JP2003192541A - Hair growth-promoting agent and skin care preparation for hair growth - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a skin care preparation (medicine or cosmetic) containing a hair growth-promoting agent which has high safety, excellent usability, and a remarkable hair-growing effect. <P>SOLUTION: This skin care preparation (cosmetic or medicine) which is used for growing hair, has high safety, excellent usability, and a remarkable hair growth-promoting effect, and is useful for preventing and improving baldness, thin hair and fallen hair, contains royal jelly or a glycoprotein obtained from the royal jelly and having a mol.wt. of 57 kilo Dalton. The royal jelly has a vascular endothelial growth factor production-promoting activity (VEGF-A, VEGF-165, VEGF-B, VEGF-C, VEGF-D, VEGF-E), a fibroblast growth factor (FGF-1: aFGF, FGF-3, FGF-2: bFGF, FGF-3: int-2, FGF-4: hst-1/kaposi-FGF, FGF-5, FGF-6: hst-2, FGF-7, FGF-8, FGF-9, FGF-10) production-promoting activity, an endothelial cell proliferation-promoting activity, and a lumen formation- promoting activity. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hair growth-promoting agent and a skin external preparation for hair growth, the mechanism of action of which is to promote vascular endothelial growth factor (VEGF) production promoting activity and fibroblast growth factor (FGF) production by royal jelly. It relates to that which is judged by the promoting activity, the endothelial cell mitogenic activity and / or the fistula formation promoting activity.

[0002]

2. Description of the Related Art It is a desire for men and women of all ages to maintain abundant black hair forever. However, aging and genetic predisposition,
Furthermore, due to social stress, etc., hair gradually falls off, causing thinning hair and baldness. Hair loss can be broadly divided into physiological natural hair loss and pathological abnormal hair loss. Natural hair removal is about 50 to 80 per day, and
Abnormal hair loss over 100 hairs is caused by stress accumulation, excessive use of nerves, insufficient maintenance of the scalp, lack of blood circulation in the scalp, disturbance of hormone balance, excessive intake of animal food, nutritional deficiency, and dietary balance. Has been done. In order to prevent such alopecia, various manufacturers have worked harder to develop hair regrowth agents. Examples of herbicide such as minoxidil, hawthorn, ginkgo and ginger extract, pantothenyl ether, alloxazine, adenosine 3 ', 5'-cyclic monophosphate (c-AMP), etc. There is.

However, the hair-growth agents that have been developed so far may be insufficient in promoting hair growth and may cause side effects such as skin irritation, so that they are sufficiently effective and safe at present. The current situation was that nothing was obtained. Therefore, there has been a demand for the development of a hair-growth promoter and a hair-growth agent which have an excellent effect of promoting hair growth and are highly safe.

On the other hand, the hair cycle is composed of three stages. The period during which hair is firmly grown is the growth period (Anagen), the period during which growth stops and the hair is regressed (Catagen), and the hair cycle. There is no hair in the capsule and the period when hair growth is stopped is called telogen. In the early stages of growth (Early Anagen), the hair bulb is closest to the epidermis, and at the peak it is farthest away. Usually, for hair, the growth period is several years, the regression period is 2-3 weeks, and the rest period is several months.
It is said that 5% is a growth period and 14% is a rest period. At the lower end of the hair root, there is a bulb-shaped bulge, which is an important part for hair generation. The bottom of the hair bulb is concave, and the papilla composed of mesenchymal cells is localized, and capillaries are stretched. From these capillaries, nutrients and oxygen necessary for hair growth are supplied to hair mother cells, and the hair mother cells proliferate and differentiate, resulting in hair growth. Thus, for the growth of hair, formation of capillary cavities is necessary for supplementing nutrients and oxygen.

Furthermore, VEGF secreted by cancer cells and the like
(Vascular Endothelial Growth Factor) and bFGF (basic fibroblast growth factor)
(r; basic fibroblast growth factor) directly acts on the membrane of vascular endothelial cells, weakens the vascular wall to allow the formation of new capillary lumens, divides cells, It moves adjacent tissue aside to secure space for blood vessels, moving new cells and shaping them into the shape of blood vessels. Up to now, as a lumen formation promoting factor, pathological levels of high-concentration glucose, insulin-like growth factor (IGF-1), insulin, leukotriene C
4, prostaglandin E2 and the like are known.

On the other hand, a protein having a molecular weight of 57 kilodalton in royal jelly promotes the production of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) in cultured hair papilla cells, and has an endothelial mitogenic activity. It is not known to promote lumen formation that leads to angiogenesis, and furthermore, royal jelly containing a protein with a molecular weight of 57 kilodaltons promotes hair growth by the above-mentioned mechanism, which results in It is also not known that a skin external preparation containing the same is a highly safe skin external composition for hair growth, which is highly effective in promoting hair growth.

[0007]

The present invention has been made under such circumstances, and provides a hair growth promoter and a skin external preparation for hair growth, which are highly safe, have excellent usability, and have a remarkable hair growth effect. The challenge is to provide.

