Preparation and application of umbilical cord extract having hair darkening effect by regulating melanin granules produced by hair follicle melanocytes

Abstract

The present invention discloses the preparation and application of an umbilical cord extract having a hair-blackening effect by regulating the production of melanin particles by hair follicle melanocytes. The preparation of the umbilical cord extract is as follows: the animal umbilical cord is washed and cut into pieces, digested with trypsin, added with serum-free mesenchymal stem cell culture medium, dispersed by blowing, and then filtered, the filtered tissue fluid is irradiated with low-energy red light and incubated for 6 to 12 hours, the tissue fluid is taken out, freeze-thawed, then ultrasonically treated, filtered, and the filtrate is diluted with sterile water to obtain the umbilical cord extract. The umbilical cord extract of the present invention has a good regulating effect on the activity of melanocytes and the ability to produce melanin, can increase the content of melanin particles in hair, and can be used as a cosmetic, raw material or medicine with a hair-blackening effect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the preparation and application of an umbilical cord extract having the effect of darkening hair by regulating hair follicle melanocytes to produce melanin particles. Background Art

[0002] Hair follicle pigment is produced by hair follicle melanocytes, which originate from embryonic neural crest cells and are then implanted in different anatomical locations in the hair follicles. There are two subpopulations of hair follicle melanocytes, namely melanocytes with melanin synthesis activity located in the hair matrix and infundibulum of the hair follicles during the growth phase, and amelanotic melanocytes that are widely distributed in the outer root sheath (ORS) of the hair during the growth phase and have no melanin synthesis activity and are not activated. The difference between hair follicle pigment units and epidermal pigment units: hair follicle pigment is mainly formed in melanosomes in the cytoplasm of hair bulb melanocytes, and then the melanosomes are secreted into the keratinocytes around the hair bulb. As the keratinocytes become keratinized, the hair becomes darker. The melanin production systems of hair follicles and epidermis are very different. Epidermal melanin synthesis is continuous, while hair follicle melanin is only synthesized during the hair growth phase. Therefore, with the cyclical changes in hair growth, the activity of hair bulb melanocytes is also periodically activated accordingly. Active melanocytes are highly expressed during the growth phase, but are almost undetectable during the regression phase. During the entire hair growth period, the ultrastructural changes that occur in the melanocytes of the hair bulb include: ① an increase in cell dendrites, ② the development of the Golgi apparatus and rough endoplasmic reticulum, ③ an increase in the number and volume of melanin granules, and ④ the transport of mature melanin granules to the prekeratinocytes of the cortex.

[0003] Pigmentation of hair: Hair color mainly depends on the content of two types of melanin, eumelanin and pheomelanin: Typical melanosomes can be seen in dark brown hair, which are mainly eumelanin. Hair color can be seen changing in many ways throughout a person's life. Children's hair color darkens with age, and can become brown hair, or yellow or auburn in middle age. Elderly gray hair is a mixture of white and black hair, which is a manifestation of aging of hair follicles, and white hair is the terminal stage of pigment loss. The exact cause of graying in the elderly is not very clear. The melanocytes in the hair bulbs of elderly gray hair are generally normal or reduced, but their melanogenesis activity is low and there are almost no melanosomes, which indicates that graying is not due to the disappearance of melanocytes, but due to melanocyte dysfunction, such as decreased tyrosinase activity.

[0004] In the process of melanin synthesis in hair follicle cells, melanin synthesis first requires direct transport of L-tyrosine from outside the cell, or L-phenylalanine hydroxylation mediated by intracellular phenylalanine hydroxylase to convert L-phenylalanine into L-tyrosine. Subsequently, tyrosinase (TYR) catalyzes the hydroxylation of L-tyrosine to L-3-4-L-DOPA, which is then oxidized to dopaquinone. Dopaquinone undergoes polymerization and oxidation reactions to generate dopachrome. Under the action of tyrosinase protein-2 (TRP-2), dopachrome is hydroxylated to 5,6-dihydroxyindolecarboxylic acid, decarboxylated to 5,6-dihydroxyindole, and oxidized to 5,6-indolequinone under the catalysis of TYR, and finally combines with other intermediates to form true melanin. Therefore, tyrosinase and tyrosinase protein-2 play a key role in the synthesis of true melanin granules in hair follicle melanocytes. Their expression in melanocytes increases and remains stable when hair follicles enter the growth phase, and decreases rapidly when entering the regression phase.

