To provide a composition having hair-growing effect, gray hair-improving effect and wrinkle-improving effect, and to provide a method for producing the composition.

Abstract

To provide a composition having a hair-growing effect, a gray hair-improving effect and a wrinkle-improving effect, and to provide a method for producing the composition.SOLUTION: The composition having hair-growing effect, gray hair-improving effect and wrinkle-improving effect comprises a culture supernatant obtained by culturing human deciduous tooth dental pulp stem cells in a serum-free medium under ultrasonic stimulation and a culture supernatant obtained by culturing dermal papilla cells in a serum-free medium under ultrasonic stimulation. A method for producing a composition having a hair growth effect, a gray hair improving effect, and a wrinkle improving effect, comprising a culture supernatant obtained by culturing deciduous tooth dental pulp stem cells in a serum-free medium under ultrasonic irradiation, and a culture supernatant obtained by culturing dermal papilla cells in a serum-free medium under ultrasonic irradiation.SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] The present invention relates to a composition having hair growth effects, anti-gray hair effects, and anti-wrinkle effects, and a method for producing the same. [Background technology]

[0002] Hair loss and gray hair are two of the distinctive features of the elderly appearance. To address this image of the elderly from a cosmetic perspective, research into hair regrowth and gray hair is being conducted around the world.

[0003] A typical form of hair loss is androgenetic alopecia (AGA). AGA alopecia is a condition in which hair matrix cells become inactive due to insufficient blood flow to the hair papilla caused by aging and stress, resulting in a shortened hair cycle, smaller hair follicles, and thinner, shorter hair. Signs of AGA alopecia begin after puberty due to elevated androgen levels, and are estimated to occur in 80% of men and 40% of women by the age of 70. Although the causes of AGA alopecia are recognized as multifactorial, including genetics, androgens, as well as inflammation, stress, and environmental factors, the only internationally approved treatment for AGA alopecia is the topical and oral minoxidil, an androgen-independent hair growth promoter. If blood flow to the hair papilla can be improved, hair matrix cells can be activated, and the transition from anagen to catagen, which repeats the cycle of cell growth, catagen, and resting phase, can be delayed, slowing down the progression of hair loss. Furthermore, if the resting phase can be shortened, the number of hairs in the growth phase can be increased, and if the hair papilla is activated, the size of the hair can be increased, which should lead to the treatment of so-called thinning hair and baldness.

[0004] Hair loss can also occur as a side effect of chemotherapy for cancer treatment. Alopecia is a well-known result of chemotherapy administered as a conditioning regimen for bone marrow stem cell transplantation for leukemia. Hair follicles are composed of actively proliferating epithelial components, with cell proliferation particularly active in the hair matrix. Anticancer drugs act on actively proliferating cells, making hair in the anagen phase susceptible to damage. Hair shafts damaged by chemotherapy narrow, and the epithelium is destroyed. Hair loss occurs 1 to 3 weeks after the start of chemotherapy. Since more than 90% of scalp hair is typically in the anagen phase, many hairs are damaged and most fall out. Hair loss also occurs in beards, eyelashes, eyebrows, axillary hair, and pubic hair. Hair loss is usually temporary, and hair regrowth occurs 3 to 6 months after the end of chemotherapy. However, the texture and color of the regrowing hair differ from that of the hair before hair loss. The hair becomes curly and may even turn white. Other reports have shown that hair loss begins an average of 18.0 days after the start of chemotherapy, with hair regrowth observed an average of 3.4 months after the end of treatment. Several previous studies have examined the impact of chemotherapy-induced hair loss on quality of life, consistently ranking it among the top three most distressing reactions. According to a 2002 report by Carelle et al., hair loss was the second most common distress felt by cancer patients undergoing chemotherapy, after negative effects on family and spouse, and was the most common distress experienced during chemotherapy. All patients were informed by their doctors in advance that hair would regrow in 3 to 6 months. However, they confessed that hair loss began 1 to 2 months after the start of treatment, and that it was a shock beyond their imagination when massive hair loss occurred after 3 months.

[0005] While gray hair, which almost inevitably occurs with aging, is not necessarily a medical condition that should be avoided, it can be a cosmetic problem for some people (especially women). However, the mechanism behind gray hair development is still not fully understood. The hair matrix cells that produce hair do not contain pigment, and black hair is colored by absorbing melanin produced by hair pigment cells (melanocytes). However, if the melanocyte stem cells in the hair root stop producing pigment for some reason, hair turns gray. It is said that the average Japanese person has 100,000 hairs on their head. The hair that grows reaches the end of its lifespan within 3 to 5 years and falls out. After a preparatory period of several months, hair regrows. During this time, if pigment cells are activated and melanin is produced, the hair will become colored; if not, the hair will turn gray.