[0008]

[Means for Solving the Problems] In view of such a situation, the present inventors have conducted diligent research efforts, and as a result, a glycoprotein having a molecular weight of 57 kilodaltons in royal jelly was found to be a vascular endothelial growth factor in vascular endothelial cell culture cells. VEGF) is promoted and endothelial cell mitogenic activity is promoted, thereby promoting lumen formation leading to angiogenesis and hair growth. By adding this to pharmaceuticals and cosmetics, excellent external skin for hair growth They found that they could provide an agent and completed the invention. That is, the present invention relates to the following technology. (1) A hair growth-promoting agent comprising royal jelly. (2) The royal jelly contains 9% by weight or more of protein shown below,
The hair growth-promoting agent according to (1). 1> Form a single band on non-denaturing polyacrylamide gel electrophoresis of proteins in royal jelly. 2> The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodaltons. 3> Includes the amino acid sequences of amino acid numbers 1 to 8 of Sequence Formula 1. (3) The hair-growth promoting agent according to (1) or (2), wherein the hair-growth promoting is based on the vascular endothelial growth factor (VEGF) production promoting activity of the 57 kilodalton protein. (4) Vascular endothelial growth factor (VEGF) is VEGF-A, VEGF16
5, one or more selected from VEGF-B, VEGF-C, VEGF-D, VEGF-E, (1) ~
The hair growth-promoting agent according to any one of (3). (5) The hair-growth promoter according to any one of (1) to (4), wherein the hair-growth promotion is due to a fibroblast growth factor (FGF) production-promoting activity by a 57-kilodalton protein. . (6) Fibroblast growth factor (FGF) is FGF-1 (acidic FGF: aFGF), FGF-3, FGF-2 (basic FGF: bFGF), FG
F-3 (int-2), FGF-4 (hst-1 / kaposi- FGF), FGF-5, FGF-6
(hst-2), FGF-7, FGF-8, FGF-9, FGF-10, which is one or more selected from (1) to
The hair growth-promoting agent according to any one of (5). (7) The hair-growth promoting agent according to any one of (1) to (6), wherein the hair-growth promoting is based on the endothelial cell division promoting activity of the 57-kilodalton protein. (8) The hair-growth promoting agent according to any one of (1) to (7), wherein the hair-growth promoting is based on the lumen formation promoting activity of the 57-kilodalton protein. (9) A skin external preparation for hair growth, containing the hair growth promoter according to any one of (1) to (8). (10) The skin external preparation for hair growth according to (9), which is a pharmaceutical product or a cosmetic product. Hereinafter, the present invention will be described focusing on the embodiments.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Qualitative Method of 57-kilodalton Protein in Royal Jelly by Electrophoresis The 57-kilodalton protein in royal jelly of the present invention was analyzed by electrophoresis for protein composition and Characterizing the molecular weight of a protein. The method of electrophoresis is not particularly limited as long as the effective protein can be specified, but a preferable method is to use a water-soluble royal jelly protein (3% royal jelly aqueous solution (W / V)) on a polyacrylamide gel (1).
It is a method of electrophoresing at a current of 20 mA at 0% homogeneity) and staining with Coomassie Brilliant Blue to identify the protein. The molecular weight of the protein of the present invention in such electrophoresis was determined to be 57 kilodaltons by SDS-polyacrylamide gel electrophoresis of the purified protein.

(2) Method for preparing 57-kilodalton protein in royal jelly 0.7% by weight of lyophilized royal jelly was added to 10 mM.
Dissolved in Tris-HCl buffer (pH 7.0) of UF10
10,000 times (Miniplate 100; ultrafiltration), concentrated 6 times, desalted 7 times, and the filtrate was further subjected to UF30,000 (Mi
The product was concentrated 8 times by niplate 30; ultrafiltration) and desalted once to obtain a fraction having a molecular weight of 100,000 to 30,000. The sample having a molecular weight of 100,000 to 30,000 can be separated by anion exchange chromatography and gel filtration chromatography to separate a protein having a molecular weight of 57 kilodaltons. The anion exchange chromatography may be performed according to a commonly known method, for example, using DEAE-Toyopearl 650M manufactured by Tosoh Corporation as a column and using 20 mM Tris-HCl buffer (pH
7.0) as the developing solution A, 20 mM Tris-HCl buffer (pH
7.0) and 1 M NaCl as a developing solution B, the flow rate was detected at an absorbance of 5 ml / min and 280 nm with a gradient, and the fraction No. fraction was 2.5 ml / tube. A protein fraction having a molecular weight of 57 kilodaltons can be detected at 119 to 127. Further, this fraction may be subjected to gel filtration chromatography according to a commonly known method. As such a preferable example, for example, HiLoa manufactured by Pharmacia Co., Ltd.
Using d16 / 60 Superdex200 as a column, a 0.15 M sodium chloride-containing 50 mM potassium phosphate buffer pH 7.0 was used as a developing solution, and the flow rate was 1.
Fraction N was set to 0 ml / min, detected by absorbance at 280 nm, and fractionated at 2.0 ml / tube
o. A protein having a molecular weight of 57 kilodaltons can be detected at 35 to 43. (The same protein was confirmed by electrophoresis.) From the result of gel filtration analysis of known molecular weight, it was confirmed that the above protein was a 57 kilodalton molecular weight protein. Also, this protein is
The present inventor has found that it is a glycoprotein because it is digested with N-glucosidase F and its molecular weight after digestion becomes 48 kilodaltons.