[0005] The most obvious sign of aging is graying hair. It takes more than 15 years for all hair to turn gray. Therefore, exploring how to maintain the activity of hair follicle melanocytes and the ability to secrete true melanin is the focus of delaying hair graying. Animal umbilical cord is an animal-derived cosmetic raw material that is easy to obtain and has relatively low immunogenicity. The biologically active substances obtained from animal raw materials vary greatly due to differences in their extraction process, extraction method, and treatment methods. The present invention provides a method for extracting animal umbilical cords. The extracted biologically active substances have a good regulating effect on the activity of hair follicle melanocytes and the ability to produce melanin, can increase the content of melanin particles in hair, and can be used as a cosmetic, raw material or medicine with a hair-blackening effect. Summary of the invention

[0006] The first object of the present invention is to provide the use of umbilical cord extract in promoting the synthesis of melanin particles by hair follicle melanocytes. The preparation steps of the umbilical cord extract are: washing and cutting the fresh animal umbilical cord, digesting it with trypsin, adding serum-free mesenchymal stem cell culture medium, blowing and dispersing the tissue, and then filtering it, irradiating the filtered tissue fluid with low-energy red light and incubating it for 6 to 12 hours, taking out the tissue fluid, performing freeze-thaw treatment, then ultrasonic treatment, filtering, and diluting the filtrate with sterile water to obtain the umbilical cord extract.

[0007] Preferably, the low-energy red light irradiation is irradiation with 620 nm low-energy red light for 10-15 min with an energy density of 0.5-1 J / cm 2 .

[0008] Preferably, the incubation is carried out in a 37°C incubator.

[0009] Preferably, the freeze-thaw treatment is specifically as follows: quick freezing at -80°C for 1 h, and thawing in warm water at 37°C; quick freezing at -80°C for 1 h, and thawing in warm water at 37°C.

[0010] Preferably, the ultrasonic treatment is specifically: ultrasonic treatment at 4° C. for 4-10 min, with an operating frequency of 80 kHz.

[0011] Preferably, the first filtration is performed using a 100-300 μm filter, and the second filtration is performed using a 10-40 μm filter.

[0012] Preferably, the trypsin digestion treatment is specifically: using 0.25% trypsin at 4°C for 8-12 h.

[0013] Preferably, the dilution with sterile water is specifically diluted to a concentration of 1-10% by mass.

[0014] The second object of the present invention is to provide the use of umbilical cord extract in preparing a product with hair darkening function, and the preparation steps of the umbilical cord extract are as described above.

[0015] Preferably, the product is an external use product.

[0016] Preferably, the products include cosmetics and medicines.

[0017] Preferably, the cosmetic comprises any one of essence, essential oil, spray, lotion, shampoo, conditioner or hair mask.

[0018] The present invention has discovered a method for processing the umbilical cord. The extract obtained by extraction has a good regulating effect on the activity of melanocytes and the ability to produce melanin, can increase the content of melanin particles in hair, and can be used as a cosmetic, raw material or medicine with the effect of blackening hair. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 EdU staining is used to detect the proliferation activity of human melanocytes. A. EdU staining is used to detect cell proliferation. Green fluorescence is EdU-positive proliferating cells; blue is the cell nucleus labeled with DAPI. B. The percentage of EdU-positive cells in different treatment groups is calculated = (number of EdU-positive cells / number of DAPI cells) × 100%. * p<0.05, ** p<0.01.

[0020] Figure 2Immunofluorescence staining is used to detect the synthesis of TYR in human melanocytes. A. Immunofluorescence staining is used to detect the expression of TYR in melanocytes (red fluorescence). Green fluorescence is phalloidin staining to show the morphology of human melanocytes; blue is the cell nucleus labeled with DAPI. B. The expression of TYR per unit area in different treatment groups is counted = (total red fluorescence intensity per unit area / area) × 100%, * p<0.05, ** p<0.01.

[0021] Figure 3 Melanin granules in human melanocytes observed under a microscope.

