROS generation. In addition, Ecklonia maxima extract containing Dieckol and Eckmaxol has strong anti-melanogenic and photoprotective activities and is an ideal raw material for the pharmaceutical and cosmeceutical industries. α-Arbutin is a bioactive substance extracted from arbutin plants and is widely used clinically as a skin depigmenting agent due to its excellent anti-melanin and antioxidant properties.

[0004] In addition, it has been found through research that the production of melanin is related to melanin tyrosinase (TYR), which is a membrane-bound enzyme located on cellular melanosomes and is associated with the albino phenotype of catalyzing L-tyrosine amino mice. Tyrosinase can convert L-tyrosine into L-dopa and then convert L-dopa into L-dopaquinone, and is considered to be the main enzyme for producing melanin. The tyrosinase family proteins consist of tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) or dopamine auto-variant enzyme (Dct). These three enzymes play important roles in controlling melanin production. The up-regulation of the expression level of tyrosinase family proteins and the activation of enzyme functions both significantly increase the production of melanin in melanocytes. Therefore, as the key enzyme accelerating the first and rate-limiting steps of melanin biosynthesis, inhibiting its activity is considered a potential mode for treating pigmentation.

[0005] Under the existing circumstances, some active ingredients in plants have been used as anti-pigmentation agents in herbal cosmetics / skin care products, such as arbutin, ascorbic acid, genistein, ellagic acid, and quercetin. However, due to many adverse reactions, none of them is completely satisfactory. Continuous therapeutic effects and good safety are the key requirements for melanin inhibitors. Therefore, there is an urgent need to find chemicals that have good control over melanin production and low side effects. Summary of the Invention

[0006] The purpose of the present invention is to provide a whitening composition and its application.

[0007] The solution of the present invention is as follows:

[0008] A whitening composition contains 3,7-dihydroxy-2,4-dimethoxyphenanthrene (DDP), and its chemical structural formula is shown as follows

[0009]

[0010] As a preferred technical solution, it is mixed with the following components in an aqueous solution of acetylated ovalbumin as a solvent:

[0011] 3,7-dihydroxy-2,4-dimethoxyphenanthrene 0.1 - 20 mg / ml.

[0012] As a preferred technical solution, it is mixed with the following components in an aqueous solution of acetylated ovalbumin as a solvent:

[0013] 3,7-dihydroxy-2,4-dimethoxyphenanthrene 20 mg / ml;

[0014] Skin care whitening complex 3 - 10 mg / ml;

[0015] The additive is 0.1 - 1 mg / ml.

[0016] As a preferred technical solution, the skin - care and whitening complex includes modified pearl powder, tea polyphenols, centella asiatica extract and angelica dahurica powder, and their mass ratio is 1 - 1.5:0.1 - 0.2:0.05 - 0.1:1.

[0017] As a preferred technical solution, the additive includes silk peptide, trehalose and shea butter, and their mass ratio is 1:2 - 3:4 - 5. The molecular weight of the silk peptide is 500 - 1000 Daltons.

[0018] The present invention also discloses a preparation method of a whitening composition, which includes the following steps:

[0019] 1) Extraction: The whole plant of dendrobium wardianum is crushed after cleaning and drying, and then extracted 3 times with 95% ethanol at room temperature. The obtained solution is subjected to reduced - pressure evaporation. The concentrated extract is suspended in a liquid and fractionated with ethyl acetate. The ethyl acetate layer is evaporated to dryness to obtain an extraction material. The extraction material is subjected to gradient elution with a first eluent, and silica gel column chromatography is used to obtain fractions A, B, C, D and E. Fraction A is subjected to silica gel column chromatography and eluted step - by - step with a second eluent to obtain sub - fractions A1, A2 and A3. Sub - fraction A3 is subjected to silica gel column chromatography and eluted step - by - step with a third eluent, and repeatedly separated with glucosyl resin and methanol to obtain 3,7 - dihydroxy - 2,4 - dimethoxyphenanthrene;

[0020] 2) Gel preparation: Under magnetic stirring, ovalbumin (OVA) is dissolved in deionized water for 2 h, and the pH value of the dispersion solution is adjusted to 8.0. Then succinic anhydride is added, and an acylation reaction is carried out for 1 h while maintaining the pH value at 8.0 during the acylation reaction. After the acylation reaction is completed, dialysis is carried out with deionized water at 2 - 6 °C for 40 - 50 h, and acetylated ovalbumin gel is obtained after freeze - drying;

[0021] 3) Preparation: Take the acetylated ovalbumin gel obtained in step 2), add phosphate - buffered saline (PBS) while stirring until it reaches 60 - 100 mg / ml, keep it at 2 - 6 °C overnight, and then heat it at 80 - 95 °C for 8 - 12 minutes to obtain an aqueous solution of acetylated ovalbumin. Add the 3,7 - dihydroxy - 2,4 - dimethoxyphenanthrene to the aqueous solution of acetylated ovalbumin to make the final concentration of 3,7 - dihydroxy - 2,4 - dimethoxyphenanthrene 20 mg / ml, stir overnight, and obtain the whitening composition.

[0022] As a preferred technical solution, NaOH is used to adjust the pH.

[0023] As a preferred technical solution, in the step (3), an aqueous solution of acetylated ovalbumin is added with 3,7-dihydroxy-2,4-dimethoxyphenanthrene, a skin care and whitening complex and an additive.

[0024] As a preferred technical solution, in the step (1), each liter of 95% ethanol contains 0.1 - 0.2 kg of the whole powder of Dendrobium wardianum.

[0025] Extract with 95% ethanol at room temperature for 3 times, once every 5 days.

[0026] The first eluent is one or both of petroleum ether and ethyl acetate; the second eluent is a mixture of petroleum ether and acetone with a volume ratio of 1 - 9:1; the third eluent is a mixture of CHCl 3 and ethyl acetate with a volume ratio of 15 - 50:1.

[0027] As a preferred technical solution, the skin care and whitening composition includes modified pearl powder, tea polyphenols, Centella asiatica extract and Angelica dahurica powder; the preparation method of the modified pearl powder is as follows:

[0028] Grind the pearl powder into powder with a particle size of 0.1 - 2.0 microns, then mix it with deionized water, stir at 120 - 150 r / min, and at the same time, perform ultrasonic treatment with an ultrasonic power of 1.5 - 2.0 kw and an ultrasonic time of 60 - 90 min to obtain a pearl powder aqueous solution with a mass percentage of 10 - 15 wt%. Add 2 - 3% of monoalkyl phosphate based on the dry weight of the pearl powder to the slurry, stir magnetically for 10 - 20 min, dropwise add glycerol and lauroyl lysine respectively, complete the dropping within 20 - 30 min, heat and control the temperature to 60 - 70 °C, control the temperature for 60 - 100 min, then age for 2 - 3 h, and perform washing, drying and pulverization to obtain the modified pearl powder; the mass ratio of monoalkyl phosphate, glycerol and lauroyl lysine is 1:0.5 - 2:0.1 - 1.

[0029] The present invention also discloses the application of a whitening composition in skin care and cosmetics.

[0030] The present invention discloses a whitening composition and its application, which contains 3,7-dihydroxy-2,4-dimethoxyphenanthrene.

[0031] Advantages of the present invention:

[0032] In the invention, 3,7-dihydroxy-2,4-dimethoxyphenanthrene has a significant effect of inhibiting melanin, and can inhibit the melanin content and tyrosinase activity. At the same time, 3,7-dihydroxy-2,4-dimethoxyphenanthrene can reduce the level of reactive oxygen species (ROS) in cells irradiated by UV-A.

[0033] The whitening composition prepared by the present invention has good biocompatibility. Experimental results show that the whitening composition can effectively reduce the accumulation of skin melanin caused by UVB irradiation, and has no side effects such as inflammation. It absorbs ultraviolet rays, inhibits inflammatory factors, repairs damaged DNA, scavenges free radicals, inhibits tyrosinase, and inhibits melanin accumulation, thereby achieving the purpose of whitening. At the same time, it can also act synergistically in other aspects to enhance the whitening and anti-wrinkle effects, achieving both prominent whitening effects and balanced cooperation without causing negative feedback phenomena. It provides scientific support for the development of 3,7-dihydroxy-2,4-dimethoxyphenanthrene (DDP) as a clinical drug or cosmetic ingredient, and lays a theoretical foundation for the scientific research of products with both food and drug functions.