(3) Examination of VEGF production promotion by glycoprotein having a molecular weight of 57 kilodaltons: VEGF production promotion from human hair papilla cells The glycoprotein having a molecular weight of 57 kilodaltons is a smooth muscle cell (aortic smooth muscle cell, Bronchi / tracheal smooth muscle cells, coronary artery smooth muscle cells, pulmonary artery smooth muscle cells, umbilical artery smooth muscle cells, uterine smooth muscle cells, etc., epithelial cells (mammary gland epithelial cells,
VEGF (VEGF-from renal cortical epithelial cells, bronchial / tracheal epithelial cells, prostate epithelial cells, renal proximal tubular epithelial cells, alveolar epithelial cells, small airway epithelial cells, etc.), macrophages, hepatocytes, dermal papilla cells, etc. A, VEGF165, VEGF-B, VEGF
-C, VEGF-D, VEGF-E) production is significantly promoted. Here, human hair hair papilla cells (THPC-001) total kit (HDPC total kit: THPCK-00)
1. Manufacturer: Cell Applications Inc. USA, import distributor: Toyobo Co., Ltd.) was used to cultivate human hair papilla cells by a conventional method, and the activity of promoting VEGF production by a glycoprotein having a molecular weight of 57 kilodalton was examined. . PCGM medium for suspending the thawed cells is dispensed into 10 mL and 15 mL centrifuge tubes, and ice-cooled. That is, a vial containing frozen human hair papilla cells (THPC-001) is rapidly thawed in a constant temperature bath at 37 ° C. About 1 mL of PCGM medium is gradually dropped into this vial to dilute DMSO, and then the whole is transferred to a centrifuge tube containing PCGM medium and suspended. Suspended cells are chilled in a low centrifuge at 4 ° C for 100
Centrifuge at 0 rpm for 5 minutes. Aspirate the supernatant, being careful not to suck up the precipitated cells, and resuspend in 1 mL PCGM medium. This whole amount was placed in a T-75 flask coated with a collagen solution, and 5% CO2 and 3 were added under humidification.
Place in an incubator kept at 7 ° C and perform static culture. After 1 day, the medium is replaced. Thereafter, the medium is replaced every other day and subcultured. The PCGM medium component is 250 mL of PCGM basal medium containing 1% FBS.
2.5m of 100 times diluted pituitary gland extract (BPE)
L, 2.5m of 100-fold diluted fetal calf serum (FCS)
L, 1.25 mL of insulin transferrin triiodothyronine solution (ITT) 200 times diluted,
Siloprotein solution (Cyp) 200 times diluted solution 1.
The one to which 25 mL was added was used. In addition, the addition and non-addition system of glycoprotein of molecular weight 57 kilodalton
The F production promotion was examined.

(4) Examination of FGF production promotion by glycoprotein of molecular weight 57 kilodalton: FGF production promotion from human hair hair papilla cells The glycoprotein of molecular weight 57 kilodalton is an endothelial cell (aortic endothelial cell, coronary artery). Endothelial cells, iliac endothelial cells,
Skin microvascular endothelial cells, pulmonary microvascular endothelial cells, pulmonary artery endothelial cells, umbilical artery endothelial cells, umbilical vein endothelial cells, myometrial microvascular endothelial cells, etc., epithelial cells (mammary epithelial cells,
Renal cortical epithelial cells, bronchial / tracheal epithelial cells, prostatic epithelial cells, renal proximal tubular epithelial cells, alveolar epithelial cells, small airway epithelial cells, etc.), fibroblasts (skin fibroblasts, lung fibroblasts etc.) , Smooth muscle cells (aortic smooth muscle cells, bronchi /
Tracheal smooth muscle cells, coronary artery smooth muscle cells, pulmonary artery smooth muscle cells, umbilical artery smooth muscle cells, uterine smooth muscle cells, etc.), skeletal muscle cells, macrophages, astrocytes, epidermal keratinocytes, epidermal melanocytes, mesangial cells, FGF (FGF-1 (acidic FGF: aFGF), FGF-3, FGF-2) from hepatocytes, dermal papilla cells (mesenchymal cells), various tumor cells (cancer cells), etc.
(Basic FGF: bFGF), FGF-3 (int-2), FGF-4 (hst-1 / kapo
si- FGF), FGF-5, FGF-6 (hst-2), FGF-7, FGF-8, FGF-
9, FGF-10) production is significantly promoted. Here, human hair papilla cells (THPC-001) total kit (H
DPC Total Kit: THPCK-001, Manufacturer:
Cell application cells, USA, import distributor: Toyobo Co., Ltd.) was used to cultivate human hair papilla cells by a conventional method, and a FGF production promoting activity was observed by a glycoprotein addition system and a non-addition system with a molecular weight of 57 kilodaltons. Study was carried out.

(5) Method for measuring vascular endothelial growth factor (VEGF) The vascular endothelial growth factor (VEGF) in the culture supernatant of human hair and hair papilla cells (THPC-001) was analyzed by Genzyme Human VEGF Immunoassay Kit (Genzyme TECHNE, AN'ALYZATM, Immunoassay
System, human VEGF).

