JPH07196529A - Wound healing agent, cosmetics and hair nourishing agent - Google Patents

Abstract

(57) [Summary] [Objective] To provide a wound healing agent, a cosmetic and a hair nourishing agent which are safe and have excellent effects. [Structure] A wound healing agent, a cosmetic and a hair nourishing agent containing lactoferrin, a hydrolyzate of lactoferrin, or a mixture thereof and epidermal growth factor.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a wound healing agent, a cosmetic and a hair restorer. More specifically, the present invention relates to a wound healing agent, a cosmetic and a hair nourishing agent containing lactoferrins, a hydrolyzate of lactoferrins or a mixture thereof and epidermal growth factor as active ingredients.

[0002]

2. Description of the Related Art Epidermal growth f
actor. (Hereinafter sometimes referred to as EGF) has a molecular weight of about 6,000, which has a cell proliferating effect on a wide variety of cells.
Peptide contained in all mammalian body fluids. Since the discovery of EGF was extremely old in 1962, various applied studies have been actively conducted since then, and EGF has a wound healing effect [Journal of Surgery Research (Jour.
nal of Surgical Research), Volume 33, Page 164
J., 1982, and the Journal of Experimental Medi
cine), volume 163, page 1319, 1986.
, EGF is effective in healing corneal injury [Experimental Eye R
esearch), Volume 40, Page 47, 1985, and Investigative Ophthalm of Investigative Ophthalmology and Visual Science.
& Visual Science), Volume 26, Page 105
J., 1985] and the like have been clarified.

Based on these findings, examples of applying EGF to skin preparations are also known. That is, for skin, skin cells are activated, metabolism is promoted, and a wound healing effect is given to give a smooth, moist and youthful skin, and for hair, activation of hair mother cells, hair There has been proposed a dermatological agent which has effects of promoting hair growth, preventing hair loss, and preventing hair nourishment, hair growth and hair loss (JP-A-615006).
Issue).

[0004] On the other hand, lactoferrin (hereinafter sometimes referred to as Lf) is an iron-binding protein having a molecular weight of about 80,000, which is contained in an extremely large amount in human milk, and is Escherichia coli, Candida, Clostridium. Fungus,
It is known to show antibacterial action against harmful microorganisms such as Staphylococcus [Journal of Pediatrics, Vol. 94, page 1, 1
979, and Journal of Dairy Science, Vol. 67, page 60, 1984].

Recently, DNA of rat small intestinal epithelial crypt cells and mouse fibroblast Balb / c3T3
The synthesis was shown to be facilitated by Lf [Pediatic Research, 21st.
Vol. 563, 1987, and Agricultural and Biological Chemistry.
lturaland Biological Chemistry), 53, 31
Page, 1989] as a new cell growth stimulating factor.

Regarding Lf and its degradation products, antibacterial activity and inhibition of tyrosinase activity (European Patent Publication No. 43).
8750), prevention of adhesion of pathogenic bacteria to cells (JP-A-3-220130), antiviral action (JP-A-1-233226), and the like.

[0007]

Conventionally, wound healing agents, dermatological agents and the like containing EGF as an active ingredient have been known, but their effects have not always been sufficient.

On the other hand, the inventors of the present invention have been studying the biological activity of lactoferrins and their hydrolysates, but have newly discovered the following facts.

Lactoferrins act synergistically with EGF to have a stronger cell growth or proliferation promoting action than lactoferrin alone and EGF alone.

Generally, a protein loses its biological activity when hydrolyzed, but a hydrolyzate of lactoferrin retains the action of lactoferrin,
To exert a cell growth or proliferation promoting action synergistically with EGF.

The present invention has been made on the basis of the novel facts as described above, and is more effective than the conventional EGF-only drug or dermatological agent for wound healing, cosmetics, and The purpose is to provide a hair restorer.

[0012]

Means for Solving the Problems The present invention, as a solution to the above problems, comprises a lactoferrin, a hydrolyzate of lactoferrin or a mixture thereof and an epidermal growth factor as active ingredients. I will provide a.

The present invention also provides a cosmetic containing lactoferrins, a hydrolyzate of lactoferrins or a mixture thereof and epidermal growth factor as active ingredients. The present invention also provides a hair nourishing agent containing lactoferrins, a hydrolyzate of lactoferrins or a mixture thereof and epidermal growth factor as active ingredients.

The present invention will be described in detail below.