[0034] While the inventive composition has a significant whitening effect, it also has good skin care effects such as antioxidant, anti-wrinkle and repair effects through synergistic cooperation. Brief Description of the Drawings

[0035] Figure 1 It is a diagram of the chemical structure of DDP in Example 1 of the present invention and the cytotoxicity determination of B16-F10 cells for 72 hours; where A is the chemical structure of DDP; B is the dose-response curve; C is the morphological observation under a bright-field microscope; the results are expressed as the average of three independent experiments (SEM±SD);

[0036] Figure 2 It is a diagram for detecting reactive oxygen species (ROS) in B16-F10 cells using the H2DCFDA fluorescent probe in the present invention; where A shows that B16-F10 cells exhibit green fluorescence after 30 minutes of UV-A irradiation; B shows that the fluorescence inverted microscope can quantitatively measure the ROS level in cells; the cell fluorescence intensity is corrected using ImageJ, and the average fluorescence intensity is calculated. The results are expressed as the average of three independent experiments (SEM±SD), *p<0.05; **p<0.01; ***p<0.005; ****p<0.001 compared with untreated cells (Ctrl);

[0037] Figure 3This is a graph showing the effect of DDP on melanogenesis in B16-F10 cells; among them, in A and B, B16-F10 cells were treated with α-MSH and DDP (5 - 20 μg / ml) or the positive control kojic acid for 48 h, and the cell culture supernatant and cell sediment were collected respectively; in C and D, the melanin content in B16-F10 cells was measured and analyzed; in E and F, DDP (20 μg / ml) and α-MSH were continuously cultured for 24 - 72 h, and the cell culture supernatant and cell sediment were collected at different time periods for comparison; in G and H, the corresponding cell melanin content was measured and analyzed; the melanin content was normalized to the total amount of cell protein and expressed as a value relative to untreated control cells; the results are expressed as the mean of three independent experiments (SEM ± SD); *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001 compared with untreated cells (Ctrl);

[0038] Figure 4 This is a graph showing the effect of DDP on tyrosinase activity and the expression level of melanin formation-related proteins in B16-F10 cells. Among them, A shows the effect of DDP on tyrosinase activity in B16-F10 cells, using a cell-free tyrosinase activity assay; in B, C, D, and E, Western blotting was used to detect the protein levels of TYR, MITF, TRP-1, and TRP-2 in B16-F10 cells after treatment with DDP for 72 h, and the results were the mean of three independent experiments; the results are expressed as the mean of three independent experiments (SEM ± SD). *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001 compared with untreated cells (Ctrl);

[0039] Figure 5 This is a molecular docking analysis graph of DDP with melanin formation-related proteins TYR and MC1R of the present invention. Among them, A is TYR-DDP; B is MC1R-DDP; C is tyr-kojic acid; D is mc1r-kojic acid; E is the binding affinity result of DDP compared with the standard tyrosinase inhibitor kojic acid;

[0040] Figure 6 This is a graph showing the characterization and biocompatibility of Example 2 of the present invention in the test;

[0041] Figure 7 This is a graph showing the effect evaluation of Example 2 of the present invention in the test. Detailed implementation mode

[0042] In order to make the technical means, creative features, achieved purposes, and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0043] Unless otherwise specified, the raw materials and reagents used in the following examples are all chemically pure, commercially available products, without specifying the manufacturer. Or they can be prepared by known methods.

[0044] Example 1:

[0045] The whole dried Dendrobium wardianum (6.3 kg) was pulverized and extracted three times (5 days each time) with 95% ethanol (40 L) at room temperature. After the combined solvent was evaporated under reduced pressure, the concentrated extract (361 g) was suspended in water and fractionated with ethyl acetate. The ethyl acetate layer was evaporated to dryness, and the resulting residue (204 g) was eluted with a gradient of petroleum ether / ethyl acetate (1:1, v / v) by silica gel column chromatography (CC) to obtain 5 fractions (A - E).

[0046] Using silica gel column chromatography, stepwise gradient elution with petroleum ether / acetone (9:1, v / v) was carried out to obtain 3 sub - fractions (A1 - A3). Silica gel column chromatography was performed on sub - fraction A3, and gradient elution was carried out step by step with CHCl 3 / ethyl acetate (50:1, v / v). Repeated separation with Sephadex LH - 20 and MeOH gave the compound 3,7 - dihydroxy - 2,4 - dimethoxyphenanthrene (3,7 - dihydroxy - 2,4 - dimethoxyphenanthrene; hereinafter referred to as DDP).

[0047] Example 2:

[0048] Ovalbumin (purity > 90%, molecular weight 45 kDa) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China); succinic anhydride (Sigma - Aldrich Corporation, Shanghai, China)

[0049] At room temperature, under magnetic stirring (the rotational speed of the magnetic stirrer used for magnetic stirring was 200 revolutions per minute), ovalbumin (1%, w / v) was dissolved in deionized water for 2 hours, and then 2 mol / L NaOH was added to adjust the pH value of the dispersion solution to 8.0. Succinic anhydride (20% w / w) was added to the subsequent ovalbumin (OVA) solution, and the acylation reaction was carried out for 1 hour. 2 mol / L NaOH was added during the acylation reaction to maintain the pH value at 8.0. Then the solution was dialyzed against deionized water at 4°C for 48 hours to remove impurities and excessive reagents, and then freeze - dried for the preparation of protein gel (AOVA). AOVA was stirred with PBS (PH7.0) to reach 80 mg / ml protein, incubated at 4°C overnight, then heated at 90°C for 10 minutes, and finally DDP was added to make the final DDP concentration 20 mg / ml, and stirred overnight to form an acetylated ovalbumin hydrogel loaded with DDP (AOVA / DDP), that is, the whitening composition.

[0050] Example 3:

[0051] The present invention also discloses a preparation method of a whitening composition, comprising the following steps:

[0052] 1) Extraction: The whole Dendrobium wardianum was crushed after cleaning and drying, and then extracted 3 times with 95% ethanol at room temperature. The obtained solution was subjected to reduced pressure evaporation, and the concentrated extract was suspended in a liquid and fractionated with ethyl acetate; the ethyl acetate layer was evaporated to dryness to obtain an extraction material. The extraction material was subjected to gradient elution with a first eluent, and fractions A, B, C, D, and E were obtained by silica gel column chromatography. Fraction A was subjected to silica gel column chromatography and eluted stepwise with a second eluent to obtain sub-fractions A1, A2, and A3; sub-fraction A3 was subjected to silica gel column chromatography and eluted stepwise with a third eluent, and 3,7-dihydroxy-2,4-dimethoxyphenanthrene was obtained by repeatedly separating with glucosyl resin and methanol.

[0053] 2) Gel preparation: Ovalbumin (OVA) was dissolved in deionized water under magnetic stirring for 2 h, and the pH value of the dispersion solution was adjusted to 8.0. Then succinic anhydride was added, and an acylation reaction was carried out for 1 h, and the pH value was maintained at 8.0 during the acylation reaction. After the acylation reaction was completed, dialysis was carried out with deionized water at 2°C for 40 h, and acetylated ovalbumin gel was obtained after freeze-drying.

[0054] 3) Preparation: The acetylated ovalbumin gel obtained in step 2) was added with phosphate buffered saline (PBS) while stirring until reaching 60 mg / ml, and left overnight at 2°C. Then it was heated at 80°C for 8 - 12 minutes to obtain an aqueous solution of acetylated ovalbumin. The 3,7-dihydroxy-2,4-dimethoxyphenanthrene, skin care whitening complex, and additive were added to the aqueous solution of acetylated ovalbumin so that the final concentration of 3,7-dihydroxy-2,4-dimethoxyphenanthrene was 20 mg / ml, the concentration of the skin care whitening complex was 3 mg / ml, and the concentration of the additive was 0.1 mg / ml. Stir and leave overnight to obtain the whitening composition.