[0022] Figure 4 Umbilical cord extract promotes the change of human hair from brown to black. A. A digital camera takes pictures of the color change of the entire hair. B. A hair mirror observes the color change of a single hair shaft. DETAILED DESCRIPTION

[0023] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0024] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0025] Example 1

[0026] Preparation of umbilical cord extract:

[0027] In the clean bench, take fresh pig umbilical cord and wash it in PBS containing penicillin (100 U / mL) and streptomycin (0.1 mg / mL) for 3 times, 5 min each time; then use sharp scissors to cut the tissue into 1 mm 2 About 100 μm, wash twice with calcium-free and magnesium-free PBS, 5 min each time; then add an equal volume of 0.25% trypsin and shake at 4°C for 12 h. Add an equal volume of serum-free mesenchymal stem cell culture medium (serum-free culture medium produced by Ecosai Biotechnology (Taicang) Co., Ltd., with a catalog number of ME000-N023), blow and disperse the tissue for 2 h, and filter the tissue liquid with a 100 μm filter; the filtered tissue liquid is placed on an open plate and irradiated with 620 nm low-energy red light for 10 min, with an energy density of 0.5-1 J / cm 2, then place the tissue fluid in a 37℃ incubator with 5% CO2 for 6 h, take out the tissue fluid, bottle it, 10 mL / bottle, and freeze it at -80℃ for 3 h until it becomes solid; thaw it in warm water at 37℃; freeze it at -80℃ for 3 h until it becomes solid, and thaw it in warm water at 37℃; then ultrasonically treat it at 4℃, using a 500W ultrasonic cell disruptor with an operating frequency of 80 kHz for 4 min; filter the obtained tissue fluid using a 40 μm filter; then dilute the filtered tissue fluid with sterile water to a concentration of 5% by mass, and the obtained liquid is the umbilical cord extract (denoted as RL-UCE), which is stored at -80℃.

[0028] The umbilical cord extract (denoted as UCE) without irradiation with 620 nm low-energy red light was prepared according to the above method. The specific steps are substantially the same as the above steps, except that the step of irradiation with 620 nm low-energy red light is omitted.

[0029] Example 2

[0030] Umbilical cord extract promotes the proliferation of human melanocytes:

[0031] Human primary melanocytes (HEMn) were purchased from Shanghai Jitai Yikesai Biotechnology Co., Ltd., an authorized distributor of ATCC official website.

[0032] Human primary melanocytes were cultured with complete medium (dermal cell basal medium (Dermal cell basal medium, PCS-200-030) + melanocyte growth factor kit (Melanocyte growth kit, PCS-200-041), purchased from Shanghai Jitai Yikesai Biotechnology Co., Ltd.) and the experiment was carried out when the cell confluence was 50%-70%. The cells were starved and incubated with dermal cell basal medium without melanocyte growth factor for 12 h, and the cells were treated according to the grouping:

[0033] Control group: cultured with complete medium only for 3 days;

[0034] UCE group: cultured with complete medium supplemented with 0.9% UCE (umbilical cord extract without 620 nm low-energy red light irradiation) for 3 days;

[0035] RL group: cultured in complete medium for 3 days, irradiated with 620 nm low-energy red light for 10 min on the first day, with an energy density of 0.5-1 J / cm 2 ;

[0036] RL-UCE group: cultured with complete medium supplemented with 0.9% RL-UCE (umbilical cord extract irradiated with 620 nm low-energy red light) for 3 days;

[0037] Three days later, the proliferation rate of human primary melanocytes was detected according to the detection steps of the EdU cell proliferation detection kit (Cell-Light EdU Apollo488 In VitroKit) (Guangzhou Ruibo Biotechnology Co., Ltd.), and statistical analysis was performed.

[0038] The results showed that the effect of RL-UCE group in promoting the proliferation of human melanocytes was significantly better than that of the control group, UCE group and RL group ( Figure 1 ), the results show that the umbilical cord extract prepared by the method of the present invention can significantly enhance the proliferation activity of human melanocytes.

[0039] Example 3

[0040] Umbilical cord extract promotes the expression of tyrosinase (TRY) in human melanocytes:

[0041] Human melanocytes were cultured with complete medium. Experiments were performed when the cell confluence was 50%-70%. They were starved for 12 h with dermal cell basal medium without melanocyte growth factor. The cells were treated according to the grouping:

[0042] Control group: cultured with complete medium only for 3 days;

[0043] UCE group: cultured with complete medium supplemented with 0.9% UCE for 3 days;

[0044] RL group: cultured in complete medium for 3 days, irradiated with 620 nm low-energy red light for 10 min on the first day, with an energy density of 0.5-1 J / cm 2 ;

[0045] RL-UCE group: cultured with complete medium supplemented with 0.9% RL-UCE for 3 days;

