CN118319828B - Hair-blacking and hair-growing composition and method of making same shampoo preparation and preparation method thereof - Google Patents

Abstract

The present invention belongs to the field of stem cell technology, and more specifically, relates to a hair darkening and growth promoting composition obtained by comprising a mesenchymal stem cell secretory group treated with psoralen, and a shampoo preparation and preparation method thereof. Experiments have shown that the composition of the present invention has a promoting effect on the proliferation of melanocytes, and can activate tyrosinase, and has excellent hair darkening and growth promoting effects.

Description

A black hair composition, a shampoo preparation containing the same, and a preparation method

Technical Field

The present invention belongs to the technical field of stem cells. More specifically, it relates to a hair darkening composition obtained by secretory groups of mesenchymal stem cells cultured by treating psoralen, and a shampoo preparation and preparation method thereof.

Background Art

Melanocytes at the root of hair slow down the rate of pigment production. When melanocytes completely stop producing pigment, white hair grows. The synthesis of melanin in human melanocytes is the result of the separate or synergistic action of three enzymes: tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and tyrosinase-related protein 2 (TYRP2). Tyrosinase is the main rate-limiting enzyme for the production of melanin, and its expression and activity determine the rate and output of melanin synthesis. Therefore, a large proportion of drugs that can activate tyrosinase activity can also activate the synthesis of melanin. By activating or inhibiting the activity of tyrosinase, the formation of hair melanin can be regulated to achieve the purpose of black hair.

The nutrition and immunomodulatory factors secreted by mesenchymal stem cells (MSC) are collectively referred to as the MSC secretome. Currently, the mesenchymal stem cell secretome has been used to promote hair follicle repair and/or hair regeneration, such as CN 110540958 A and CN 115537393 A, but no research on tyrosinase activity is involved.

Psoralidin belongs to the coumarin class of compounds, with CAS number 18642-23-4, molecular formula C ₂₀ H ₁₆ O ₅ , and structural formula as shown below. Psoraleadin is derived from the seeds of Psoralea corylifolia Linn. It is known that Psoraleadin has multiple biological activities such as anticancer, antioxidant, antibacterial, antidepressant, anti-inflammatory, and insulin signaling regulation. In addition, Psoraleadin is considered to be a potential candidate drug for the treatment of postmenopausal osteoporosis ^[1]. Furthermore, Psoraleadin exhibits strong inhibitory activity against SARS-CoVPLpro (IC ₅₀ = 4.2 μmol/L), and is a very promising antiviral candidate drug ^[2] . However, there is no report on the use of the secretome obtained by treating mesenchymal stem cells with Psoralea corylifolia for the development of black hair. In view of this, the present invention is proposed.

Reference 1: CAO H J, LI C R, WANG L Y, et al. Effect and mechani sm ofpsoralidin on promoting osteogenesis and inhibiting adipogenesis[J]. Phytomedicine, 2019, 61:152860.

Reference 2: KIM D W, SEO K H, CURTIS-LONG M J, et al.Phenolicphytochemical displaying SARS-CoV papin-like protease inhibition from the seeds of Psoralea corylifolia[J].J Enzyme Inhib Med Chem, 2014, 29(1) :59.

Summary of the invention

In view of the deficiencies in the prior art, the present invention provides a tyrosinase activator, a hair darkening composition and a preparation method thereof, and a shampoo containing the composition.

In one aspect, the present invention relates to a tyrosinase activator, which particularly comprises a secretome obtained by culturing mesenchymal stem cells in a medium supplemented with psoralen.

Prior to the application date, there have been reports on the use of mesenchymal stem cell secretomes for promoting hair follicle repair and/or hair regeneration, such as CN 110540958 A and CN 115537393 A, but no research on tyrosinase activity was involved.

The psoralen described in the present invention has a chemical structure as shown in the following formula:

In the use as a tyrosinase activator, in the process of obtaining the secretory group of mesenchymal stem cells, the concentration of psoralen in the culture medium is 1 to 10 nmol/L, for example, preferably 1 nmol/L or 10 nmol/L. The results of Study 1 show that different concentrations of psoralen show great differences in the proliferation activity of umbilical cord mesenchymal stem cells. When the concentration of psoralen is within the range of 1 to 10 nmol/L, it promotes the proliferation of hUCMSCs, and the peak value appears at 10 nmol/L, when the cell activity rate is as high as 120.37%, which is significantly promoted compared with the control group (no drug addition). When the concentration of psoralen continues to increase to more than 1×10 ³ nmol/L, it shows the effect of inhibiting hUCMSCs, and as the concentration of psoralen increases, the inhibitory effect becomes more obvious.