The lactoferrins used in the wound healing agent, cosmetics and hair nourishing agent of the present invention are commercially available Lf, animal milk,
Lf separated from human milk by a conventional method, apolactoferrin obtained by removing iron from these Lf by a conventional method, and apolactoferrin containing a metal such as iron, copper, zinc and manganese completely or partially chelated by a conventional method. It may be either saturated metal or partially saturated lactoferrin, or a mixture thereof. The hydrolyzate of lactoferrin used in the wound healing agent, cosmetics and hair nourishing agent of the present invention is a hydrolyzate obtained by hydrolyzing the lactoferrin with an acid or an enzyme by a conventional method. The lactoferrin and the hydrolyzate thereof may be mixed and used.

The EGF used in the wound healing agent, cosmetics and hair nourishing agent of the present invention is human urine, animal milk or human milk isolated by a conventional method, one produced by a recombinant DNA technique, or chemically. It may be any of those synthesized above, commercial products or a mixture thereof.

The wound healing agent of the present invention should be put to practical use as a general pharmaceutical preparation containing lactoferrin, a hydrolyzate of lactoferrin or a mixture thereof and EGF as active ingredients, for example, an ophthalmic external preparation. You can Various dosage forms can be selected appropriately according to the purpose of use,
For example, it can be used as a liquid coating agent, lotion, aerosol (spray), ointment, suppository, injection, etc.

The cosmetics and hair nourishing agents of the present invention are in various forms like ordinary cosmetics or hair nourishing agents except that they contain lactoferrin, a hydrolyzate of lactoferrin or a mixture thereof and EGF as active ingredients. It can be prepared. For example, as a cosmetic applied to the skin such as a lotion, a cream, a foundation, an emulsion, a hair cream, a hair liquid, a hair roll.
It can be used as a cosmetic for hair applied to the scalp and hair of hair, hair rinse, hair tonic, pomade, shampoo and the like.

The amount of lactoferrin and hydrolyzate of lactoferrin which are the active ingredients of the wound healing agent, cosmetics and hair nourishing agent of the present invention is not particularly limited and can be appropriately selected from a wide range, but is preferably 0.1 μg to 100
It is in the range of mg / g. The amount of EGF is 0.0
A preferable range is 1 ng to 10 μg / g.

Since lactoferrins, their hydrolysates and EGF are natural products, it is clear that there is no problem with their safety.

Next, the operation and effect of the present invention will be described with reference to test examples. Test Example 1 In this test, mouse fibroblasts Swiss3T3 (Amer
(purchased from ican Type Culture Collection) was used to examine the effect of coexistence of lactoferrin and EGF and the effect of coexistence of lactoferrin hydrolyzate and EGF on cell proliferation. (1) Preparation of sample Commercially available mouse submandibular gland-derived EGF (Takara Shuzo) and beef lactoferrin (bLf; Snow Brand Dairy Co., Ltd.) were used.

Hydrolyzate of lactoferrin (bLf-H
y) was prepared from bLf as follows. Commercial b
Lf (Oleofina) 500 g, purified water 9.5 l
And add hydrochloric acid to adjust the pH to 3.0.
Adjust to 1 and then add commercially available pig pepsin (manufactured by Wako Pure Chemical Industries)
0 g was added and hydrolyzed at 37 ° C. for 6 hours. Next, the pH was adjusted to 7.0 with 6N sodium hydroxide, the enzyme was inactivated by heating at 80 ° C for 10 minutes, cooled to room temperature, filtered through Celite, and the filtrate was freeze-dried, and lactoferrin pepsin. A hydrolyzate was obtained. (2) Test method Swiss3T3 cells were placed in a 24-well culture plate 1
Sows 3000 seeds per hole and includes the following test specimens.
0.5 ml of Dulbecco's modified method containing 2% fetal bovine serum
It was cultured for 6 days in a glu culture solution (Nissui Pharmaceutical Co., Ltd.). During this period, the culture medium was exchanged on the second and fourth days of culture. On the 6th day, the cells were treated with a 0.25% trypsin solution, the cells were suspended, and the cell number was measured using a hemocytometer.

The test sample was a control sample to which nothing was added, b
Sample with Lf added at a rate of 50 μg / ml (Sample 1), sample added with bLf-Hy at a rate of 50 μg / ml (Sample 2), sample added with 2 ng / ml of EGF (Sample 3), 2 ng of EGF / Ml and bLf 50
Sample added with μg / ml (Sample 4), 2 ng of EGF /
It is a sample (sample 5) to which 50 μg / ml of ml and bLf-Hy was added. (3) Test results The results of this test are shown in Table 1. Table 1 shows
It is a relative value showing the cell number of each test sample when the cell number on the 6th day of culture of the control sample is 100%.