[0006] Although the causes are different, the mechanisms of hair loss and graying are nearly identical. While there are various causes of hair loss, the majority is due to the reduction or disappearance of hair matrix cells in the hair follicles at the base of the hair, while graying is caused by the deterioration of hair root melanocyte stem cells. While various hair growth agents have been proposed, none are truly effective, and many have side effects. Transplantation of hair matrix stem cells or dermal papilla cells has been investigated as one solution. However, cell therapy is too complicated to handle and has not produced the expected results (Non-Patent Document 1). Research into the treatment of wrinkles, a symptom of skin aging, is also being actively conducted. However, although attempts have been made to use drugs that activate epidermal cells or dermal fibroblasts, or even transplantation of the cells themselves, the effectiveness of these approaches has been limited (Non-Patent Document 2). To date, no satisfactory treatment has been developed in the fields of hair regeneration or aging skin.

[0007] Signaling molecules produced by a series of stem cells (hair root melanocyte stem cells and hair matrix stem cells) involved in normal hair formation and coloring regulate the hair cycle. The most important signals in hair formation are those from hair follicle papilla cells (HFPDCs), which are deeply involved in hair coloration and elongation. Dermal papilla cells are known as the control center for hair formation, and various signaling molecules (secretome) secreted by these cells regulate the proliferation and differentiation of hair follicle cells (Non-Patent Documents 3 and 4). These signaling molecules include IGF-1, HGF, VEGF, and FGF, but FGF7 (fibroblast growth factor 7) in particular is thought to be particularly important among the signals emitted by HFPDCs throughout the entire process of hair follicle development and growth. Stem Cell Human Exfoliated Deciduous Teeth (SHED) are a type of mesenchymal stem cell known to produce thousands of signaling molecules (cytokines and growth factors). Representative examples include growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), hepatocytokinase growth factor (HGF), transforming growth factor (TGF), and vascular endothelial growth factor (VEGF), as well as monocyte chemotactic protein-1 (MCP-1), sialic binding immunoglobulin-type lectins-9 (Siglec-9), and exosomes (Non-Patent Documents 5 and 6). These are known to have various biological effects, such as regulating the microenvironment for tissue regeneration, including angiogenesis, cell regeneration, and protection against apoptosis. Among these, HGF and Siglec-9 are thought to be particularly important for hair regeneration.

[0008] Patent Document 1 proposes a cosmetic product containing culture supernatant of porcine dental pulp stem cells that has wrinkle-reducing effects and hair growth and hair restoration effects (Patent Document 1), Patent Document 2 proposes a hair growth promoting sheet containing culture supernatant of human deciduous dental pulp stem cells (Patent Document 2), Patent Document 3 proposes a topical skin composition for hair restoration containing culture supernatant of mammalian dermal papilla cells (Patent Document 3), and in Patent Document 4 the inventor of the present application proposes a hair restoration agent containing culture supernatant of dental pulp stem cells and culture supernatant of dermal papilla cells (Patent Document 4). However, the effects of these products, including the hair restoration agent of the present inventor in Patent Document 4, are considered insufficient and have not yet become widespread. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2013 / 118877 [Patent Document 2] International Publication No. 2016 / 175164 [Patent Document 3] Japanese Patent Application Laid-Open No. 2003-146894 [Patent Document 4] Japanese Patent Publication No. 2020-164473 [Non-patent literature]

[0010] [Non-Patent Document 1] Anil Kumar Garg, Seema Garg: Complications of Hair Transplant Procedures-Causes and Management. Indian J. Plast. Surg., 2021 Dec 31;54(4):477-482. [Non-patent document 2] Kerure A. S, Patwardhan N., Complications in hair transplantation. J. Cutan Aesthet Surg. 2018;11(04):182-189. [Non-patent document 3] Itami, S. et al: Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth gactpr-1 from dermal papilla cells. Biochem. Biophys. Res. Commun., 212; 988-, 1995. [Non-patent document 4] Shimaoka, S. et al: Hepatocyte growth factor / scatter factor expressed in follicular papilla cells stimulates human hair growth in vitro. J. Cell Physiol. 165;333-, 1995 [Non-patent document 5] Matsubara, K., et al: Secreted ectodmain of sialic acid-binding Ig-like lectin and monocyte chemoattractant protein-1 promote recovery after rat spinal cord injury by altering macrophage polarity., J.Neuroscience 35(2015):2452-2464. [Non-patent document 6] Drago, D., et al: The stem cell secretome and its role in brain repair.,Biochimie 95.12(2013):2271-2285. Summary of the Invention [Problem to be solved by the invention]

[0011] The problem to be solved by the present invention is to provide a composition having hair growth effects, anti-graying effects, and anti-wrinkle effects, and a method for producing the same. [Means for solving the problem]

[0012] The inventors of the present application suspected that the insufficient effect of hair growth agents containing dental pulp stem cell culture supernatant and hair papilla cell culture supernatant was due to an insufficient secretome concentration in the culture supernatant. After extensive research into methods for increasing the secretome concentration in the culture supernatant, they came up with the idea of ​​applying ultrasound during dental pulp stem cell and hair papilla cell culture. After examining various ultrasound irradiation conditions during dental pulp stem cell and hair papilla cell culture, they discovered ultrasound irradiation conditions that can increase the secretome concentration in the culture supernatant. Furthermore, they discovered that compositions containing culture supernatants obtained by applying ultrasound under suitable ultrasound irradiation conditions during dental pulp stem cell and hair papilla cell culture have hair growth effects, anticancer drug-induced hair loss prevention effects, anti-cancer drug-induced hair loss prevention effects, and anti-wrinkle effects.