The test results showed that the hUCMSCs secretion groups treated with 1 nmol/L and 10 nmol/L psoralen had the highest activation rates for tyrosinase, which were 259.82% and 363.41% respectively, which was 2 to 3 times higher than the secretion group without psoralen treatment.

In one of the more preferred embodiments of the invention, the mesenchymal stem cells are cultured in a medium supplemented with psoralen for 12 to 36 hours, preferably 24 hours.

As mentioned above, tyrosinase is the main rate-limiting enzyme for the production of melanin, and its expression and activity determine the speed and output of melanin synthesis. By activating the activity of tyrosinase, the formation of hair melanin can be regulated to achieve the purpose of dark hair. Therefore, on the other hand, the present invention relates to the use of the activator in the preparation of a dark hair composition.

The test results showed that the hUCMSCs secretion groups treated with 1nmol/L and 10nmol/L psoralen, which had a higher activation rate for tyrosinase activity, also exhibited higher proliferation-promoting activity on melanocytes (121.75% and 134.28%, while the blank group was 100.00%), indicating that the two hUCMSCs secretion groups had ideal hair blackening effects.

The "dark hair" mentioned in this article refers to increasing the melanin content in the hair, reducing the amount of white hair, preventing the formation of white hair, and accelerating the formation of black hair.

On the other hand, the present invention relates to a black hair composition, which comprises a secretome obtained by culturing mesenchymal stem cells in a culture medium supplemented with psoralen.

Prior to the application, there were no reports on the use of psoralen or the secretory group obtained by treating stem cells with psoralen for black hair growth. Experimental evidence (Studies 2 and 4) showed that compared with psoralen, the hUCMSCs secretory group obtained by adding psoralen to treat cells has a stronger effect of promoting melanocyte proliferation and hair growth, which was unexpected by technicians in this field.

In one of the more preferred embodiments of the invention, the concentration of psoralen in the culture medium is 1-10 nmol/L, for example, preferably 1 nmol/L or 10 nmol/L.

In one of the more preferred embodiments of the invention, the mesenchymal stem cells are cultured in a culture medium supplemented with psoralen for 12 to 36 hours, preferably 24 hours.

In one of the more preferred embodiments of the invention, the mesenchymal stem cells are umbilical cord mesenchymal stem cells or adipose mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells.

In one of the preferred embodiments of the invention, the secretome is prepared by the following specific steps:

Obtaining mesenchymal stem cells that meet the requirements; culturing the mesenchymal stem cells for 12 to 36 hours using a culture medium supplemented with 1 to 10 nmol/L of psoralen; collecting the culture fluid, centrifuging it, filtering it, and obtaining a secretory group.

On the other hand, the hair darkening composition of the present invention can be formulated in the form of shampoo, scalp care lotion, hair care essence, hair mask, and hair darkening medicine.

Preferably, the hair darkening composition can be made into a shampoo, which can theoretically contain the composition and additives acceptable in the art or other active ingredients with hair darkening effects. The additives can be exemplified by moisturizers, surfactants, thickeners, chelating agents, preservatives, fragrances, etc.

The moisturizers include glycerin, butylene glycol, squalane, sodium hyaluronate, trehalose, etc.; the surfactants include sodium laureth sulfate, cocamidopropyl betaine, cocamidopropyl hydroxysulfobetaine, sodium methyl cocoyl taurate; the thickeners include hydroxyethyl cellulose, carbomer, acrylic acid (esters)/C10-30 alkyl acrylate crosspolymer, xanthan gum, sodium acrylate/sodium acryloyldimethyl taurate copolymer; the chelating agents include EDTA-2Na, tetrasodium glutamate diacetate, trisodium ethylenediamine disuccinate; the preservatives include phenoxyethanol, methylisothiazolinone, methylparaben, ethylparaben, ethylhexylglycerin, sodium benzoate.

Most importantly, the composition may be present in the shampoo at a weight ratio of 0.1 to 99.0% or 1 to 55% or 1 to 32% or 1 to 25% or 1 to 15% or 1 to 10%.

Beneficial effects of the present invention:

The present invention relates to a secretome obtained by treating mesenchymal stem cells with Psoralidin, which exhibits obvious characteristics of black hair growth: higher tyrosinase activity activation rate; higher melanocyte proliferation promoting ability; stronger effect of promoting hair growth, and can be used as an ideal black hair growth active ingredient.