[0055] NaOH was used to adjust the pH.

[0056] In step 1), each liter of 95% ethanol contained 0.1 kg of the whole powder of Dendrobium wardianum.

[0057] It was extracted 3 times with 95% ethanol at room temperature, and extracted once every 5 days.

[0058] The first eluent was petroleum ether; the second eluent was a mixture of petroleum ether and acetone with a volume ratio of 1:1; the third eluent was a mixture of CHCl 3 and ethyl acetate with a volume ratio of 15:1.

[0059] The skin care and whitening composition includes modified pearl powder, tea polyphenols, centella asiatica extract and angelica dahurica powder, and their mass ratio is 1:0.1:0.05~1.

[0060] The additive includes silk peptides, trehalose and shea butter, and their mass ratio is 1:2:4. The molecular weight of the silk peptides is 500 Daltons.

[0061] The preparation method of the modified pearl powder is as follows:

[0062] Grind the pearl powder into powder with a particle size of 0.1~2.0 microns, then mix it with deionized water, stir at 120 r / min, and at the same time, perform ultrasonic treatment with an ultrasonic power of 1.5 kw and an ultrasonic time of 60 min to obtain a pearl powder aqueous solution with a mass percentage of 10 wt%. Add monoalkyl phosphate accounting for 2% of the dry weight of the pearl powder to the slurry, stir magnetically for 10 min, dropwise add glycerol and lauroyl lysine respectively, complete the dropping within 20 min, heat and control the temperature to 60 °C, control the temperature for 60 min, then age for 2 h, and perform washing, drying and pulverization to obtain modified pearl powder; the mass ratio of monoalkyl phosphate, glycerol and lauroyl lysine is 1:0.5:0.1.

[0063] Example 4:

[0064] The present invention also discloses a preparation method of a whitening composition, including the following steps:

[0065] 1) Extraction: Crush the whole part of dendrobium wardianum after cleaning and drying, then extract it 3 times with 95% ethanol at room temperature. The obtained solution is subjected to reduced pressure evaporation, and the concentrated extract is suspended in a liquid and fractionated with ethyl acetate; the ethyl acetate layer is evaporated to dryness to obtain an extract material, and the extract material is subjected to gradient elution with a first eluent, and A fraction, B fraction, C fraction, D fraction and E fraction are obtained by silica gel column chromatography. The A fraction is subjected to silica gel column chromatography and eluted step by step with a second eluent to obtain A1 sub-fraction, A2 sub-fraction and A3 sub-fraction; the A3 sub-fraction is subjected to silica gel column chromatography and eluted step by step with a third eluent, and 3,7-dihydroxy-2,4-dimethoxyphenanthrene is obtained by repeatedly separating with glucosyl resin and methanol.

[0066] 2) Gel preparation: Dissolve ovalbumin (OVA) in deionized water under magnetic stirring for 2 h, adjust the pH value of the dispersion solution to 8.0, then add succinic anhydride, and perform acylation reaction for 1 h. During the acylation reaction, keep the pH value at 8.0. After the acylation reaction is completed, dialyze with deionized water at 6 °C for 50 h, and obtain acetylated ovalbumin gel after freeze-drying.

[0067] 3) Preparation: Take the acetylated ovalbumin gel from step 2), add phosphate buffered saline (PBS) while stirring until it reaches 100 mg / ml, incubate overnight at 6°C, and then heat at 95°C for 12 minutes to obtain an aqueous solution of acetylated ovalbumin. Add the 3,7-dihydroxy-2,4-dimethoxyphenanthrene, skin whitening complex, and additive to the aqueous solution of acetylated ovalbumin to make the final concentration of 3,7-dihydroxy-2,4-dimethoxyphenanthrene 20 mg / ml, the concentration of the skin whitening complex 10 mg / ml, and the concentration of the additive 1 mg / ml. Stir and incubate overnight to obtain a whitening composition.

[0068] The skin whitening complex includes modified pearl powder, tea polyphenols, centella asiatica extract, and angelica dahurica powder, and their mass ratio is 1.5:0.2:0.1:1.

[0069] The additive includes silk peptide, trehalose, and shea butter, and their mass ratio is 1:3:5. The molecular weight of the silk peptide is 1000 daltons.

[0070] NaOH is used to adjust the pH.

[0071] In step 1), each liter of 95% ethanol contains 0.2 kg of the whole powder of dendrobium wardianum.

[0072] Extract with 95% ethanol at room temperature 3 times, once every 5 days.

[0073] The first eluent is ethyl acetate; the second eluent is a mixture of petroleum ether and acetone with a volume ratio of 9:1; the third eluent is a mixture of CHCl 3 And ethyl acetate with a volume ratio of 50:1.

[0074] The preparation method of the modified pearl powder is as follows:

[0075] Grind the pearl powder into powder with a particle size of 0.1 - 2.0 microns, then mix it with deionized water, stir at 150 r / min, and simultaneously perform ultrasonic treatment. The ultrasonic power is 2.0 kw, and the ultrasonic time is 90 min to obtain an aqueous solution of pearl powder with a mass percentage of 15 wt%. Add 3% of the dry weight of the pearl powder of monoalkyl phosphate to the slurry, stir magnetically for 20 min, dropwise add glycerol and lauroyl lysine respectively, complete the dropping in 30 min, heat and control the temperature to 70°C, control the temperature for 100 min, then age for 3 h, and perform washing, drying, and pulverization to obtain modified pearl powder; the mass ratio of monoalkyl phosphate, glycerol, and lauroyl lysine is 1:2:1.

[0076] Example 5:

[0077] The present invention also discloses a preparation method of a whitening composition, including the following steps:

[0078] 1) Extraction: The whole Dendrobium wardianum after cleaning and drying is pulverized, and then extracted 3 times with ethanol at a concentration of 95% at room temperature. The obtained solution is subjected to reduced pressure evaporation, and the concentrated extract is suspended in a liquid and fractionated with ethyl acetate. The ethyl acetate layer is evaporated to dryness to obtain an extraction material, and the extraction material is subjected to gradient elution with a first eluent, and silica gel column chromatography is used to obtain fraction A, fraction B, fraction C, fraction D and fraction E. Fraction A is subjected to silica gel column chromatography and eluted stepwise with a second eluent to obtain fraction A1, fraction A2 and fraction A3. Fraction A3 is subjected to silica gel column chromatography and eluted stepwise with a third eluent, and repeatedly separated with glucosyl resin and methanol to obtain 3,7-dihydroxy-2,4-dimethoxyphenanthrene;

[0079] 2) Gel preparation: Under magnetic stirring, ovalbumin (OVA) is dissolved in deionized water for 2 h, the pH value of the dispersion solution is adjusted to 8.0, and then succinic anhydride is added for acylation reaction for 1 h. During the acylation reaction, the pH value is maintained at 8.0. After the acylation reaction is completed, dialysis is carried out with deionized water at 4 °C for 48 h, and acetylated ovalbumin gel is obtained after freeze-drying;

[0080] 3) Preparation: Take the acetylated ovalbumin gel obtained in step 2), add phosphate buffered saline (PBS) while stirring until reaching 80 mg / ml, keep it at 4 °C overnight, and then heat it at 90 °C for 10 minutes to obtain an aqueous solution of acetylated ovalbumin. The 3,7-dihydroxy-2,4-dimethoxyphenanthrene, skin care and whitening complex and additive are added to the aqueous solution of acetylated ovalbumin so that the final concentration of 3,7-dihydroxy-2,4-dimethoxyphenanthrene is 20 mg / ml, the concentration of the skin care and whitening complex is 8 mg / ml, and the concentration of the additive is 0.5 mg / ml, stir overnight to obtain a whitening composition.