[0013] The first invention is a composition having a hair-growth effect, which contains a culture supernatant obtained by culturing human deciduous dental pulp stem cells in a serum-free medium and applying ultrasonic stimulation during the culture, wherein the ultrasonic irradiation conditions for the ultrasonic stimulation are an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition cycle of 1.0 kHz, an ultrasonic output of 50 to 150 mW / cm2, and an irradiation time of 30 minutes; and further, the composition contains a culture supernatant obtained by growing hair papilla cells in a medium containing basic fibroblast growth factor, culturing them in a serum-free medium, and applying ultrasonic stimulation during the culture, wherein the ultrasonic irradiation conditions for the ultrasonic stimulation are an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition cycle of 1.0 kHz, an ultrasonic output of 50 to 150 mW / cm2, and an irradiation time of 30 minutes.

[0014] The second invention is a composition having a hair-growth effect according to the first invention, which contains a culture supernatant obtained by culturing human deciduous dental pulp stem cells in a serum-free medium and applying ultrasonic stimulation during the culture, wherein the ultrasonic irradiation conditions for the ultrasonic stimulation are an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition cycle of 1.0 kHz, an ultrasonic output of 100 mW / cm2, and an irradiation time of 30 minutes; and further contains a culture supernatant obtained by growing hair papilla cells in a medium containing basic fibroblast growth factor, culturing them in a serum-free medium, and applying ultrasonic stimulation during the culture, wherein the ultrasonic irradiation conditions for the ultrasonic stimulation are an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition cycle of 1.0 kHz, an ultrasonic output of 100 mW / cm2, and an irradiation time of 30 minutes; and is characterized in that in addition to the hair-growth effect, the composition also has anti-canice effects and anti-wrinkle effects.

[0015] The third invention is a method for producing a composition having a hair-growth effect, which comprises culturing human deciduous dental pulp stem cells in a serum-free medium and applying ultrasonic stimulation during the culture, thereby obtaining a culture supernatant, and the ultrasonic irradiation conditions for the ultrasonic stimulation are an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition cycle of 1.0 kHz, an ultrasonic output of 50 to 150 mW / cm2, and an irradiation time of 30 minutes.Furthermore, the third invention is a method for producing a composition having a hair-growth effect, which comprises culturing human deciduous dental pulp stem cells in a serum-free medium, thereby obtaining a culture supernatant, and the ultrasonic irradiation conditions for the ultrasonic stimulation are an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition cycle of 1.0 kHz, an ultrasonic output of 50 to 150 mW / cm2, and an irradiation time of 30 minutes.

[0016] The fourth invention is a method for producing a composition having hair growth effects, anti-canity effects, and anti-wrinkle effects, which comprises culturing human deciduous dental pulp stem cells in a serum-free medium and adding a culture supernatant obtained by applying ultrasonic stimulation to the culture, wherein the ultrasonic irradiation conditions for the ultrasonic stimulation are an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition cycle of 1.0 kHz, an ultrasonic output of 100 mW / cm2, and an irradiation time of 30 minutes; and further comprising growing hair papilla cells in a medium containing basic fibroblast growth factor, culturing them in a serum-free medium, and adding a culture supernatant obtained by applying ultrasonic stimulation to the culture, wherein the ultrasonic irradiation conditions for the ultrasonic stimulation are an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition cycle of 1.0 kHz, an ultrasonic output of 100 mW / cm2, and an irradiation time of 30 minutes. [Effects of the Invention]

[0017] The composition of the present invention has hair growth effects, anti-gray hair effects, and anti-wrinkle effects. [Brief explanation of the drawings]