As is clear from Table 1, the cell number on the 6th day of culture of Sample 3 to which EGF was added alone increased to 280%. On the other hand, the cell numbers on day 6 of sample 1 containing bLf alone and sample 2 containing bLf-Hy alone were 475% and 315%, respectively.
BLf and bLf-Hy were found to be effective in cell proliferation as compared to

Furthermore, sample 4 containing EGF and bLF added
, The number of cells on the 6th day of culture was significantly increased to 801%. Similarly, in the sample 5 to which EGF and bLF-Hy were added, the cell number increased, and it was 580% on the 6th day of culture. As described above, coexistence of EGF and bLf,
It was revealed that the cells proliferate remarkably by coexisting with EGF and bLf-Hy. Although the test was performed by changing the types of bLf and bLf-Hy, similar results were obtained.

[0026]

[Table 1]

Test Example 2 This test was carried out to examine the effective amount of the Lf degradation product on the cell growth activity. The cell proliferation activity was ³ H-
The amount of thymidine incorporated was evaluated. (1) Preparation of sample bLf-Hy was prepared according to Test Example 1. (2) Test Method Swiss3T3 cells were placed in a 48-well culture plate 1
Dulbecco's modified Eagle culture medium (manufactured by Nissui Pharmaceutical Co., Ltd .; hereinafter F)
The cells were cultivated in BS-DMEM) until they became confluent (0.5 ml of the culture medium was used per well). The culture solution was exchanged with 1% FBS-DMEM, and the cells were further cultured for 2 days to introduce the cells into the stationary phase. DM cells
After washing once with EM (0.5 ml), DMEM (0.3 ml) containing bLf-Hy at the concentration shown in Table 2 and 10 n
D containing g / ml EGF and various concentrations of bLf-Hy
The medium was replaced with MEM (0.3 ml), and the cells were cultured for 19 hours. The culture solution was replaced with DMEM (0.15 ml) containing ³ H-thymidine (manufactured by ICN Biomedicals, 1 μC / ml), and further cultured for 2 hours. The cells were washed twice with phosphate buffer [PBS (-), 1 ml] and then ice-cooled 5%.
Trifluoroacetic acid (TFA, 1 ml) was added and left in the refrigerator for 1 hour. The cells were washed twice with 5% TFA (1 ml), 1N sodium hydroxide (0.2 ml) was added, and the mixture was left at 37 ° C. for 1 hour to lyse the cells. 6N Hydrochloric acid (0.04 ml) was added to neutralize, and then the amount of ³ H-thymidine incorporated into DNA was measured with a liquid scintillation counter (manufactured by LKB). (3) Test results The results of this test are shown in Table 2. As is clear from Table 2, in the group to which EGF was added at 10 ng / ml,
^The amount of ³ H-thymidine incorporated was 2119, which was 126% of the amount of ³ H-thymidine incorporated (1677) in the non-added control group. bLf-Hy at 50 μg / ml, 1
The 3 H-thymidine uptake in the group added with 50 μg / ml was 2013 and 2315, respectively, which corresponded to 120% and 138% in the control group without addition, and EGF.
Similar to the above, cell proliferation activity was observed. 10 ng of EGF
/ Ml and bLf-Hy 17 μg / ml, 50 μg / m
The amounts of ³ H-thymidine incorporated in the groups to which 1 and 150 μg / ml were added were 2284, 2955 and 2969, respectively.
Met. These values are 13 of the additive-free symmetric group, respectively.
6%, 176%, 177%, in the presence of EGF, ³
It was revealed that the amount of H-thymidine uptake dramatically increased corresponding to the dose of bLf-Hy. Note that b
Almost similar results were obtained in the test in which the type of Lf-Hy was changed and the test in which bLF was used instead of bLf-Hy.

[0028]

[Table 2]

Reference Example 1 (Preparation of Apo (Deferred Iron) Lactoferrin) Apolactoferrin was obtained from commercially available beef lactoferrin (manufactured by Olefina), Suzuki et al. (Nutrition and Food, Volume 31,
It was prepared as follows by the method of page 395, 1978).