[0081] The skin care and whitening complex includes modified pearl powder, tea polyphenols, Centella asiatica extract and Angelica dahurica powder, and their mass ratio is 1.5:0.1:0.05:1.

[0082] The additive includes silk peptide, trehalose and shea butter, and their mass ratio is 1:2:5. The molecular weight of the silk peptide is 500 daltons.

[0083] NaOH is used to adjust the pH.

[0084] In step 1), the whole Dendrobium wardianum is 6.3 kg, and the ethanol at a concentration of 95% is 40 L;

[0085] It is extracted 3 times with ethanol at a concentration of 95% at room temperature, and extracted once every 5 days;

[0086] The first eluent is petroleum ether and ethyl acetate, and their volume ratio is 1:1; the second eluent is a mixture of petroleum ether and acetone, and their volume ratio is 4:1; the third eluent is a mixture of CHCl 3 and ethyl acetate, and their volume ratio is 30:1.

[0087] The preparation method of the modified pearl powder is as follows:

[0088] Grind the pearl powder into a powder with a particle size of 0.1 - 2.0 microns, then mix it with deionized water, stir at 140 r / min, and at the same time, perform ultrasonic treatment with an ultrasonic power of 2.0 kw and an ultrasonic time of 80 min to obtain an aqueous pearl powder solution with a mass percentage of 12 wt%. Add monoalkyl phosphate accounting for 3% of the dry weight of the pearl powder to the slurry, stir magnetically for 15 min, dropwise add glycerol and lauroyl lysine respectively, complete the dropping within 30 min, heat and control the temperature to 60 °C, control the temperature for 80 min, then age for 3 h, and perform washing, drying and pulverization to obtain the modified pearl powder; the mass ratio of monoalkyl phosphate, glycerol and lauroyl lysine is 1:1:0.5.

[0089] I. Take 3,7-dihydroxy-2,4-dimethoxyphenanthrene (DDP) prepared in Example 1 for testing

[0090] Based on the B16-F10 melanoma cell model, detect the effects of DDP on the intracellular melanin content, extracellular tyrosinase activity and intracellular antioxidant activity of the cells. At the same time, perform molecular docking of DDP with tyrosinase (TYR) and Melanocortin1 Receptor (MC1R) to evaluate the melanin inhibition potential of DDP.

[0091] The test shows that DDP can effectively reduce the production of melanin and the activity of extracellular tyrosinase in a concentration-dependent manner within the safe concentration range, and its effect is equivalent to that of the standard tyrosinase inhibitor Kojicacid. At the same time, DDP can also effectively reduce the level of intracellular reactive oxygen species (ROS) induced by UV-A. Further findings show that these effects of DDP may be related to the reduction of the levels of intracellular melanin production-related proteins (TYR, TRP-1, TRP-2 and MITF).

[0092] On the other hand, the binding of DDP to melanogenesis-related receptors was compared, with Kojic acid as a positive control. It was found that compound DDP had the highest binding activities to TYR (-6.28 kcal / mol) and MC1R (-6.74 kcal / mol), which were significantly higher than those of the positive control kojic acid, with binding affinities for the target proteins TYR and MC1R of -4.94 and -5.66 kcal / mol, respectively. This indicated that DDP binds to TYR and MC1R more stably and strongly than kojic acid. In conclusion, DDP is an efficient and safe melanin synthesis inhibitor with the potential to become a highly effective skin whitening agent of plant origin.

[0093] Materials and Methods

[0094] 1) Chemicals and Reagents

[0095] Koji cacid and α-MSH were purchased from MCE (China). 3,4-Dihydroxyphenylalanine (L-DOPA), tyrosine, WST-8 (CCK8), and dimethyl sulfoxide (DMSO) were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Organic solvents were purchased from Aladdin Biotechnology Co., Ltd. in HPLC grade. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin, penicillin, and streptomycin were purchased from Thermo Scientific Co. (Shanghai, China). The primary and secondary antibodies were both purchased from proteintech (China).

[0096] 2) Cell Culture

[0097] B16-F10 melanoma cells were obtained from the stem cell bank (Chinese Academy of Sciences, Shanghai, China) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NV, USA) and antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin). They were grown in a humidified atmosphere containing 5% CO 2 until 70 - 80% confluent and then used for further experiments.

[0098] 3) CCK8 Cytotoxicity Assay

[0099] WST-8 is a compound similar to MTT that can be reduced by some dehydrogenases in mitochondria to produce orange-yellow formazan in the presence of an electron coupling reagent. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. The CCK8 cytotoxicity assay was used to evaluate the cytotoxic activity of DDP using a WST-8-based assay. B16-F10 cells were seeded into 96-well plates at a density of 0.8×10 4 cells / well.

[0100] After 24 hours, the cell culture medium was replaced with a treatment medium containing different concentrations of DDP (1–80 μg / mL). Then the cells were cultured for another 72 hours in a humidified CO 2 incubator. After the treatment, the medium was replaced with CCK8 solution and incubated at 37 °C for 1 hour. Finally, the absorbance was measured at 450 nm using a UV-VIS spectrophotometer. The dose-response curve and IC50 value were generated using GraphPad software version 10.

[0101] 4) Determination of cellular antioxidant activity

[0102] The cellular antioxidant activity of DDP was determined by labeling the cellular ROS level with the fluorescent probe H2-DCFDA. B16-F10 melanoma cells were seeded at 4×10 4 cells / well in a 24-well plate. The cells were maintained at 37 °C and 5% CO 2 for 24 hours to adhere. Then the cells were pretreated with increasing concentrations of DDP for 2 hours before UV-A irradiation. UV-A radiation (8 J / cm 2 ) for 30 minutes could induce an increase in cellular ROS. After UV-A irradiation, the cells were washed with serum-free medium and analyzed with 20 μM dichlorofluorescein diacetate (DCF-DA) at 37 °C for 30 minutes. Intracellular reactive oxygen species could oxidize non-fluorescent DCFH to generate green-fluorescent DCF. Therefore, the cellular ROS level could be quantified using a fluorescence inverted microscope. The fluorescence intensity of the cells was corrected using ImageJ and the mean fluorescence intensity was calculated.

[0103] 5) Determination of melanin content

[0104] B16-F10 melanoma cells were seeded in a 6-well plate at a density of 6×10 4 cells / well. After 24 hours, α-melanocyte-stimulating hormone (α-MSH; 100 nM) was injected into the cells, and then kojic acid or DDP was added. The cells were cultured in a CO 2 incubator at 37 °C for 72 hours. The cells were washed with PBS and the melanin-containing granules were collected by centrifugation at 12,000 rpm for 10 minutes at 4 °C using RIPA buffer containing 1% protease inhibitor and 1% phosphatase inhibitor, and further dissolved at 80 °C in 200 μL of 1 mol / L NaOH / 10% DMSO for 3 hours. The absorbance value of the obtained melanin was measured at 490 nm. The melanin content was normalized to the total amount of cellular protein and expressed as a relative value compared to untreated control cells.

[0105] 6) Measurement of cellular tyrosinase activity

[0106] The direct inhibition of tyrosinase activity was evaluated. B16-F10 cells were cultured until 70 - 80% confluent, trypsinized and collected, and centrifuged at 5000 rpm for 5 minutes (4 °C). The cell pellet was lysed in 1% Triton X-100 in PBS containing 1% protease inhibitor and 1% phosphatase inhibitor at 4 °C with continuous rotation for 1 hour at 10-minute intervals. Then, the cell lysate was centrifuged at 12000 rpm for 15 minutes (4 °C), and the supernatant was collected. DDP at final concentrations of 0, 5, 10, and 20 μg / ml was added to PBS (pH 6.8), and an equal volume of 100 μg protein supernatant was added simultaneously. The reaction mixture was incubated at 37 °C for 10 min, 2 mM L-DOPA was added, and the mixture was incubated at 37 °C for 2 h. The absorbance of dopamine formed was measured at 490 nm using a microplate reader. The direct effect of DDP on tyrosinase activity was determined by the following formula:

[0107]

[0108] 7) Western blot analysis

[0109] B16-F10 cells (1×105 cells / well) in 6-well plates were cultured in complete DMEM with or without different concentrations of DDP or α-MSH for 72 h or 24 - 72 h. Cells were harvested and lysed on ice for 45 minutes in RIPA buffer containing 1% protease inhibitor and 1% phosphatase inhibitor. Then, equal protein samples determined by the BCA assay kit were mixed with loading buffer, heated at 95 °C for 5 minutes, and subjected to 6% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Proteins separated in the polyacrylamide gel were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% non-fat milk in TBST (25 mmol / L Tris-HCl; pH 7.4, 125 mmol / L NaCl, 0.1% Tween 20) for 2 hours at room temperature. The membrane was incubated with specific primary antibody overnight at 4 °C. Subsequently, the PVDF membrane was incubated with the corresponding secondary antibody for 2 hours at room temperature. Signals were detected using a chemiluminescence imaging system. The intensity of protein signals was quantified using ImageJ and plotted using GraphPad software version 10.