(6) Method of measuring fibroblast growth factor (FGF) Fibroblast growth factor (FGF) in the culture supernatant of human hair hair papilla cells (THPC-001) was tested by using Funakoshi's human FGF immunoassay kit, human (FGF). basic, Human, ELISA Kit, Qua
ntikine (96 well) ； R & D SYSTEMS, INC. ； Funakoshi）
Was measured using.

(7) Study on promotion of endothelial cell proliferation by glycoprotein having molecular weight of 57 kilodaltons The glycoprotein having molecular weight of 57 kilodaltons is bovine carotid artery-derived endothelial cells, human aorta-derived endothelial cells, bovine aorta-derived endothelial cells, human coronary arteries. -Derived endothelial cells, rat coronary artery-derived endothelial cells, human umbilical artery-derived endothelial cells, human umbilical vein-derived endothelial cells, human iliac artery-derived endothelial cells, human pulmonary artery-derived endothelial cells, human pulmonary vein-derived endothelial cells, etc. Cells, human skin microvascular-derived endothelial cells, human lung microvascular-derived endothelial cells, human myometrium microvascular-derived endothelial cells,
It shows proliferative activity of endothelial cells such as human corneal endothelial cells, bovine corneal endothelial cells, and liver sinusoidal endothelial cells. Furthermore, human aorta-derived endothelial cells (HAEC, Code: C2535, Bio Whittaker Co. Lt.
d.), human coronary artery-derived endothelial cells (HCAEC, Code: C2585, Bi
o Whittaker Co. Ltd.), human iliac artery-derived endothelial cells (HIAEC, Code: C2545, Bio Whittaker Co. Ltd.), human skin microvessel-derived endothelial cells (HMVEC-d, Code :, Bio Whitta
ker Co. Ltd.), human lung microvessel-derived endothelial cells (HMVEC-
L, Code: C2527, Bio Whittaker Co. Ltd.), human pulmonary artery-derived endothelial cells (HPAEC, Code: C2530, Bio Whittaker Co. Ltd.
Ltd.), human umbilical artery-derived endothelial cells (HUAEC, Code: C252
0, Bio Whittaker Co. Ltd.), human umbilical vein-derived endothelial cells (HUVEC, Code:, Bio Whittaker Co. Ltd.), human myometrium microvessel-derived endothelial cells (UtMVEC, Code: C2564, Bio W
Hittaker Co. Ltd.), normal human skin microvascular endothelial cell total kit (manufacturer: Cell Applications Inc. USA, import distributor: Toyobo Co., Ltd.) are commercially available and convenient. Here, using a normal human skin microvascular endothelial cell total kit (manufacturer: Cell Applications Inc. USA, import distributor: Toyobo Co., Ltd.), normal human skin microvascular endothelial cells were cultured by a conventional method to determine the molecular weight. The proliferative activity of the 57 kilodalton glycoprotein was examined. An ampoule containing frozen normal human skin microvascular endothelial cells is immersed in a 37 ° C. thermostat for 1 minute to rapidly thaw the cells. 500 μL of the ampoule cell suspension was added to an Ixtra cellular matrix protein-coated flask (7.5 mL of CADMEC Growth Medium).
um) and shake it so that the cells are evenly dispersed. Place the flask in an incubator (37
(C), 5% CO2, humidified conditions), the cap of the flask is loosened to allow gas exchange, and static culture is performed. After culturing for 8 hours or overnight, the medium is replaced to eliminate the influence of DMSO in the medium. In addition, the promotion of endothelial cell growth was examined by a glycoprotein addition system with a molecular weight of 57 kilodaltons and a glycoprotein addition system.

(8) Method for measuring growth promoting action on normal human dermal microvascular endothelial cells A protein having a molecular weight of 57 kilodalton was added up to 95% concentration to CADMEC Growth Medium, which is a culture medium for normal human dermal microvascular endothelial cells. Then
Normal human skin microvascular endothelial cells were cultured as described in (5) of the embodiment of the invention, and the proliferation promoting action on normal human skin microvascular endothelial cells after 48 hours was analyzed by the MTT method.

(9) Method for measuring cell proliferation by MTT method Normal human dermal microvascular endothelial cells were made to have a cell density of 500 or 5000 / well by CADMEC Grow.
Seed in a 96-well plate containing th Medium, and when the medium was replaced the next day, a glycoprotein having a molecular weight of 57 kilodalton was added and cultured for 3 days, and the growth of normal human skin microvascular endothelial cells when the additive-free system was 100. M activity
It was measured by the TT method. Specifically, as a procedure for measuring cell proliferation by the MTT method, a cell suspension (100 μL) having an appropriate concentration was seeded on a 96-well microplate, and 95% at the maximum of dermal papilla cell culture supernatant was CADMEC.
Add to Growth Medium (control: add the same amount of fresh PCGM medium as a non-added system) and culture for 3 days. Then 100 μL CADM
Replace the medium with EC Growth Medium, and
μL MTT solution (MTT: [3- (4,5-dimethyltiazol-2
-yl) -2,5-diphenyl tetrazolium bromide] 50mg and PBS
(10 mL added solution) was added, and after the reaction, measurement was performed with a microplate reader (BioRad) to determine the cell proliferation activity. (As the target wavelength of 630 nm, the absorbance at a wavelength of 570 nm was measured)

(10) Glycoprotein having a molecular weight of 57 kilodalton which is a luminal cavity formation promoting agent of the present invention The term "luminal cavity formation promoting agent" as used herein refers to a tubular structure differentiated during the process of culturing mammalian endothelial cells. Induced, which means that it has the same effect as an angiogenesis promoter and a lymphangiogenesis promoter. Particularly, in vitro system, it has an action of promoting lumen formation during the culture of bovine carotid artery-derived endothelial cells, bovine aorta-derived endothelial cells, large blood vessel-derived endothelial cells such as rat coronary artery endothelial cells, and human umbilical vein-derived endothelial cells. is there.