1 liter of 1% lactoferrin aqueous solution was dialyzed against 0.1 molar citric acid solution (pH 2.2) containing 20 times amount of 0.05% EDTA at 4 ° C. for 30 hours, and deionized. It was dialyzed against water for 24 hours and freeze-dried to obtain about 10 g of apolactoferrin. Reference Example 2 (Preparation of EGF) EGF was prepared by the method of Coene and Carpenter [S.
Cohen and G. Carpenter: S. Cohen and G. Carpenter: Proceedings of National Academy of Sciences USA
Proceedings of the Natlional Academy of Sciences
USA), 72, 1317, 1975], Savage and Harper's method [Analytical Biochemistry, 111, 195, 1981]. )] And Nishimuro et al. [Chemical and Pharmacuetical Bulletin, 33rd.
Vol. 4037, 1985] and prepared from human urine as follows.

To approximately 20 liters of human raw urine, 1 liter of glacial acetic acid was added to make it acidic, and concentrated hydrochloric acid was added to adjust the pH to 3.0-.
Adjusted to 3.3. Ion exchange resin Bio-Rex70
(BioRad) was adjusted to pH 3.1 with glacial acetic acid, washed with 5% acetic acid, added to urine, and stirred at 4 ° C. for 18 hours. After standing for 2 to 4 hours, the supernatant is discarded and the resin is washed with 0.0
After washing with 1N hydrochloric acid, 1 molar ammonium acetate (pH
The EGF was eluted at 8.0). Lyophilize the eluate,
The dried product was dissolved in 50 ml of distilled water, pepstatin (0.5 mg), 2 mmol of arginine, 200 mg.
Bovine serum albumin (BSA) was added.

To this solution, 1,500 ml of ethanol was added, and the mixture was stirred, allowed to stand for 30 minutes, and kept at 1,000 × g for 20 minutes.
After centrifuging for 15 minutes, 15 ml of 2 mmol arginine was added to the precipitate, the pH was adjusted to 3.0 with hydrochloric acid, and further 30,0.
Centrifugation at 00 × g for 20 minutes gave a supernatant.

A column (4 × 14) packed with DEAE cellulose (manufactured by Whatman) equilibrated with 0.05% formic acid.
cm), and the EGF that elutes without adsorbing to the column is collected, and the eluate is freeze-dried.
It was dissolved with 1 L of 0.05 N hydrochloric acid, the pH was adjusted to 1.5, and the mixture was centrifuged at 3,000 × g for 30 minutes to obtain a supernatant.

A column (4 × 90 cm) packed with Bio-GelP-10 (manufactured by Bio-Rad) was equilibrated with 0.05N hydrochloric acid and eluted at a flow rate of 42 ml / hour. Most UV absorbers elute in 1.5 column volumes,
EGF was eluted after 1.7 column volume. Active fractions were collected, adjusted to pH 6.0 with aqueous ammonia, and lyophilized. The dried product was added with 0.04 mol ammonium acetate (pH
3.9) Dissolved in 50 ml, ultrafiltered and concentrated to 5 ml.

A column (0.9.times.10 cm) packed with CM cellulose (manufactured by Whatman) was equilibrated with 0.04 mol ammonium acetate, and the above concentrate was added to give 0.04.
Wash with molar ammonium acetate, elute with 14 ml of 1 molar ammonium acetate, freeze-dry the eluate and
It was dissolved in 5 ml of 02 molar ammonium acetate (pH 5.3).

DE-52 Cellulose (manufactured by Whatman)
The column (0.9 x 10 cm) packed with was equilibrated with 0.02 mol ammonium acetate (pH 5.3), and 8 m / h
elution at a flow rate of 1 l, washing with 0.02 mol ammonium acetate after addition of the above solution, elution with a concentration gradient of 0.02 to 0.3 mol ammonium acetate, and three peaks (1,
2, 3) were obtained. Among these, the main peaks of EGF activity were 1 and 3, and about 150 to 250 μg of EGF was obtained. Reference Example 3 (Preparation of iron-saturated lactoferrin) Iron-saturated lactoferrin was prepared by the method of Suzuki et al. (Nutrition and Food, Vol. 31, p. 395, 1978) from commercially available beef lactoferrin as follows. Was prepared.

To 1 liter of a 1% aqueous solution of lactoferrin was added 0.2 liter of a 0.1 mol sodium citrate solution containing 3 mmol of ferric chloride, and the mixture was gently stirred for 1 hour, and then deionized water was added. It was dialyzed for 24 hours and freeze-dried to obtain about 10 g of iron-saturated lactoferrin.