[0110] 8) Molecular docking

[0111] DDP was molecularly docked with the melanogenesis-related proteins tyrosinase (TYR, PDBID: 2Y9X) and melanin-concentrating hormone receptor 1 (MC1R, PDBID: 7F41). In addition, DDP and kojic acid were prepared as ligands in 2D form in ChemDraw Ultra 12.0 and further converted to 3D form in Chem3D Pro 17.0. The protein receptors were prepared using OpenBabel-3.1.1. The grid box was determined by redocking with AutoDockTools-1.5.6. The grid box of the TYR receptor was located at the center x,y,z (22.548, 2.483, -93.071) with a size of The grid box of the MC1R receptor was located at the center position of x,y,z (92.688, 80.386, 111.935) with a size of Molecular docking of all ligands was performed using AutoDock 4.0. The interactions between the ligands and the receptors were studied using DS BIOVIA Discovery Studio 2016 v16.1.0 x64. Visualization between the ligands and the receptors was generated using PyMOL software v.2.4.1.

[0112] 9) Statistical analysis

[0113] All data were expressed as mean ± SD. One-way analysis of variance was used for the data. A p < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism software, Windows version 9.4.1.

[0114] Results:

[0115] 1. Structural identification of DDP and its effects on the viability / proliferation of B16-F10 melanoma cells.

[0116] DDP is a natural compound extracted from Dendrobium officinale. The structure of this compound was identified. The structure of DDP is as follows:

[0117]

[0118] 1H nuclear magnetic resonance [(CD 3 ) 2 CO, 600 MHz]: δ 9.33 (1H, d, J = 8.6, H-5), 7.55 (1H, d, J = 8.6, H-9), 7.43 (1H, d, J = 8.6, H-10), 7.23 (1H, s, H-8), 7.19 (1H, s, H-1), 7.17 (1H, d, J = 8.6, H-6), 3.92 (3H, s, 2-OCH 3 ), 3.89 (3H, s, 4-OCH3 );

[0119] Carbon spectrum [(CD 3 ) 2 CO, 125 MHz]: δ 155.00 (s, C-7), 147.54 (s, C-2), 144.41 (s, C-4), 140.21 (s, C-3), 119.18 (s, C-4a), 128.24 (d, C-5), 127.26 (d, C-10), 134.17 (s, C-8a), 124.44 (d, C-9), 123.09 (s, C-4b), 125.58 (s, C-10a), 116.50 (d, C-6), 111.35 (s, C-8), 105.08 (d, C-1), 55.45 (q, 4-OCH 3 ), 58.74 (q, 2-OCH 3 ).

[0120] Before studying the anti-melanogenesis effect, to detect the cytotoxic activity of DDP, B16-F10 melanoma cells were exposed to DDP at concentrations between 0 and 80 μg / mL for 72 hours. DDP-treated cells did not show morphological changes at concentrations of 5 - 20 μg / mL ( Figure 1 C). The cell viability of melanoma B16-F10 cells treated with DDP (0 - 80 μg / mL) for 72 hours was further evaluated by the CCK8 method. The results showed that compared with the untreated control group, treating cells with 5 - 20 μg / mL of DDP did not cause a significant change in the viability of B16-F10 cells ( Figure 1 B). However, DDP at concentrations greater than 20 μg / mL inhibited the proliferation activity of B16-F10 cells in a concentration-dependent manner within 72 hours and gradually caused morphological changes in the cells ( Figure 1 C).

[0121] Therefore, the concentration range of 0 - 20 μg / mL, which had no obvious effect on the proliferation of B16-F10 cells, was finally selected for subsequent experiments, meeting the two key requirements of neither affecting the proliferation of B16-F10 cells nor significantly regulating melanogenesis, laying a foundation for the following research.

[0122] 2. Cellular antioxidant activity of DDP

[0123] Reactive oxygen species (ROS) have been shown to significantly cause excessive oxidative damage, leading to disease progression, including melanogenesis. In addition, ROS are also produced in melanoma affected by high ultraviolet radiation.

[0124] Therefore, reducing the intracellular ROS level will be beneficial to inhibiting the melanogenesis pathway, thereby affecting melanin synthesis in melanocytes. In the detection, cells were exposed to UV-A radiation to induce a B16-F10 melanoma cell model. Cells exposed to UV-A radiation usually showed an increase in cellular reactive oxygen species (ROS). B16-F10 cells were loaded with the DCFH-DA probe to detect the effect of DDP on the intracellular ROS level in B16-F10 cells. The esterified form of DCFH can penetrate the cell membrane and produce green fluorescence ( Figure 2 A). The fluorescence intensity was quantified to determine the cellular ROS level, and the intracellular ROS level in DDP-treated B16-F10 cells decreased in a concentration-dependent manner ( Figure 2 B). A previous study showed that Dendrobium has strong cytoprotective activity, and polysaccharides from Dendrobium officinale inhibit oxidative damage of glial cells in aging mice by regulating the intestinal flora. Other Dendrobium extracts, such as the Dendrobium biphenyl new compound dendrobine, have antioxidant activity and protective effects on hydrogen peroxide-induced oxidative stress in HaCaT keratinocytes.

[0125] 3. Inhibitory effect of DDP on melanogenesis in B16-F10 cells

[0126] To investigate the effect of DDP on melanogenesis in B16-F10 cells, B16-F10 cells were first treated with α-melanocyte-stimulating hormone (α-MSH) as a positive control for stimulating melanogenesis. In normal physiological processes, α-MSH secreted by keratinocytes induces melanin synthesis, and α-MSH binds to the melanocortin-1 receptor (MC1R) on melanocytes to trigger melanin synthesis.

[0127] B16-F10 cells were cultured in DDP effective concentrations (5 - 20 μg / ml) or α-MSH for 72 h, and co-cultured in the highest DDP concentration of 20 μg / ml and α-MSH for 24 - 72 h to detect their ability to produce melanin. First, the most intuitive method was used to evaluate the ability of DDP to inhibit melanogenesis. By observing the color change of the culture medium to judge the amount of melanin secreted, the culture medium in the positive control group was the darkest, and the cells secreted a large amount of melanin into the culture medium. As the concentration of DDP increased, the color of the culture medium became lighter ( Figure 3 A). Subsequently, cell pellets were collected separately, and it was intuitively found that the production of this melanin in B16-F10 cells could be inhibited by increasing the concentration of DDP ( Figure 3B), indicating that DDP has an inhibitory effect on melanin production. It is worth noting that when B16-F10 cells were treated with a lower concentration (5 μg / ml) of DDP, although the generation of melanin granules decreased slightly, significant anti-melanogenesis effect was only shown when the treatment concentration of DDP was 20 μg / ml ( Figure 3 C). Kojic acid is a natural metabolite derived from fungi, which can inhibit tyrosinase activity and plays an important role in melanin synthesis. Similar results were also observed in α-MSH-induced B16-F10 cells treated with the standard tyrosinase inhibitor kojic acid (200 μM). Figure 3 A-C). Figure 3 E-G describe the comparable anti-melanogenesis effects in B16-F10 cells treated with α-MSH and DDP (20 μg / ml) at different time points (24 - 72 hours), further demonstrating that DDP can significantly inhibit melanin production in B16-F10 cells. On this basis, the melanin content in B16-F10 cells was measured by the NaOH alkaline lysis method. Figure 3 D, H), and the corresponding experimental results indicate that DDP can significantly inhibit melanin production and can significantly reduce the melanin content induced by melanogenic agents.