(11) Method for measuring luminal formation promoting action of bovine carotid artery-derived endothelial cells of glycoprotein having a molecular weight of 57 kilodaltons: Sandwich culture method between collagen gels (Atherosclerosis, 92, 141, 1992 or Virchows Arc)
h. B Cell Pathol., 60, 245, 1991) A glycoprotein having a molecular weight of 57 kilodalton was aseptically administered to the cells of the following tube formation experimental system, and tube formation was observed. Bovine carotid artery-derived endothelial cells were cultured in Eagle's minimal medium (MEM: Gibco Laboratory) supplemented with 10% FBS.
es, NY), 37 ° C., 5% CO 2 in the incubator. The cells used in this experiment were cultured 6 to 10 times. The lumen formation experiment
Using a 12-well plate with a diameter of 22 mm, 10% FB
Bovine carotid artery-derived endothelial cell density was 1 × 1 in S-containing MEM
05 cells / 1.5 mL of Vitrogen 100 (C
ollagen CO. , CA) is 8, 0.1N Na
OH was seeded in a well plate containing 0.75 mL of collagen gel consisting of a mixture of 1 × 10 MEM (pH 7.4) at a volume ratio of 1 and incubated for 24 hours. This medium was aspirated and layered on bovine carotid artery-derived endothelial cells with 0.5 mL of collagen gel, 2% FBS was added thereto, and MEM 1.5 m containing glycoprotein having a molecular weight of 57 kilodalton was added and incubated. The medium was exchanged once every 3 days and the cells were cultured. This allows
The influence of lumen formation of a glycoprotein having a molecular weight of 57 kilodalton was observed. As a negative control, a molecular weight of 57
As a positive control, 100 μU / mL of insulin was added to the system in which no kilodalton glycoprotein was added. The morphological changes were observed by a phase contrast optical microscope (Olympu
s phase contrast IMT2), and a micrograph was taken at 33 times. Indiscriminately selecting 5 locations from each culture vessel, the tubular structure was traced from 10 photographs, and the length of the tube was determined by the tissue analysis system (TAS p
lus, Ernst Leitz Wetzlar G
mbH, F.I. R. G. ). The length of the tube is given by the following equation. Specific length (mm-1) = total tube length (mm) / total area (mm2)

(12) Evaluation method by mouse hair growth test:
Effect of glycoprotein with molecular weight of 57 kilodaltons (hair growth evaluation method using C3H mouse) Male C3 at 7 weeks of age
H mouse (Charles River) was purchased, acclimated and bred for 2 weeks, and then used for the experiment. The back surface of the mouse was shaved with an electric clipper, and the half surface facing the tail was shaved with a shaver (National High Spin ES467). 1
As a group of 7 animals, 40 μl of sample was shaved at 1
It was applied for 5 days a week. The growth of hair was evaluated by visual observation and objective measurement by measuring the lightness value (L) with a colorimeter (Minolta CR-200). The results were judged from the results of 5 animals excluding the animals showing the minimum and maximum effects in consideration of the variation among the 7 animals.

(13) External Agent for Hair Growth of the Present Invention The external agent for skin growth of the present invention contains a glycoprotein having a molecular weight of 57 kilodaltons, and has a vascular endothelial growth factor (VEGF) production promoting activity and fibroblast. Cell growth factor (FGF) production promoting activity,
It has endothelial cell division promoting activity and lumen formation promoting activity, and is highly safe, and its remarkable hair growth promoting effect is useful for preventing and improving baldness and thinning hair. Here, the skin external preparation for hair growth referred to in the present invention is a general term for a composition applied externally to the skin, and a skin external medicine containing a patch and a cosmetic containing a detergent can be preferably exemplified. Among these, tonic dosage forms, lotion dosage forms, and cream dosage forms are particularly preferable. The skin external preparation for hair growth of the present invention has high safety, excellent usability, and a remarkable hair growth effect. In the skin external preparation for hair growth of the present invention, the preferred content of the lumen formation promoter composed of a glycoprotein having a molecular weight of 57 kilodalton is 0.001 to 30% by weight based on the total amount of the skin external preparation. ,
More preferably, it is 0.1 to 10% by weight. This is because, if the amount is too small, the hair-growth promoting action may not be exhibited, and if the amount is too large, the effect may reach the ceiling and the freedom of other prescription ingredients may be impaired. It also differs depending on the form of the fistula formation promoting agent composed of the treated dermal papilla cell culture supernatant.