[0046] After 3 days, the cells were fixed with 4% paraformaldehyde, and the expression of TYR in the cells was detected by immunofluorescence staining; phalloidin showed the morphological skeleton of melanocytes, and DAPI marked the cell nucleus;

[0047] Specific steps: After the cells were fixed with 4% paraformaldehyde, they were washed with PBS three times for 5 minutes each time, and then blocked with 1% BSA blocking solution containing 10% goat serum at room temperature for 30-40 minutes. The blocking solution was removed and incubated with TYR primary antibody for 24 hours. The TYR primary antibody was removed and washed with PBS three times for 5 minutes each time. The cells were incubated with red fluorescent secondary antibody for 1 hour and washed with PBS three times for 5 minutes each time. The cells were incubated with phalloidin staining solution and DAPI at room temperature in the dark for 30-40 minutes and washed with PBS three times for 5 minutes each time. Anti-fluorescence quencher was used, the slides were sealed, and the immunofluorescence microscope was used to take pictures. The average red fluorescence intensity per unit area was statistically analyzed using Image software to calculate the TYR expression level.

[0048] The results showed that the expression level of TYR in melanocytes of the control group, UCE group and RL group was low, indicating that the synthesis of melanin granules was not active; while the expression level of TYR in melanocytes of the RL-UCE group was high, indicating that the synthesis of melanin granules was active ( Figure 2 ).

[0049] Example 4

[0050] Umbilical cord extract promotes melanin granule formation in human melanocytes:

[0051] Human melanocytes were cultured with complete medium. Experiments were performed when the cell confluence was 50%-70%. They were starved for 12 h with dermal cell basal medium without melanocyte growth factor. The cells were treated according to the grouping:

[0052] Control group: cultured with complete medium only for 3 days;

[0053] UCE group: cultured with complete medium supplemented with 0.9% UCE for 3 days;

[0054] RL group: cultured in complete medium for 3 days, irradiated with 620 nm low-energy red light for 10 min on the first day, with an energy density of 0.5-1 J / cm 2 ;

[0055] RL-UCE group: cultured with complete medium supplemented with 0.9% RL-UCE for 3 days;

[0056] Then observe the number of melanin granules in the cells under a microscope.

[0057] The results showed that the melanin granules in the melanocytes of the control group, UCE group and RL group were less, while the melanin granules in the melanocytes of the RL-UCE group were more ( Figure 3 ).

[0058] Example 5

[0059] Umbilical cord extract promotes the formation of melanin particles in human hair:

[0060] Three people with gray-black hair were selected and 3 mL of RL-UCE was applied to the scalp every day for 3 consecutive months.

[0061] Before the first use and after the last use, take photos with a digital camera in the same location and environment to observe the overall hair color; Figure 4 In A, you can see that the hair is grayish black before use; after 3 months of use, the hair is black.

[0062] Before the first use and after the last use, take photos with a hair mirror at the same position (red mark point) to observe the color of the local single hair; Figure 4 In B, you can see that the three hairs around the red mark were brown before use, while after three months of use, the three hairs around the red mark were black.

[0063] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of umbilical cord extract in the preparation of a product that promotes the synthesis of melanin particles by hair follicle melanocytes, characterized in that: The preparation steps of the umbilical cord extract are as follows: washing and chopping the fresh animal umbilical cord, digesting it with trypsin, adding serum-free mesenchymal stem cell culture medium, blowing and dispersing the tissue, and then filtering it. The filtered tissue fluid is irradiated with 620 nm low-energy red light for 10-15 min with an energy density of 0.5-1 J / cm 2 , then incubate in a 37°C incubator for 6-12 h, take out the tissue fluid, perform freeze-thaw treatment, then perform ultrasonic treatment, filter, and dilute the filtrate with sterile water to obtain the umbilical cord extract; The freeze-thaw treatment is specifically as follows: quick freezing at -80°C for 3 h, thawing in warm water at 37°C; quick freezing at -80°C for 3 h, thawing in warm water at 37°C; The ultrasonic treatment is specifically: ultrasonic treatment at 4°C for 4-10 min, with an operating frequency of 80 kHz.

2. Use of umbilical cord extract in preparing a product with hair darkening function, characterized in that: The preparation of the umbilical cord extract is as described in claim 1.

3. The use according to claim 2, characterized in that: The product described is a product for external use.

4. The use according to claim 3, characterized in that The products include cosmetics and medicines.

5. The use according to claim 4, characterized in that The cosmetics include any one of essence, shampoo, conditioner or hair mask.

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