[0128] 4. Effects of DDP on tyrosinase in B16-F10 cells and melanogenesis-related proteins.

[0129] Since tyrosinase is the key enzyme for melanin formation, the effect of DDP on tyrosinase activity was evaluated based on extracellular experiments. According to previous reports on the inhibitory effect of tyrosinase, DDP with a final concentration of 0 - 20 μg / ml was directly added to the mixture of tyrosinase extracted from B16-F10 cells and its substrate L-DOPA, and the effect of DDP on tyrosinase activity was directly evaluated by measuring the conversion of L-DOPA catalyzed by tyrosinase into melanin. Figure 4 A shows that after the cell extract was treated with DDP for 1 h, the tyrosinase activity in the protein lysate decreased significantly and reached the lowest level when DDP was 20 μg / ml.

[0130] Subsequently, to study whether the reduction of melanin in DDP-treated B16-F10 cells is related to the alteration of key melanogenic enzymes, the protein expression levels of TYR, MITF, TRP-1, and TRP-2 were detected by western blot in B16-F10 cells cultured with DDP at effective concentrations (5 - 20 μg / ml) and α-MSH for 72 h, and in B16-F10 cells co-cultured with DDP at the highest concentration of 20 μg / ml and α-MSH for 24 - 72 h. Figure 4In B, after treating α-MSH-induced melanogenic cells with increasing concentration gradients of DDP (compared to α-MSH treatment alone), DDP effectively reduced the protein expression levels of TYR, MITF, TRP-1, and TRP-2 in B16F10 cells. In B16-F10 cells cultured with 20 μg / ml of the effective concentration of DDP and α-MSH for 24 - 72 h, the protein expression levels of TYR, MITF, TRP-1, and TRP-2 increased with the induction time, but the presence of DDP significantly weakened this trend( Figure 4 C). These results suggest that the inhibitory effect of DDP on melanogenesis is related to downregulating the expression of melanogenesis-related proteins.

[0131] 5. DDP Molecular Docking

[0132] Docking studies were conducted to gain an in-depth understanding of the possible binding conformations of DDP and to compare the binding of DDP and kojic acid to the relevant receptors. In Autodock, DDP or kojic acid was stably bound to tyrosinase and the MC1R receptor for ligand-receptor complex docking. Figure 5 A shows the binding of DDP to the following amino acid residues at the tyrosinase binding site: LEU-59, ASN-57, ASP-348, GLU-377, LYS-376. Figure 5 B shows the binding sites of DDP to MC1R residues: LEU-646, ASN-530, LYS-547, ASP-645. Figure 5 C-D show the binding conformations of the positive control kojic acid to tyrosinase and MC1R. The compound DDP has the highest binding activity to TYR (-6.28 kcal / mol) and MC1R (-6.74 kcal / mol), which is significantly higher than that of the positive control kojic acid. The binding affinities of kojic acid for the target proteins TYR and MC1R are -4.94 and -5.66 kcal / mol, respectively( Figure 5 E). The greater the negative value of the total net charge, the higher the binding affinity of the ligand for the target protein. According to ligand-protein interaction analysis, the compound formed conventional hydrogen bonds with TYR and MC1R Figure 5 C, Figure 5 D). This chemical bond is considered much stronger than other chemical bonds (such as metallic bonds, ionic bonds, and covalent bonds). These results indicate that the binding of DDP to tyrosinase and MC1R is more stable and stronger than that of kojic acid (a tyrosinase inhibitor).

[0133] Discussion

[0134] Continuous ultraviolet irradiation induces DNA damage, gene mutations, and even the occurrence and development of cancer and immune system damage. Under normal physiological conditions, melanogenesis promotes pigmentation and has a photoprotective effect on the human skin, especially protecting against ultraviolet-induced damage. However, the abnormal accumulation of melanin will also cause a series of skin problems, and the existing drugs that inhibit melanin activity have not become the optimal choice for various reasons.

[0135] Therefore, new compounds that can inhibit melanogenesis are explored for use as new drugs and drug precursors. Dendrobium is one of the most important genera of medicinal plants. Dendrobium has attracted attention for its potential inhibition and prevention of cancer, stimulation of the immune system, and antioxidant activity. It is reported that Dendrobium has high antioxidant activity, similar to vitamin C, and can prevent cell damage and destruction. Gigantol is a bibenzyl compound isolated from Dendrobium, which acts as an antioxidant, helps inhibit the occurrence of cataracts, and exhibits anti-lung cancer and anti-platelet aggregation properties.

[0136] DDP, a natural phenanthrene compound extracted from Dendrobium nobile Lindl., was tested for its effect on melanin production induced by α-MSH in cells, indicating that DDP has the potential to inhibit melanogenesis. Given these results, the anti-melanogenic effect of DDP and the molecular mechanisms involved in this activity at the cellular level were elucidated. Tyrosinase plays a crucial role as the rate-limiting enzyme controlling melanin production. The increase in melanogenesis is directly related to tyrosinase activity. Therefore, it was investigated whether DDP could inhibit melanin synthesis by inhibiting tyrosinase activity in α-MSH-induced melanoma cells. It was found that DDP significantly inhibited melanin synthesis and tyrosinase activity in a dose-dependent manner and had no cytotoxic effect on melanoma cells.

[0137] In addition, DDP was able to significantly inhibit the expression of melanogenesis-related proteins (TYR, TRP-1, TRP-2, and MITF) at the protein level, indicating that it may inhibit the related signaling pathways of melanogenesis. Although the key role of DDP in the melanogenic signaling network was not further verified on the related signaling pathways. However, molecular docking of DDP with melanin-concentrating hormone receptor 1 (MC1R) and tyrosinase (TYR) showed that DDP bound more tightly to MC1R and TYR compared with the commercial tyrosinase inhibitor Kojic acid. In addition, DDP could also inhibit the increase in intracellular reactive oxygen species induced by UVA irradiation, showing strong antioxidant properties. In summary, DDP is an efficient and safe melanin synthesis inhibitor and has the potential to become an efficient skin whitening agent derived from plants.

[0138] Conclusion

[0139] In summary, the DDP proposed by the present invention has the activity of inhibiting melanin, and can inhibit the melanin content and tyrosinase activity. At the same time, DDP can reduce the level of reactive oxygen species (ROS) in cells irradiated with UV-A. Regarding its related mechanism, DDP can significantly reduce the expression of melanogenesis-related proteins induced by α-MSH. At the same time, based on molecular docking analysis, DDP has a higher binding affinity for the melanin-forming proteins TYR and MC1R than kojic acid (a commercial tyrosinase inhibitor). In summary, DDP shows the potential to be developed into a skin UV protectant for cosmetic or pharmaceutical industrial applications.

[0140] Second, the whitening composition (AOVA / DDP protein gel) of Example 2 was involved in the following test experiments

[0141] 1 Materials and Methods

[0142] 1.1 Hemolysis experiment

[0143] Collect blood from the guinea pig heart into a heparin sodium blood collection tube, centrifuge at 3000 rpm for 15 min at 4°C. Discard the supernatant, resuspend the red blood cells with an appropriate amount of phosphate buffer (PBS), continue to centrifuge for 5 min, repeat the washing until the supernatant is not significantly red, and dilute the red blood cells with PBS to a 2% red blood cell suspension for standby. At the same time, prepare hydrogel materials with different concentrations, mix 1 mL of the sample with 1 mL of red blood cells, and incubate at 37°C for 3 h. The experiment also includes a negative control (PBS) and a positive control (H 2 O) to calculate the hemolysis rate. Centrifuge all samples at 3000 rpm for 15 min, take the upper layer solution and detect the absorbance at 540 nm by an enzyme-linked immunosorbent assay (ELISA) reader, and use GraphPad Prism 10 software for data plotting.