DETAILED DESCRIPTION

The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

Study 1: Effects of different concentrations of Psoralidin on the proliferation activity of umbilical cord mesenchymal stem cells

Human umbilical cord mesenchymal stem cells (hUCMSCs) in the logarithmic growth phase were obtained, digested with trypsin, and prepared into a cell suspension with a concentration of 5×10 ⁴ cells/mL using complete culture medium (human MSCs serum-free culture medium, purchased from Wuhan Punosai Company), and inoculated in a 96-well culture plate at 100 μL/well, with 6 duplicate wells set for each group. After the cells adhered to the wall, the old culture medium was discarded, and each group of the drug-treated group was added with 100 μL of complete culture medium containing different concentrations of Psoralidin and cultured for 24 hours, and the control group was added with 100 μL of 1% volume fraction of DMSO, and the culture medium was discarded, and 20 μL of MTT solution (5g/L) was added to each well, incubated in a 37°C incubator for 4 hours, and the culture medium containing MTT in the well was discarded, and 200 μL of DMSO was added to each well, and the mixture was slightly shaken for 5 minutes. The absorbance was detected by an enzyme marker (wavelength of 570 nm), and calculated according to the following formula:

Activity rate = absorbance of _{drug administration group} / absorbance _{of control group} × 100%

Table 1: Comparison of the effects of different concentrations of Psoralidin on the activity of hUCMSCs ( n＝6)

Psoralidin concentration (nmol/L) Activity rate (%) Control group 100.00±7.36 0.1 103.27±9.18 1 113.63±5.31 ^* 10 120.37±10.24 ^** 100 101.82±6.78 1×10 ³ 95.42±2.65 1×10 ⁴ 90.33±4.04

Note: Compared with the control group, ^* P＜0.05, ^** P＜0.01.

From the above results, it can be seen that when the concentration of Psoralidin is within the range of 0.1-10 nmol/L, it promotes the proliferation of hUCMSCs, and the peak value appears at 10 nmol/L, when the cell activity rate is as high as 120.37%, which is significantly promoted compared with the control group (without drug addition) (P<0.01). When the concentration of Psoralidin continues to increase to above 1×10 ³ nmol/L, it inhibits the proliferation of hUCMSCs, and the inhibitory effect becomes more obvious as the concentration of Psoralidin increases.

According to the above research results, the culture supernatants of the 1 nmol/L and 10 nmol/L groups were collected, centrifuged at a low speed of 1500 rpm/min for 10 min, and filtered through a 0.22 μm microporous filter membrane to obtain the hUCMSCs secretion groups treated with 1 nmol/L and 10 nmol/L concentrations of psoralen, and the following research was continued.

Study 2: Effects on melanocyte proliferation activity

Human melanocytes in the logarithmic growth phase were digested with trypsin and prepared into a cell suspension with a concentration of 5×10 ³ cells/ml using RPMI 1640 medium. The cells were inoculated in a 96-well plate at a rate of 100 μL/well per well. Six replicate wells were set up for each group. After culturing for 24 hours, the old culture medium was aspirated and discarded. 100 μL of the sample solution of each group was added to each well. After incubation for 24 hours, the old culture medium was aspirated and discarded. 20 μL of MTT solution (5 g/L) was added to each well. The cells were incubated in a 37°C incubator for 4 hours. The culture medium containing MTT in the wells was aspirated and discarded. 200 μL of DMSO was added to each well. The plates were gently shaken for 5 minutes. The absorbance was detected using an enzyme reader (wavelength of 570 nm) to calculate the activity rate.

The composition of each group of sample solutions: control group: 100 μL of 1% volume fraction DMSO was added; normal group: hUCMSCs secretion group obtained by culturing without adding psoralen (the preparation method refers to study 1, the only difference is that psoralen was not added during the culture process); 1nmol/L secretion group: hUCMSCs secretion group obtained by culturing with 1nmol/L psoralen; 10nmol/L secretion group: hUCMSCs secretion group obtained by culturing with 10nmol/L psoralen; control group: 10nmol/L psoralen aqueous solution.

Table 2: Comparison of effects on melanocyte proliferation activity ( n＝6)

Group Activity rate (%) Blank group 100.00±1.26 Normal group 109.27±3.18 ^* 1nmol/L secretion group 121.75±5.44 ^**# 10nmol/L secretion group 134.28±9.67 ^**## Control group 102.34±0.93

Note: Compared with the blank group, ^* P＜0.05, ^** P＜0.01; compared with the normal group, ^# P＜0.05, ^## P＜0.01.