[0018] [Figure 1] 1 is a graph showing the relationship between the duration of ultrasound irradiation during culturing of stem cells derived from deciduous dental pulp and the content of HGF in the resulting culture supernatant. [Figure 2] 1 is a graph showing the relationship between the output of ultrasound irradiated during culture and the content of HGF, Siglec-9, or FGF7 in the resulting culture supernatant. [Figure 3] 1 is a graph showing the hair growth effect on patients with AGA alopecia of a composition containing a culture supernatant obtained by irradiating with ultrasound and a composition containing a culture supernatant obtained without irradiating with ultrasound. [Figure 4] These are photographs of the head of a patient (male, 38 years old) with AGA alopecia, taken before (left) and after (right) treatment, who was administered a composition made by mixing non-ultrasound-irradiated R-SHEDCM and R-HFCM. [Figure 5]These are photographs of the head of a patient (male, 50 years old) with AGA alopecia, taken before (left) and after (right) treatment, who was administered a composition made by mixing non-ultrasound-irradiated R-SHEDCM and R-HFCM. [Figure 6] These are photographs of the head of a patient (male, 60 years old) with AGA alopecia, taken before (left) and after (right) treatment, who was administered a composition made by mixing non-ultrasound-irradiated R-SHEDCM and R-HFCM. [Figure 7] These are photographs of the head of a patient (male, 38 years old) with AGA alopecia, taken before (left) and after (right) treatment, who was administered a composition made by mixing non-ultrasound-irradiated R-SHEDCM and R-HFCM. [Figure 8] These are photographs of the head of a patient with AGA alopecia (male, 48 years old) before (left) and after (right) treatment, who was administered a composition made by mixing non-ultrasound-irradiated R-SHEDCM and R-HFCM. [Figure 9] These are photographs of the head of a patient (male, 74 years old) with AGA alopecia who was administered US2, taken before (left) and after (right) treatment. [Figure 10] These are photographs of the head of a patient (male, 58 years old) with AGA alopecia who was administered US1, taken before (left) and after (right) treatment. [Figure 11] These are photographs of the head of a patient (45-year-old male) with AGA alopecia who was administered US3, taken before (left) and after (right) treatment. [Figure 12] 1 is a graph showing the improving effect on anticancer drug-induced alopecia of a composition containing a culture supernatant obtained by ultrasonic irradiation and a composition containing a culture supernatant obtained without ultrasonic irradiation. [Figure 13] 1 is a table showing the improvement results of anticancer drug-induced alopecia for compositions US1, US2, and US3 containing culture supernatants obtained by ultrasonic irradiation, or composition R containing culture supernatants obtained without ultrasonic irradiation. [Figure 14] 1 is a graph showing evaluation of hair color using a color difference meter. [Figure 15] These are photographs of the head of a 47-year-old man before (left) and after (right) treatment after US2 was administered. [Figure 16] These are microscopic images of the cheek and forehead texture of the control and treatment groups before and after 8 weeks of treatment. [Figure 17] This is a graph quantitatively showing skin texture using skin images taken with a digital microscope. [Figure 18] These are photographs of the area sprayed with US2 in a 34-year-old woman, taken before treatment (left) and 6 months after 8 weeks of treatment (right). [Figure 19] These are photographs of the area sprayed with US2 in a 75-year-old woman, taken before treatment (left) and after 8 weeks of treatment (right). [Figure 20] These are photographs of the area where US2 was sprayed on a 42-year-old woman before treatment (left) and 6 months after treatment (right). [Figure 21] These are photographs of the area sprayed with US2 in a 62-year-old woman, taken before treatment (left) and after 8 weeks of treatment (right). DETAILED DESCRIPTION OF THE INVENTION

[0019] Next, an embodiment of the present invention will be described, but it goes without saying that various changes and modifications can be made without departing from the technical scope of the present invention. [Example]

[0020] Cultivation of deciduous dental pulp-derived stem cells (SHED) and preparation of culture supernatant: (1)Cell culture Exfoliated primary teeth obtained from healthy children were disinfected with isodine solution, and the crowns were cut horizontally using a dental diamond point. Pulp tissue was then extracted using a dental reamer. The extracted pulp tissue was digested for 1 hour at 37°C in a solution of 3 mg / ml type I collagenase and 4 mg / ml dispase. After filtering through a 70 mm cell strainer (Falcon; BD Labware, Franklin Lakes, NJ), cells were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Rockville, MD) containing 20% ​​mesenchymal cell growth supplement (Lonza Inc., Walkersville, MD) and antibiotics (100 U / ml penicillin, 100 mg / ml streptomycin, and 0.25 mg / ml amphotericin B; GIBCO) at 37°C under 5% CO2. The culture supernatant was removed, and cells with adhesive properties were selected as dental pulp stem cells. Approximately 1 × 10 4 The cells were subcultured at 1 to 3 passages. The cells were used to prepare the culture supernatant. (2) Preparation of culture supernatant SHED(4×10 5 The cells were cultured in serum-free DMEM / F12 (Invitrogen-Gibco-BRL, Grand Island, NY) at 37°C under 5% CO2. During this time, ultrasonic stimulation was applied using an ultrasonic generator attached to the bottom of the culture dish. The ultrasonic irradiation conditions were: ultrasonic frequency 1.5 MHz, burst width 200 μsec, repetition period 1.0 kHz, ultrasonic output 50, 100, or 150 mW / cm 2 After 72 hours of culture, the SHED culture supernatant was collected, centrifuged at 300 × g for 5 minutes, and filtered using a 0.22 mm syringe filter. To examine the effect of ultrasonic stimulation, a control culture was also performed without ultrasonic irradiation, and the culture supernatant was similarly collected. Hereinafter, the culture supernatant obtained by ultrasonic irradiation will be referred to as US-SHEDCM, and the culture supernatant obtained by culturing without ultrasonic irradiation will be referred to as R-SHEDCM.