[0144] 1.2 Animal experiment

[0145] 1.2.1 Establish a guinea pig model of epidermal pigmentation induced by UVB

[0146] Healthy female brown guinea pigs, 4 months old, weighing 300 - 350 g, of ordinary clean grade, were purchased from the Experimental Animal Department of Kunming Medical University (Kunming, China). The brown guinea pigs were housed in an air-conditioned room at a temperature of 24 ± 1°C with a 12-hour light / dark cycle. Shave the back hair 48 hours before UVB irradiation, and use a marker pen to divide the experimental areas on the bare back skin area of the guinea pigs. The area of each experimental area is 1.5 × 1.5 cm 2 , and the back area of the guinea pigs was randomly divided into 6 groups:

[0147] (1) Normal group (UVB-), without UV irradiation and any treatment;

[0148] (2) UVB irradiation group (UVB+), i.e., the group that received only UVB irradiation;

[0149] (3) Positive control group (UVB+HQ), treated with hydroquinone cream before UVB irradiation;

[0150] (4) Positive control group (UVB+Arb), treated with arbutin cream before UVB irradiation;

[0151] (5) Negative control group (UVB+AOVA), treated with AOVA gel before UVB irradiation;

[0152] (6) Experimental group (UVB+AOVA / DDP), treated with AOVA / DDP protein gel before UVB irradiation.

[0153] Place the narrow-spectrum UVB ultraviolet light instrument (290 - 320 nm, 5 mW / cm 2 ) 10 cm above the skin for irradiation, once every other day, for 3 weeks, a total of 10 irradiations, and the irradiation dose is 40 - 440 mJ / cm 2 . After the UVB irradiation is completed, the guinea pigs are sacrificed by cervical dislocation, and the back skin tissues are taken for subsequent experimental analysis.

[0154] 1.2.2 Detection of skin color changes in guinea pigs by skin chromometer

[0155] After grouping and labeling before the experiment and after the UVB ultraviolet irradiation is completed, randomly select 4 sites in each experimental area, and detect the L value (Light) reflecting skin brightness through a skin chromometer. The value ranges from 1 to 100. The difference in L value before and after the experiment is ΔL, and its calculation method is: ΔL = L value (before the experiment) - L value (after the UVB irradiation is completed).

[0156] 1.2.3 Hematoxylin and eosin staining (H&E) experiment

[0157] After dehydrating the skin tissue, make paraffin sections. After dewaxing and rehydrating, perform H&E staining (Beijing Solarbio Science & Technology Co., Ltd.) according to the steps in the instruction manual. The sections are stained with hematoxylin solution to label the cell nuclei and washed with running water; then differentiated in 1% hydrochloric acid ethanol solution for 8 seconds, and then blued in ammonia water. After that, stained with eosin solution to label the cytoplasm and washed with running water. Dehydrate in gradient, make transparent and mount the slides, and observe under an optical microscope.

[0158] 1.2.4 Masson-Fontana staining experiment

[0159] After dewaxing and rehydrating the paraffin sections, immerse the sections in Fontana ammoniacal silver solution and stain them in the dark at room temperature for 12 to 24 hours or incubate them in an incubator at 56 °C for 30 minutes. Then wash them thoroughly with distilled water (5 to 6 times). Subsequently, immerse the sections in hypo solution for 2 minutes and then rinse them with tap water for 5 minutes. Then immerse the sections in neutral red staining solution and lightly counterstain for 5 minutes, and rinse with distilled water. After dehydration and clearing of the sections, mount the slides and then analyze them with an optical microscope.

[0160] 1.2.5 Statistical analysis

[0161] All experiments were performed at least three times, and the results are expressed as mean ± SEM, corresponding to 3 or more replicates. Student's t-test or one-way analysis of variance (ANOVA) was used for statistical analysis. A P value of < 0.05 was considered statistically significant.

[0162] 2 Results

[0163] 2.1 Characterization and biocompatibility of AOVA / DDP protein gel

[0164] Previous studies have shown that OVA is a food globular protein, and the AOVA protein gel prepared by the thermal-induced self-assembly method has excellent physicochemical properties and stability. On this basis, the present invention prepared an AOVA / DDP protein gel (i.e., the whitening composition prepared in Example 2), and the results showed that the gel was semi-transparent and had good adhesion ( Figure 6 A). The results of FT-IR spectroscopy ( Figure 6 B) showed that there was no obvious change in the peak shapes of NOVA and AOVA. The FTIR spectra of OVA and AOVA had 5 characteristic peaks, showing stretching vibrations at amide I (1635 cm-1), amide II (1532 cm-1), amide III (1397 cm-1), amide A (3273 cm-1), and amide B (2961 cm-1), respectively. Compared with OVA, the peak intensities of AOVA in multiple characteristic bands were enhanced. At 1635 cm-1 and 1532 cm-1, the two enhanced peaks of AOVA should be attributed to the stretching vibrations of C-O and C-N, respectively. The enhanced peak of AOVA at 1397 cm-1 was attributed to C-N stretching and N-H deformation. In addition, the bands at 3273 cm-1 (N-H stretching vibration) and 2961 cm-1 (C-H antisymmetric stretching vibration) were stronger than those of OVA. The above characteristics indicated that succinic anhydride successfully modified the OVA molecule. To observe the internal structure of the AOVA / DDP protein gel, the microscopic morphology of the gel was observed by scanning electron microscopy (SEM) ( Figure 6C). The AOVA protein gel loaded with DDP showed a tightly arranged porous network structure, which was beneficial to increasing the drug loading capacity and improving the delivery efficiency of the gel as a drug carrier. In addition, the dense network structure could slow down the diffusion rate of drug molecules, thus effectively controlling the drug release.

[0165] For the application of clinical skin drugs, good biocompatibility is a basic requirement. Therefore, the cytotoxicity of the AOVA / DDP protein gel (i.e., the whitening composition prepared in Example 2) against keratinocytes (HaCaT) and B16-F10 cells was detected. Different groups of materials were co-cultured with cells for 4 days, and the cell viability of different groups was measured on the designated 1st day, 2nd day, and 4th day. The results showed that compared with the blank control group, AOVA / 20DDP had a certain inhibitory effect on cell viability on the fourth day of co-culture with cells, while no obvious cytotoxicity was shown in other groups. Figure 6 D, E). Meanwhile, the live / dead cell staining further supported this result. After the cells were co-cultured with different groups of materials for 4 days, no obvious cell death was found. Figure 6 G, H). In addition, the hemolysis rate is an important index for evaluating the safety of materials. The protein gels of different groups were incubated with red blood cells for 3 hours, and their hemolysis rates were all less than 5%. Figure 6 F). The above results indicate that the AOVA / DDP protein gel (i.e., the whitening composition prepared in Example 2) prepared by the present invention has good biocompatibility and can be used for the next in vivo experiment verification.

[0166] 2.2 Evaluation of the in vivo effect of the AOVA / DDP protein gel

[0167] Brown guinea pigs are ideal animal models for evaluating natural products. The distribution of skin melanocytes and melanosomes in brown guinea pigs is similar to that of human skin. After their skin is irradiated with UVB ultraviolet rays, skin pigmentation can be induced, simulating the pigment increase in human skin caused by ultraviolet irradiation. Therefore, UVB irradiation was used to irradiate the skin of brown guinea pigs to construct a pigmentary skin disease model to evaluate the therapeutic potential of the AOVA / DDP protein gel. After three weeks of UVB irradiation, compared with the non-irradiated group UVB(-), obvious pigmentation appeared in the epidermis of UVB(+) guinea pigs. In addition, the positive treatment groups UVB(+)HQ and UVB(+)Arb both significantly reduced skin pigmentation. The AOVA protein gel did not show an obvious improvement effect, but the AOVA / DDP protein gel could significantly improve the skin pigment accumulation caused by UVB irradiation, and its effect was better than that of the Arb group and comparable to that of the HQ group. Figure 7A). The colorimeter was used to measure the L value (Light) of each experimental area of ​​the guinea pig skin before and after the experiment. The larger the value, the brighter the skin. Correspondingly, before UVB irradiation, there was no significant difference in the L value of each experimental area, but after UVB irradiation and different drug treatments, the L value changed differently ( Figure 7 B). Among them, the treatment with HQ and DDP resulted in the smallest change in ΔL (L before experiment - L after experiment), which was close to that of the UVB non-irradiated group ( Figure 7 C). These results indicate that AOVA / DDP protein gel can significantly improve UVB-induced skin pigmentation.