The skin external preparation for hair growth of the present invention comprises minoxidil, stigmasteranol maltoside, pantothenyl ethyl ether, alloxazine, adenosine 3 ', 5'-cyclic monophosphate (known as a hair growth promoter). c-AMP), vitamin E acetate, carpronium chloride, benzyl nicotinate, nicotinic acid, D
When combined with L-α-tocopherol, DL-α-tocopherol nicotinate ester, methyl nicotinate, and cepharanthin, the hair growth promoting effect is excellent due to the synergistic effect. In addition to the above-mentioned essential components, any components usually used in cosmetics and external medicines for skin can be contained. Such optional components include, for example, royal jelly, hawthorn, ginkgo, ginger, senburi, capsicum, spruce, ginseng, dio, chorei, swordfish, licorice, rhubarb, chimpi, clove, psyllium, psyllium, peony, gem ginger, fennel, Kaikonose, Kengoshi, Keigai, Ages,
Extracts of crude drugs such as Kumazasa, Kagosou, Acacia yak, Chazenchi, Prunus vulgaris, Shirogane lacquer, Marva serrata, etc., hydrocarbons such as squalane, vaseline, microcrystalline wax, jojoba oil, carnauba wax, octyldodecyl oleate, etc. , Triglycerides such as olive oil, beef tallow, and coconut oil, fatty acids such as stearic acid, oleic acid, and lithinoleic acid, higher alcohols such as oleyl alcohol, stearyl alcohol, octyldodecanol, sulfosuccinic acid ester, sodium polyoxyethylene alkyl sulfate, etc. Anionic surfactants, amphoteric surfactants such as alkyl betaine salts, cationic surfactants such as dialkyl ammonium salts, sorbitan fatty acid esters,
Nonionic surfactants such as fatty acid monoglyceride, polyoxyethylene adducts thereof, polyoxyethylene alkyl ethers and polyoxyethylene fatty acid esters, polyhydric alcohols such as polyethylene glycol, glycerin and 1,3-butanediol, Viscous / gelling agent,
Antioxidants, ultraviolet absorbers, coloring agents, preservatives, powders and the like can be contained.

[0023]

EXAMPLES The present invention will be described in more detail below with reference to Examples, but it goes without saying that the present invention is not limited to these Examples.

<Examples 1 to 4> Human VEGF ELISA kit was used to determine the VEGF production promoting ability of a glycoprotein having a molecular weight of 57 kilodaltons.
(Genzyme) was used for quantification. That is, the molecular weight of each concentration is 5
7 kilodalton glycoprotein (1 ng / mL, 5 ng / m
L, 10 ng / mL, 20 ng / mL) was added to a human hair papilla cell line to measure the VEGF production promoting ability. The experimental results are shown in Table 1. As can be seen from Table 1, the system in which 10 ng / mL of glycoprotein having a molecular weight of 57 kilodalton was most excellent in the VEGF production promoting ability as compared with the control (no addition system). Therefore, the glycoprotein having a molecular weight of 57 kilodaltons is an excellent VEGF production promoter. (In addition, the result of Table 1 shows the average value and SEM of the results of three experiments.)

[0025]

[Table 1]

<Examples 5 to 8> The ability of a glycoprotein having a molecular weight of 57 kilodaltons to promote the production of bFGF was confirmed by using a human FGF immunoassay kit (FGF basic, Human, ELISA Kit, Quantikine (9
6 well); R & D SYSTEMS, INC .; Funakoshi). That is, each concentration of glycoprotein having a molecular weight of 57 kilodaltons (1 ng / mL, 5 ng / mL, 10 ng / mL, 2
0 ng / mL) was added to a human hair papilla cell line to measure bFGF production promoting ability. The experimental results are shown in Table 2. As can be seen from Table 2, the system in which 10 ng / mL of glycoprotein having a molecular weight of 57 kilodalton was most excellent in the bFGF production promoting ability as compared with the control (no addition system). Therefore, the glycoprotein having a molecular weight of 57 kilodalton is an excellent bFGF production promoter. (In addition, the result of Table 2 shows the average value and SEM of the results of three experiments.)

[0027]

[Table 2]

<Examples 9 to 12> Effect of glycoprotein having a molecular weight of 57 kilodalton on normal human skin microvascular endothelial cells A glycoprotein having a molecular weight of 57 kilodalton at each concentration was added to normal human skin microvascular endothelial cells (1 ng). / ML, 5ng
/ ML, 10 ng / mL, 20 ng / mL), 3 days,
After culturing normal human skin microvascular endothelial cells. Its proliferative activity was determined. (As a control, non-additive type.
Note that the effect of a glycoprotein having a molecular weight of 57 kilodalton on the proliferative activity of normal human skin microvascular endothelial cells was determined as a relative value, with the control proliferative activity value as 100).
The results are shown in Table 3. Hair papilla cell culture supernatant 1 of Example 11
The 0 ng / mL addition system promoted the proliferative activity of the most normal human skin microvascular endothelial cells. (In addition, the result of Table 3 shows the average value and SEM of the results of three experiments.)