[0021] Dermal papilla cell culture and culture supernatant preparation: (1)Cell culture Human scalp skin was used as a by-product of a surgical operation. In light of the purpose of the present invention, it is desirable to use scalp skin derived from an adult male, but this is not limited thereto, and scalp skin derived from a child or a woman may also be used. The skin used may be not only head hair but also body hair, etc., as long as the hair papilla maintains normal physiological function. Generally, scalp skin derived from the temporal or occipital regions is preferred. Isolated dermal papilla cells for culture are prepared by cutting the scalp into strips approximately 5 mm thick, washing with phosphate-buffered saline (PBS), etc., and then, if necessary, using proteolytic enzymes or surgically treating the scalp to remove the epidermal and dermal layers, leaving only the subcutaneous fat layer. The hair follicles are then physically isolated, for example, using tweezers. The hair bulb is excised from the hair follicle, and the dermal papilla is exposed from below the hair bulb to isolate the dermal papilla (cells). The isolated dermal papilla cells thus obtained can be cultured (primary culture and subculture) in commercially available nutrient media used for culturing animal cells, either directly or with modifications. Typical media that can be used for culturing dermal papilla cells include Dulbecco's Modified Eagle Medium (available from GibcoBL) containing fetal bovine serum and Chang's medium (available from Irvine Scientific). The medium can further contain cell growth factors, hormones, and other micronutrients as needed. Specific examples of these include transferrin, insulin, triiodothyronine, glucagon, hydrocortisone, testosterone, estradiol, progesterone, and selenium. Furthermore, this culture method is characterized in that the culture is carried out in the presence of basic fibroblast growth factor (bFGF). bFGF may be derived from humans or other mammals, such as cows, mice, or rats, or may be recombinant. The concentration of bFGF to be present in the culture medium is not particularly limited, but is, for example, 0.01 ng / ml to 10 μg / ml, preferably 0.1 ng / ml to 100 ng / ml, and more preferably about 10 ng / ml. Isolated dermal papilla cells are typically cultured in these media in a culture dish in an incubator at 37°C under a 5% CO2 atmosphere. Once outgrowth is confirmed, the medium (primary culture) is replaced and the culture is continued (subculture). The cultured cells obtained in this manner can be subcultured for the required number of passages. Subculture can be continued until the required number of papilla cells is reached; for example, 10 or more passages, or 15 or more, or even 20 or more passages if a larger amount is required. (2) Preparation of dermal papilla cell culture supernatant The cells collected by the above method are cultured in an incubator for adherent cell culture, and colonies of cells showing adherence are subcultured to select dermal papilla cells (HFDPCs). Alternatively, dermal papilla cells may be selected based on cell size and morphology. The selected dermal papilla cells (HFDPCs) (4 x 10 5 Cells) were cultured in serum-free DMEM / F12 (Invitrogen-Gibco-BRL, Grand Island, NY) at 37°C under 5% CO2. During this time, ultrasonic stimulation was applied using an ultrasonic generator attached to the bottom of the culture dish. The ultrasonic irradiation conditions were an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition rate of 1.0 kHz, and an ultrasonic output of 50, 100, or 150 mW / cm. 2After 72 hours of culture, the culture supernatant was collected, centrifuged at 300×g for 5 minutes, and filtered using a 0.22 mm syringe filter. To examine the effect of ultrasonic stimulation, a control culture was also carried out without ultrasonic irradiation, and the culture supernatant was similarly collected. Hereinafter, the culture supernatant obtained by ultrasonic irradiation will be referred to as US-HFCM, and the culture supernatant obtained by culturing without ultrasonic irradiation will be referred to as R-HFCM.

[0022] Preparation of the composition: 1 ml of the US-SHEDCM stock solution and 1 ml of the US-HFCM stock solution were mixed with 48 ml of purified water to obtain 50 ml of a composition (hereafter referred to as US). As a control, 1 ml of the R-SHEDCM stock solution and 1 ml of the R-HFCM stock solution were mixed with 48 ml of purified water to obtain 50 ml of a control composition (hereafter referred to as R). [Example]

[0023] US-SHEDCM and R-SHEDCM were obtained using the method described in paragraph 20. However, the ultrasonic irradiation time during culture was varied to 30, 60, 90, or 2,880 minutes. HGF content was measured using a microarray method. Figure 1 shows the HGF content in the culture supernatant after ultrasonic irradiation for 0 minutes (R-SHEDCM), 30 minutes, 60 minutes, 90 minutes, and 2,880 minutes. HGF content reached its maximum after 30 minutes of ultrasonic irradiation and did not increase further with continued ultrasonic irradiation. Because ultrasonic irradiation can potentially damage cells, a shorter production time is desirable from the standpoint of cost and risk reduction. We concluded that 30 minutes of ultrasonic irradiation was optimal. [Example]