[0168] Subsequently, the skin of each experimental area was stained with HE and Masson-Fontana to observe the pathological changes of skin tissue and the deposition of epidermal melanin particles. The results of HE staining showed that the skin structure of each experimental area was intact, with clear layers, no skin thickening, no atypical cells, and no inflammatory cell infiltration in the dermis, indicating that the topical use of AOVA / DDP protein gel would not cause side effects such as skin inflammation and hyperplasia. In addition, it also ruled out the possibility that the changes in skin pigmentation were post-inflammatory pigmentation ( Figure 7 D). The results of Masson-Fontana staining and statistical analysis of relative melanin content showed that in the group without UVB irradiation, scattered and small amounts of melanin particles were deposited in the epidermis, mainly distributed in the basal layer of the skin at the junction of the true epidermis, while in the group with UVB irradiation, a large amount of melanin particles were deposited at the junction of the true epidermis, and a large number of "melanin caps" were seen in the epidermal keratinocytes. Compared with the simple matrix AOVA group and the UVB irradiation group, the AOVA / DDP protein gel had significantly less melanin particle deposition, and the difference was statistically significant ( Figure 7 E). The above results show that AOVA / DDP protein gel can significantly improve UVB-induced epidermal melanin granule deposition without side effects such as inflammation.

[0169] 3 Conclusion

[0170] DDP is a natural active ingredient isolated and identified from Dendrobium officinale. The skin protective effect of DDP was evaluated on UVB-irradiated guinea pig skin by using acylated ovalbumin (AOVA) protein gel as a drug delivery carrier for DDP, and compared with commercial skin care products. The results showed that AOVA / DDP protein gel had good biocompatibility. More importantly, the results of in vivo experiments showed that AOVA / DDP protein gel could effectively reduce the accumulation of skin melanin caused by UVB irradiation without side effects such as inflammation. This study provides scientific support for the development of DDP as a clinical drug or cosmetic ingredient, and lays a theoretical foundation for the scientific research of food and drug homologous products.

[0171] The above has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention

[0172] will also have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A whitening composition, characterized in that: Contains 3,7-dihydroxy-2,4-dimethoxyphenanthrene, the chemical structure of which is shown below 。 2. A whitening composition according to claim 1, characterized in that: The composition is prepared by mixing the following components with an acetylated ovalbumin aqueous solution as solvent: 3,7-Dihydroxy-2,4-dimethoxyphenanthrene 0.1~20mg / ml.

3. A whitening composition as claimed in claim 2, characterized in that , which is prepared by mixing the following components with an acetylated ovalbumin aqueous solution as solvent: 3,7-dihydroxy-2,4-dimethoxyphenanthrene 20 mg / ml; Skin care and whitening complex 3-10mg / ml; Additive 0.1~1mg / ml.

4. A whitening composition as claimed in claim 3, characterized in that: The skin care and whitening complex comprises modified pearl powder, tea polyphenols, centella asiatica extract and angelica dahurica powder, and the mass ratio thereof is 1-1.5:0.1-0.2:0.05-0.1:

1.

5. A whitening composition as claimed in claim 3, characterized in that: The additives include silk peptide, trehalose and shea butter, the mass ratio of which is 1:2-3:4-5, and the molecular weight of the silk peptide is 500-1000 Daltons.

6. A method for preparing the whitening composition according to any one of claims 1 or 2, characterized in that: The following steps are involved: 1) Extraction, crushing the whole part of the washed and dried polydendrobe, then extracting it three times with 95% ethanol at room temperature, evaporating the obtained solution under reduced pressure, suspending the concentrated extract in a liquid, and fractionating it with ethyl acetate; evaporating the ethyl acetate layer to dryness to obtain an extract, gradient eluting the extract with a first eluent, and performing silica gel column chromatography to obtain an A fraction, a B fraction, a C fraction, a D fraction, and an E fraction; performing silica gel column chromatography on the A fraction, and performing step-by-step gradient elution with a second eluent to obtain an A1 pressure fraction, an A2 sub-fraction, and an A3 sub-fraction; performing silica gel column chromatography on the A3 sub-fraction, performing step-by-step gradient elution with a third eluent, and repeatedly separating the 3,7-dihydroxy-2,4-dimethoxyphenanthrene with a glucose-based resin and methanol; 2) Gel preparation: ovalbumin was dissolved in deionized water under magnetic stirring for 2 h, the pH value of the dispersed solution was adjusted to 8.0, and then succinic anhydride was added for acylation reaction for 1 h. The pH value was kept at 8.0 during the acylation reaction. After the acylation reaction was completed, the solution was dialyzed with deionized water at 2-6°C for 40-50 h, and the acetylated ovalbumin gel was obtained after freeze-drying; 3) Preparation: Take the acetylated ovalbumin gel of step 2), add phosphate saline while stirring to reach 60-100 mg / ml, keep overnight at 2-6°C, and then heat at 80-95°C for 8-12 minutes to obtain an acetylated ovalbumin aqueous solution, add the 3,7-dihydroxy-2,4-dimethoxyphenanthrene to the acetylated ovalbumin aqueous solution to make the final 3,7-dihydroxy-2,4-dimethoxyphenanthrene concentration of 20 mg / ml, stir, and keep overnight to obtain a whitening composition.

7. The method for preparing a whitening composition according to claim 6, characterized in that: In the step 3), the acetylated ovalbumin aqueous solution is added with the 3,7-dihydroxy-2,4-dimethoxyphenanthrene, the skin care and whitening complex and the additives.

8. The method for preparing a whitening composition according to claim 6, characterized in that: In the step 1), each liter of 95% ethanol contains 0.1-0.2 kg of whole polydendrobe powder; Extract with 95% ethanol at room temperature three times, once every five days; The first eluent is one or both of petroleum ether and ethyl acetate; the second eluent is a mixture of petroleum ether and acetone, with a volume ratio of 1 to 9:1; the third eluent is a mixture of CHCl3 and ethyl acetate, with a volume ratio of 15 to 50:

1.

9. The method for preparing a whitening composition according to claim 7, characterized in that: The skin care and whitening composition comprises modified pearl powder, tea polyphenols, Centella asiatica extract and Angelica dahurica powder; the preparation method of the modified pearl powder is as follows: The pearl powder is ground into powder with a particle size of 0.1 to 2.0 microns, and then mixed with deionized water, stirred at 120 to 150 r / min, and ultrasonicated at the same time, with an ultrasonic power of 1.5-2.0 kW and an ultrasonic time of 60 to 90 minutes to obtain a pearl powder aqueous solution with a mass percentage of 10 to 15 wt%, and a monoalkyl phosphate ester of 2 to 3% of the dry weight of the pearl powder is added to the slurry, and magnetic stirring is performed for 10 to 20 minutes, and glycerol and lauroyl lysine are added dropwise respectively, and the addition is completed within 20 to 30 minutes, and the temperature is controlled to 60 to 70° C. for 60 to 100 minutes, and then aged for 2 to 3 hours, washed, dried and crushed to obtain modified pearl powder; the mass ratio of monoalkyl phosphate ester, glycerol and lauroyl lysine is 1:0.5 to 2:0.1 to 1.

10. Use of the whitening composition according to any one of claims 1 to 5 in skin care and cosmetics.

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