The test results showed that the melanocyte proliferation activity of the 1 nmol/L secretion group and the 10 nmol/L secretion group was stronger, and the melanocyte activity rate of the 10 nmol/L secretion group reached 134.28%, which was significantly different from the normal group (P < 0.01). Surprisingly, the melanocyte activity rate of the melanocytes directly cultured with 10 nmol/L Psoralidin (control group) was not statistically significant compared with the blank group, which suggested that the hUCMSCs secretion group obtained by Psoralidin treatment may have produced more components that promote melanocyte proliferation.

Study 3. Determination of tyrosinase activity activation rate

The sample solutions were divided into groups as follows: according to the dosage in Table 3 below, the four solutions of C ₁ , C ₂ , T ₁ , and T ₂ were accurately drawn, kept in a water bath at 37°C for 10 minutes, 0.3 ml of tyrosinase solution was added to each solution, and after reacting for 5 minutes, the solution was quickly transferred to a cuvette, and the absorbance (OD value) was measured at 475. The activation rate of tyrosinase was calculated according to the following formula:

The composition of each group of sample solutions: normal group: hUCMSCs secretion group obtained by culturing without adding psoralen (preparation method refers to study 1); 1nmol/L secretion group: hUCMSCs secretion group obtained by culturing with 1nmol/L psoralen; 10nmol/L secretion group: hUCMSCs secretion group obtained by culturing with 10nmol/L psoralen.

Table 3: Reagent dosage table for each group

Table 4: Activation rate of tyrosinase

Group Tyrosinase activation rate (%) Normal group 96.37 1nmol/L secretion group 259.82 10nmol/L secretion group 363.41

As can be seen from Table 3, the hUCMSCs secretion group treated with 1 nmol/L and 10 nmol/L Posoralidin showed a higher tyrosinase activation rate, which was as high as 363.41%, which was about 3 times higher than that of the normal group.

Study 4: Effect on hair growth

Free hair follicles in the growth phase were taken and transferred into a 24-well culture plate, with one hair follicle in each well and three replicate wells in each group. William's E medium (Thermo Fisher Scientific (China) Co., Ltd., Catalog No.: A1217601) was added and cultured for 24 hours, then discarded by aspiration, and 200 μL of sample solution was added to each well, and the sample solutions were grouped as follows: blank group: no treatment; normal group: hUCMSCs secretion group obtained by culture without adding psoralen (preparation method refers to study 1); 1nmol/L secretion group: hUCMSCs secretion group obtained by culture with 1nmol/L psoralen; 10nmol/L secretion group: hUCMSCs secretion group obtained by culture with 10nmol/L psoralen; control group: 10nmol/L psoralen aqueous solution; after 10 days of culture, the relative length of hair growth was counted, and the results are shown in Table 4 below.

Relative hair growth length = hair length after 10 days of culture - original length

Table 4: Comparison of relative hair length ( n＝3)

Group Relative hair growth length (mm) Blank group 1.25 Normal group 1.57 1nmol/L secretion group 2.16 10nmol/L secretion group 2.54 Control group 1.49

As shown in Table 4, the hair growth rate of the 1 nmol/L and 10 nmol/L secretion groups was the fastest and the relative hair growth length was the highest, indicating that the secretion group obtained by adding psoralen contained more components that promoted hair growth, and the growth-promoting effect was stronger than that of directly adding psoralen.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims (8)

1. A tyrosinase activator, characterized in that it comprises a secretory group obtained by culturing mesenchymal stem cells in a culture medium supplemented with psoralen; in the culture medium, the concentration of psoralen is 1-10 nmol/L. 2. The activator according to claim 1, characterized in that the mesenchymal stem cells are cultured in a culture medium supplemented with psoralen for 12 to 36 hours. 3. Use of the activator according to claim 1 or 2 in the preparation of a hair darkening composition. 4. A hair darkening composition, characterized in that it comprises a secretome obtained by culturing mesenchymal stem cells in a culture medium supplemented with psoralen, wherein the concentration of psoralen in the culture medium is 1-10 nmol/L. 5. The composition according to claim 4, characterized in that the mesenchymal stem cells are cultured in a culture medium supplemented with psoralen for 12 to 36 hours. 6 . The composition according to claim 5 , wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells or adipose mesenchymal stem cells. 7. The composition according to any one of claims 4 to 6, characterized in that the secretome is prepared by the following steps: Obtaining mesenchymal stem cells that meet the requirements; culturing the mesenchymal stem cells for 12 to 36 hours using a culture medium supplemented with 1 to 10 nmol/L psoralen; collecting the culture fluid, centrifuging, filtering, and obtaining a secretory group. 8. A blackening hair shampoo, characterized in that it comprises the blackening hair composition according to any one of claims 4 to 7.