[0024] The HGF and Siglec-9 contents of US-SHEDCM and R-SHEDCM prepared by the method described in Example 1, and the fibroblast growth factor 7 (FGF7) contents of US-HFCM and R-HFCM were measured and compared. The HGF and Siglec-9 contents were measured by microarray analysis, and the FGF7 content was measured by ELISA. For US-SHEDCM and US-HFCM, ultrasound was irradiated at an ultrasound output of 50, 100, or 150 mW / cm. 2 The results of HGF, Siglec-9, and FGF7 were measured for each of the samples. Figure 2 (a) shows the results for HGF content, (b) for Siglec-9 content, and (c) for FGF7 content. The contents of HGF, Siglec-9, and FGF7 were all higher in the culture supernatants irradiated with ultrasound than in those not irradiated with ultrasound. Among the culture supernatants irradiated with ultrasound, the culture supernatant irradiated with an ultrasound output of 100mW / cm2 had the highest content, followed by the culture supernatant irradiated with an ultrasound output of 150mW / cm2, and the culture supernatant irradiated with an ultrasound output of 50mW / cm2 had the lowest content. [Example]

[0025] Effectiveness for AGA hair loss: The composition of the present invention was administered to patients with AGA alopecia, and the hair growth effect was investigated. US and R described in paragraph 0022 were used. For US, the ultrasonic output was set at 50 mW / cm. 2 The resulting mixture of US-SHEDCM and US-HFCM (referred to as US1) was subjected to ultrasonic power of 100 mW / cm 2 The mixed composition of US-SHEDCM and US-HFCM obtained as above (referred to as US2) and the ultrasonic output was 150 mW / cm 2 The mixed composition of US-SHEDCM and US-HFCM obtained as above (referred to as US3) was used. The efficacy study was conducted as an open-label, prospective observational study. Participants were adult men and women aged 18 to 75 years who had been diagnosed with androgenetic alopecia (AGA). Clinical severity ranged from mild to severe before treatment. Participants were prohibited from using oral or topical hair growth products for four weeks prior to the start of the study and throughout the study. They were also instructed not to use any hair treatments that could affect hair loss, and to maintain their usual hair hygiene habits, diet, exercise habits, and contraceptive methods. The use of anti-inflammatory drugs, antihistamines, immunosuppressive therapy, and retinoids was also prohibited during the study. The 25 subjects consisted of 14 men and 11 women, with an average age of 55.8 years (38-75 years). Among the AGA patients, 15 patients in the treatment group (9 men, 6 women) received US by applying it to the scalp twice daily for 8 weeks. The amount of US administered per treatment was 1 to 2 ml depending on the area of ​​the treatment site and the degree of hair loss. The control group of 10 patients (5 men, 5 women) received the same amount of R.

[0026] The state of hair regrowth was scored according to the following criteria: Marked improvement (5 points): Dense hair growth (almost complete coverage of the thinning area. Hair density on the scalp is almost equal to that of the non-thinning area) Moderate improvement (4 points): Moderate hair growth (thinning areas are partially covered by new hair growth, but it is less dense and easily visible than non-thinning areas) Mild improvement (3 points): slight hair growth (clear hair growth, but not enough to cover the thinning area) No change (2 points): No visible hair growth Worsening (1 point): Recession of hair growth

[0027] Of the 15 patients in the treatment group, 13 showed significant improvement (8 men and 5 women). All of these patients received US2. Two patients (1 man and 1 woman) showed moderate improvement (US1) or mild improvement (US3). The average hair improvement score for the treatment group was 4.8 points. All 10 patients in the control group showed mild improvement or no change, with an average hair improvement score of 2.6 points. A significant difference was observed between the two groups at a risk level of 1% (Figure 3). Figures 4 to 11 show representative photographs of each case. The left side of the representative case is a photograph before treatment, and the right side is a photograph after 8 weeks of treatment. Figures 4 to 8 show the control group administered a composition containing a mixture of R-SHEDCM and R-HFCM without ultrasound irradiation. In both cases, post-treatment improvement was either similar to that before treatment or only mild. Figures 9 to 11 show the treatment group administered a composition containing a mixture of US-SHEDCM and US-HFCM with ultrasound irradiation. Significant improvement was observed in both cases. As shown above, the composition according to the present invention exhibited a high hair growth effect. [Example]

[0028] Effectiveness for chemotherapy-induced hair loss: The composition of the present invention was applied at least several days after the start of anticancer drug administration, and administration was continued for 8 weeks. The subjects consisted of 20 subjects, 10 males and 10 females. The average age of the subjects was 61.8 years (range, 25-79 years). The cancer types were leukemia (15 subjects), breast cancer (4 subjects), and ovarian cancer (1 subject). The cancer stages were stage II (10 subjects), stage III (5 subjects), and stage IV (5 subjects). The anticancer drugs used were alkylating agents (CPA, CDDP, CBDCA) and antimetabolite (5-FU), and concomitant medications were taxol, bleomycin, and cisplatin. Fifteen subjects in the treatment group received US1, US2, or US3 (described in paragraph 0022) by applying it to the scalp twice daily for eight weeks. The dose per application was 1 to 2 ml depending on the area of ​​the treatment site and the degree of hair loss. Five subjects in the control group received the same amount of R.