[0029]

[Table 3]

<Examples 13 to 16> Effect of lumen formation of a glycoprotein having a molecular weight of 57 kilodaltons on bovine carotid artery-derived endothelial cells Effects of lumen formation on bovine carotid artery-derived endothelial cells of the embodiment (4) Glycoprotein having a molecular weight of 57 kilodaltons at each concentration of 1 ng / mL and 5 ng / m
L, 10 ng / mL, and 20 ng / mL were added, and the effect of promoting lumen formation on bovine carotid artery-derived endothelial cells was determined.
(In addition, as a negative control, a system to which a glycoprotein having a molecular weight of 57 kilodalton was not added was added, and as a positive control, 100 μU / mL of insulin was added.)
The results are shown in Table 4. The glycoprotein having a molecular weight of 57 kilodalton showed a remarkable luminal formation promoting action on bovine carotid artery-derived endothelial cells. The 10 ng / mL addition system of a glycoprotein having a molecular weight of 57 kilodalton promoted the lumen formation most to bovine carotid artery-derived endothelial cells. (In addition, the result of Table 4 shows the average value and SEM of the results of three experiments.)

[0031]

[Table 4]

<Examples 17 to 19> (Hair growth test using C3H mouse back) Glycoprotein having a molecular weight of 57 kilodalton was added to 70% ethanol at 10 ng / m.
L, dissolved in 50 ng / mL, 100 ng / mL concentration,
This was used as a test sample. On the other hand, as a control, vehicle 70% ethanol and comparative control example 1% minoxidil were used. The back surface of the C3H mouse was shaved with an electric clipper, and the tail half surface was shaved with a shaver (National High Spin ES467). As a group of 7 animals, 40 μl of the sample was applied to the shaved site every day for 5 days a week. The growth of hair was evaluated by visual observation and objective measurement by measuring the lightness value (L) with a colorimeter (Minolta CR-200). Table 5 shows the test results of the mouse hair nourishing effect after 20 days. As can be seen from Table 5, the molecular weight is 57
The kilodalton glycoprotein showed an excellent hair growth promoting effect. (In addition, the results of Table 5 are the average value of the results of three experiments and S.
Indicates EM)

[0033]

[Table 5]

Example 20 A tonic shown below was prepared using a glycoprotein having a molecular weight of 57 kilodaltons, and the effect of promoting hair growth in humans was examined. At the same time, a tonic excluding the glycoprotein having a molecular weight of 57 kilodalton was prepared as a control, and a tonic containing 1% minoxidil as a comparative control example was prepared. 10 glycoproteins with a molecular weight of 57 kilodaltons
% Tonic, control emulsion and tonic containing 1% minoxidil were divided into 3 groups, and the average age was 4
Thirty eight-year-old male pattern baldness subjects were collected and divided into three groups of ten persons each. I had him use it twice a day for 6 months in a row. After 6 months, the degree of improvement due to hair nourishment was examined.
The results are expressed as ++ for a group that significantly improved compared to before use, + for a group that slightly improved compared to before use, and ± for a group that did not improve compared to before use. From the results in Table 6, the molecular weight is 57
The kilodalton glycoprotein-containing tonic was found to be as effective as and more effective for androgenetic alopecia than the minoxidil-containing tonic.

Glycoprotein having a molecular weight of 57 kilodaltons
10 parts by weight 1,3 butanediol
5 parts by weight glycerin 3 parts by weight citric acid
0.1 parts by weight sodium citrate
0.1 parts by weight methylparaben
0.1 parts by weight ethanol
45 parts by weight water
36.7 parts by weight

[0036]

[Table 6]

Example 21 An emulsion containing 10% of a glycoprotein having a molecular weight of 57 kilodaltons was prepared by treating the components of the emulsion base shown below by a conventional method, and 5 groups of panelists suffering from thinning hair were selected. For 6 months, they were used twice a day in the morning and evening to evaluate the effect of preventing and improving thinning hair. The criteria for evaluation are as follows: grade 2: significant improvement, grade 1: clear improvement, grade 0.5: slight improvement, grade 0: no improvement. The average score was 0.86. It was found that the emulsion containing the glycoprotein having a molecular weight of 57 kilodaltons, which has the effect of promoting hair growth of the present invention, has an excellent effect of improving thinning hair. Behenyl alcohol 0.2 parts by weight 1,3-butylene glycol 8 parts by weight cetyl 2-ethylhexanoate 3 parts by weight squalane 8 parts by weight dipotassium glycyrrhizinate 0. 02 parts by weight methyl paraoxybenzoate 0. 3 parts by weight Lipophilic glyceryl monostearate 2.5 parts by weight polyoxyethylene hydrogenated castor oil (50 E.O.) 1.6 parts by weight beeswax 1.5 parts by weight glycoprotein having a molecular weight of 57 kilodaltons 10 parts by weight Fragrance 0. 3 parts by weight purified water 57. 76 parts by weight

Example 22 A lotion-type drug was prepared according to the following formulation. That is, the formulation ingredients were stirred and solubilized at room temperature to obtain a lotion. About this lotion-type drug, 5 panelists from 1 group suffering from thinning hair were asked to use it twice a day in the morning and evening for 6 months, and their prevention and improvement effects of thinning hair were evaluated. The evaluation criteria are as follows: score 2: significant improvement, score 1: clear improvement, score 0.5: slight improvement,
Score 0: No improvement standard. The average score was 0.94. It was found that the addition of the glycoprotein having a molecular weight of 57 kilodaltons having a hair growth promoting effect of the present invention and minoxidil has a synergistic effect in promoting hair growth, and that the lotion type drug has an excellent effect in improving thinning hair. . Glycoprotein with a molecular weight of 57 kilodaltons 10 parts by weight minoxidil 1 part by weight 1,3 butanediol 8 parts by weight glycerin 4 parts by weight citric acid 0.1 parts by weight sodium citrate 0.1 parts by weight methylparaben 0.2 parts by weight ethanol 25 parts by weight polyoxyethylene Hydrogenated castor oil (40E.O.) 1 part by weight water 50.6 parts by weight