Next, the present invention will be described more specifically by showing examples, but the present invention is not limited to the following examples.

[0039]

[Examples] Example 1 (hydrophilic ointment) Lactoferrin (manufactured by Oleofina) 10 (g) EGF 0.01 white petrolatum 250 stearyl alcohol 220 propylene glycol 120 sodium ularyl sulfate by the same method as in Reference Example 2 15 Methyl paraoxybenzoate 0.25 Propyl paraoxybenzoate 0.15 Purified water 384.59 The above ingredients were mixed to produce 1000 g of a hydrophilic ointment. In addition, as raw materials other than EGF, commercially available products were used. Example 2 (Cream) Apolactoferrin by the same method as in Reference Example 1.0 (g) EGF 0.001 by the same method as in Reference Example 2 Polyoxyethylene stearyl ether 2.0 Polyoxyethylene cetyl Ether 3.0 Beeswax 4.0 Cetanol 3.0 Lanolin 1.0 Isopropyl palmitate 2.0 Liquid paraffin 15.0 Polyethylene glycol monostearate 0.5 Methyl paraoxybenzoate 0.1 Purification Water 68.399 The above components were blended to produce 100 g of a cream.
In addition, as raw materials other than apolactoferrin and EGF, commercially available products were used. Example 3 (Foundation) Iron-saturated lactoferrin by the same method as Reference Example 1.0 (g) EGF by the same method as Reference Example 0.001 Stearic acid 2.4 Propylene glycol monostearate 2 0.0 Cetostearyl alcohol 0.2 Liquid lanolin 2.0 Liquid paraffin 3.0 Isopropyl myristate 8.5 Preservative 63.399 Carboxymethylcellulose sodium 0.2 Bentonite 0.5 Propylene glycol 4. 0 Triethanolamine 1.0 Titanium oxide 8.0 Talc 4.0 Colorant Appropriate amount Purified water Appropriate amount The above components were blended to produce a foundation of about 100 g. As raw materials other than iron-saturated lactoferrin and EGF, commercially available products were used. Example 4 (Emulsion) Lactoferrin hydrolyzate by the same method as Test Example 2.0 (g) EGF by the same method as Reference Example 0.001 Self-emulsifying glycerol monostearate 1.1 Polyoxyethylene Cetyl ether 1.9 MC stearic acid 2.0 Cetanol 1.0 Isopropyl myristate 2.0 Methyl paraoxybenzoate 0.1 Perfume 0.1 Purified water 89.799 Blend the above ingredients to obtain 100 g of An emulsion was produced. In addition, as the raw materials other than the lactoferrin hydrolyzate and EGF, commercially available products were used. Example 5 (pack) Lactoferrin hydrolyzate obtained by the same method as in Test Example 1.0 (g) EGF by the same method as in Reference Example 0.001 Ethanol 3.0 Methyl paraoxybenzoate 0. 1 Carboxyvinyl polymer-1.0 Calcium carbonate 0.3 Purified water 94.599 The above components were blended to produce a 100 g pack. In addition, as the raw materials other than the lactoferrin hydrolyzate and EGF, commercially available products were used. Example 6 (Pomado) Lactoferrin (manufactured by Oleofina) 1.0 (g) Lactoferrin hydrolyzate by the same method as in Test Example 1.0 EGF 0.001 Mokuru by the same method as in Reference Example 13. 0 Castor oil 84.799 Perfume 0.2 The above ingredients were blended to produce 100 g of pomade.
In addition, as the raw materials other than the lactoferrin hydrolyzate and EGF, commercially available products were used.

[0040]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides a wound healing agent, cosmetics and hair nourishing agent which are safe and have excellent effects.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 38/00 38/22 ADA A61K 37/24 ADA (72) Inventor Tomo Hagiwara Asahi, Yokohama City, Kanagawa Prefecture 1-89-33 Tsurugamine, Ward Ma Maison V2-C Room

Claims (3)

[Claims] 1. A wound healing agent containing lactoferrins, a hydrolyzate of lactoferrins or a mixture thereof and epidermal growth factor as active ingredients. 2. A cosmetic containing lactoferrin, a hydrolyzate of lactoferrin or a mixture thereof and epidermal growth factor as active ingredients. 3. A hair nourishing agent containing lactoferrins, a hydrolyzate of lactoferrins or a mixture thereof and epidermal growth factor as active ingredients.