[0029] The results of the treatment were obtained by interviewing the participants themselves. Of the 15 people in the treatment group, 6 reported a significant improvement, 9 reported a moderate improvement, and the average hair improvement score was 4.4 points. On the other hand, all 5 people in the control group reported no change, with an average hair improvement score of 2 points. A significant difference was observed between the two groups at a risk level of 1% (Figure 12). Figure 13 shows the results of improving anticancer drug-induced alopecia using compositions US1, US2, or US3 containing culture supernatant obtained with ultrasound irradiation, and composition R containing culture supernatant obtained without ultrasound irradiation. Four of the five subjects administered US2 showed a significant improvement, and one showed a moderate improvement. Of the five subjects administered US1 or US3, one showed a significant improvement, and four showed a moderate improvement. Meanwhile, none of the five subjects in the control group administered R showed any change. These results indicate that culture supernatant R without ultrasound irradiation showed no improvement in anticancer drug-induced alopecia, whereas culture supernatants US1, US2, and US3 irradiated with ultrasound showed a significant improvement. [Example]

[0030] Gray hair improvement effect: The study involved 17 subjects, all male. Ten subjects in the treatment group received US2 (described in paragraph 0023) by applying it to the scalp twice daily for eight weeks. The dose per application was 1 to 2 ml depending on the area of ​​the treatment site. Seven subjects in the control group received the same amount of R.

[0031] The degree of improvement in gray hair was evaluated by measuring hair color with a color difference meter and by observation by a dermatologist and a hair diagnostician for 6 months after use of the composition.

[0032] Objective evaluation using a colorimeter confirmed significant darkening starting one month after use, with further darkening confirmed after three and six months. Figure 14 shows colorimeter measurements of the subject's hair before treatment and six months after the end of US2 or R treatment. Compared to pretreatment, the subjects' hair darkened in both R and US2 treatments, but the subject's hair after US2 treatment was the darkest. Significant differences were observed at a 0.1% risk level between the subject's hair darkness before treatment and the subject's hair darkness after R treatment, and between the subject's hair darkness after R treatment and the subject's hair darkness after US2 treatment. Furthermore, observational evaluation by a dermatologist and a hair diagnostician confirmed improvement in 33.3% of subjects after three months of use and 63.0% after six months. Figure 15 shows photographs of a 47-year-old man's head before treatment (left) and six months after treatment (right). In this case, it was confirmed that approximately 3 cm from the root had turned black. This demonstrates that US2 is significantly more effective at preventing gray hair than R. [Example]

[0033] Wrinkle improvement effect: The subjects consisted of 20 women. The subjects' ages ranged from 29 to 60, with an average age of 48. Ten subjects in the treatment group were administered the composition described in paragraph 0022, US1, US2, or US3, by spraying it onto the skin surface within 30 minutes of bathing each day, after removing all makeup, and spreading it over the entire face with the palm of their hand. After the composition had dried (approximately 15 minutes later), a moisturizing cream was applied. Ten subjects in the control group were administered R in the same manner. Administration of the composition continued for 8 weeks.

[0034] To evaluate the degree of wrinkle improvement, images of the same area of ​​the face were taken using a digital microscope (DINO-LITE PRO POLARIZER, Sanko Co., Ltd., Tokyo). Measurements were taken at three locations: the forehead, cheeks, and chin. The degree of wrinkle improvement was subjectively categorized into three levels (good, medium, poor). When taking the images, makeup was removed only from the areas near the measurement locations.

[0035] The visual impression of human skin varies from person to person and is determined by factors such as the contours and color of the skin surface. The skin surface has relatively large contours formed by skin grooves and relatively small contours formed by skin ridges, but fine wrinkles and texture are important from a cosmetic standpoint because they affect the appearance of the skin. These characteristics vary not only between individuals, but also with the environment surrounding the skin, such as the season, aging, and changes in physical condition. While changes in skin are often obvious to the human eye, the evaluation is subjective and subject to significant individual variation, making methods for appropriately assessing skin condition extremely limited. Figure 16 shows images of the cheek and forehead texture of the control group (R group) and treatment group (US group) taken before and after 8 weeks of treatment using a microscope with excellent reproducibility and ease of use. The quality of the texture can be visually confirmed. Figure 17 shows a graph quantitatively representing the skin texture before and after 8 weeks of treatment using skin images taken with a digital microscope. Compared to before treatment, the average brightness increased in both the control group (R group) and treatment group (US group), but the treatment group had the highest brightness, and a significant difference from the control group was observed at a risk level of 1% (Figure 17a). Furthermore, when comparing the effects of US1, US2, and US3, US2 had the greatest effect on increasing average brightness, and a significant difference from the effects of US1 and US2 was observed at a risk level of 1% (Figure 17b).