Example 23 A scalp cream was prepared according to the following formulation. That is, (a), (b) and (c) were each heated and dissolved at 80 ° C., (a) and (b) were gradually added, and after further adding (c), the emulsified particles were homogenized with a homomixer and cooled to obtain a scalp cream. . This scalp cream had an excellent effect in preventing and improving hair loss. B) Squalane 8 parts by weight Cetanol 5 parts by weight Sorbitan sesquistearate 2 parts by weight Polyoxyethylene (20) behenyl ether 2 parts by weight Vitamin E acetate 0. 2 parts by weight b) 1,3-butanediol 7 parts by weight Glycoprotein having a molecular weight of 57 kilodalton 5 parts by weight Carboxyvinyl polymer 0.3 parts by weight Methylparaben 0.2 parts by weight water 40 parts by weight c) Water 30. 1 part by weight Potassium hydroxide 0.2 part by weight

[0040]

INDUSTRIAL APPLICABILITY According to the present invention, royal jelly or a molecular weight obtained from royal jelly having vascular endothelial growth factor production promoting activity, fibroblast growth factor production promoting activity, endothelial cell growth promoting activity or lumen formation promoting activity. By adding a 57-kilodalton glycoprotein to a skin external preparation for hair growth, it is highly safe, has excellent usability, and has a remarkable hair growth effect. Can be provided.

[0041]

[Sequence list]

[0042] &#60; 210 &#62; 1 &#60; 211 &#62; 25 &#60; 212 &#62; PRT &#60; 213 &#62; Apis mellifera &#60; 220 &#62; &#60; 221 &#62; UNSURE &#60; 222 &#62; (24) &#60; 223 &#62; Xaa = unknown &#60; 400 &#62; 1 Asn Ile Leu Arg Gly Glu Ser Leu Leu Lys Lys Leu Pro Ile Leu His  1 2 10 10 Glu Met Lys Phe Phe Asp Tyr Xaa Asp              20 25

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // C07K 14/435 ZNA A61K 37/02 F term (reference) 4C083 AA071 AA072 AA082 AB032 AC022 AC072 AC102 AC122 AC182 AC302 AC302 AC352 AC422 AC432 AC442 AC482 AC852 AD092 AD411 AD412 AD532 AD662 CC37 DD23 DD31 EE22 4C084 AA02 AA03 BA01 BA02 BA17 BA19 BA34 CA49 MA17 MA22 MA28 MA63 NA14 ZA922 ZC022 4C087 AA01 AA02 02 AA10 AA30 BA10 CA51 EA28 HA06

Claims (10)

[Claims] 1. A hair-growth promoting agent comprising royal jelly. 2. The hair growth-promoting agent according to claim 1, wherein the royal jelly contains 9% by weight or more of protein shown below. 1) Form a single band on non-denaturing polyacrylamide gel electrophoresis of proteins in royal jelly. 2) The molecular weight measured by SDS-polyacrylamide gel electrophoresis under reducing conditions is about 57 kilodaltons. 3) Includes the amino acid sequences of amino acid numbers 1 to 8 in Sequence Formula 1. 3. The hair-growth promoting agent according to claim 1, wherein the hair-growth promoting is based on the activity of promoting the production of vascular endothelial growth factor (VEGF) by 57-kilodalton protein. 4. The vascular endothelial growth factor (VEGF) is VEGF-A or VEGF.
165, VEGF-B, VEGF-C, VEGF-D, VEGF-E consisting of one or two or more selected from the above, the hair growth promoter according to any one of claims 1 to 3. . 5. The promotion of hair growth according to any one of claims 1 to 4, wherein the promotion of hair growth is based on fibroblast growth factor (FGF) production promoting activity by the 57 kilodalton protein. Agent. 6. The fibroblast growth factor (FGF) is FGF-
1 (acidic FGF: aFGF), FGF-3, FGF-2 (basic FGF: bFG
F), FGF-3 (int-2), FGF-4 (hst-1 / kaposi- FGF), FGF-5,
6. One or two or more selected from FGF-6 (hst-2), FGF-7, FGF-8, FGF-9, and FGF-10, characterized in that: A hair growth promoter according to item. 7. The hair-growth promoting agent according to claim 1, wherein the hair-growth promoting is based on the endothelial cell division promoting activity of the 57-kilodalton protein. 8. The hair growth-promoting agent according to claim 1, wherein the promotion of hair growth is based on the activity of promoting lumen formation by the 57-kilodalton protein. 9. A skin external preparation for hair growth, which comprises the hair growth promoter according to any one of claims 1 to 8. 10. The skin external preparation for hair growth according to claim 9, which is a pharmaceutical or cosmetic product.