[0036] 18 to 21 show photographs of the areas sprayed with US2 for each case before administration (pre-treatment) and after 8 weeks of US2 administration (post-treatment). Significant improvement was observed in both cases. As shown above, the composition of the present invention demonstrated a high wrinkle improvement effect. [Industrial Applicability]

[0037] The composition of the present invention contributes to the creation of cosmetics having hair growth agents, agents for preventing gray hair, and anti-wrinkle effects.

Claims

1. A composition having a hair growth effect, The present invention comprises a culture supernatant obtained by culturing human deciduous dental pulp stem cells in a serum-free medium and applying ultrasonic stimulation during the culture, The ultrasound irradiation conditions for the ultrasound stimulation were an ultrasound frequency of 1.5 MHz, a burst width of 200 μsec, a repetition period of 1.0 kHz, an ultrasound output of 50 to 150 mW / cm, and an irradiation time of 30 minutes. The present invention further comprises a culture supernatant obtained by growing hair papilla cells in a medium containing basic fibroblast growth factor, culturing the cells in a serum-free medium, and applying ultrasonic stimulation during the culture, The ultrasonic irradiation conditions for the ultrasonic stimulation are an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition period of 1.0 kHz, an ultrasonic output of 50 to 150 mW / cm2, and an irradiation time of 30 minutes. A composition having a hair growth effect characterized by:

2. The composition having a hair growth effect according to claim 1, The present invention comprises a culture supernatant obtained by culturing human deciduous dental pulp stem cells in a serum-free medium and applying ultrasonic stimulation during the culture, The ultrasound irradiation conditions for the ultrasound stimulation were an ultrasound frequency of 1.5 MHz, a burst width of 200 μsec, a repetition period of 1.0 kHz, an ultrasound output of 100 mW / cm, and an irradiation time of 30 minutes. The present invention further comprises a culture supernatant obtained by growing hair papilla cells in a medium containing basic fibroblast growth factor, culturing them in a serum-free medium, and applying ultrasonic stimulation during the culture, wherein the ultrasonic stimulation is performed under ultrasonic irradiation conditions of an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition cycle of 1.0 kHz, an ultrasonic output of 100 mW / cm2, and an irradiation time of 30 minutes; In addition to hair growth effects, it also has anti-gray hair and anti-wrinkle effects. A composition characterized by:

3. A method for producing a composition having a hair growth effect, comprising: Human deciduous dental pulp stem cells are cultured in a serum-free medium, and a culture supernatant obtained by applying ultrasonic stimulation during the culture is added to the medium. The ultrasound irradiation conditions for the ultrasound stimulation were an ultrasound frequency of 1.5 MHz, a burst width of 200 μsec, a repetition period of 1.0 kHz, an ultrasound output of 50 to 150 mW / cm, and an irradiation time of 30 minutes. Furthermore, the method comprises growing hair papilla cells in a medium containing basic fibroblast growth factor, culturing the cells in a serum-free medium, and then adding a culture supernatant obtained by applying ultrasonic stimulation during the culture; The ultrasonic irradiation conditions for the ultrasonic stimulation are an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition period of 1.0 kHz, an ultrasonic output of 50 to 150 mW / cm2, and an irradiation time of 30 minutes. A method for producing a composition having a hair growth effect, characterized by:

4. A method for producing a composition having a hair growth effect, a gray hair prevention effect, and a wrinkle prevention effect, comprising: Human deciduous dental pulp stem cells are cultured in a serum-free medium, and a culture supernatant obtained by applying ultrasonic stimulation during the culture is added to the medium. The ultrasound irradiation conditions for the ultrasound stimulation were an ultrasound frequency of 1.5 MHz, a burst width of 200 μsec, a repetition period of 1.0 kHz, an ultrasound output of 100 mW / cm, and an irradiation time of 30 minutes. Furthermore, the method comprises growing hair papilla cells in a medium containing basic fibroblast growth factor, culturing the cells in a serum-free medium, and then adding a culture supernatant obtained by applying ultrasonic stimulation during the culture; The ultrasonic irradiation conditions for the ultrasonic stimulation were an ultrasonic frequency of 1.5 MHz, a burst width of 200 μsec, a repetition period of 1.0 kHz, an ultrasonic output of 100 mW / cm2, and an irradiation time of 30 minutes. A method for producing a composition having hair growth effects, anti-gray hair effects, and anti-wrinkle effects, characterized by:

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