WO2008032847A1

WO2008032847A1 - External preparation for skin

Info

Publication number: WO2008032847A1
Application number: PCT/JP2007/068013
Authority: WO
Inventors: Hiroyoshi Inoue
Original assignee: Up Well Co. Ltd.
Priority date: 2006-09-11
Filing date: 2007-09-10
Publication date: 2008-03-20
Also published as: JPWO2008032847A1

Abstract

Disclosed is an external preparation for the skin, which has a high antibacterial activity against a bacterium that normally colonizes on the skin surface. The external preparation comprises an apolactoferrin. The apolactoferrin has a degree of binding to iron of 5% or less. When an aqueous solution containing the apolactoferrin at a concentration of 1 w/v% is prepared, the aqueous solution has a total cation concentration of 5 mmol/L or less. The external preparation has a high antibacterial activity against bacteria that normally colonize on the skin surface, is highly safe, and causes no adverse side effect. Therefore, the external preparation can be used suitably in a product for daily use, including a cosmetic product and a toiletry product.

Description

Memo book Skin topical preparation Technical field

The present invention relates to an external preparation for skin. More specifically, the present invention relates to an external preparation for skin containing antibacterial apo-actoferrin. Background art

Living organisms are resistant to invasion of microorganisms in the skin by physical methods or by chemical methods such as bactericidal substances or mucus secretion. However, if the infectious ability of a bacterium exceeds the resistance of a living body, an infectious disease appears in the living body. Such infectious symptoms include, for example, acne, atopic dermatitis, and pyoderma. The causative organism for each of these symptoms can be a bacteria that is resident in the skin. Various drugs have been used for the prevention or treatment of such symptoms, most of which are antibiotics or synthetic antibacterials, which have the problem of strong antibacterial but side effects.

Lactoferrin is present in body fluids of many mammals, such as milk. In particular, it is known that colostrum of breast milk contains 5 to 10 g Z L and occupies 30 to 70% of the total protein contained therein. Ratatofurin is an important protein for maintaining and developing infant health, and has recently been shown to have antibacterial and antibacterial effects, and is used in various fields in addition to the food industry. .

Lactofurin is an iron-binding glycoprotein with a molecular weight of about 80,00, which binds two irons in one molecule. Lactofurin releases iron under acidic conditions such as pH 2 and becomes apolatatoferrin. Bacteriostatic bacteriostatic Bacteria) Regarding the action, it was considered as follows. The chelating action of apolactoferrin deprives the iron necessary for the growth of microorganisms and restricts its growth. For this reason, microorganisms that strongly require iron during growth are subject to the bacteriostatic (antibacterial) action of ratatopherin. Such a bacteriostatic (antibacterial) action of lactoferrin has been considered particularly in the intestinal environment. Thus, the chelating action of apolatatopherin is attracting attention regarding the bacteriostatic (antibacterial) action of ratatopherin. However, the nature of apolactoferrin itself is still unknown.

Japanese Patent Application Laid-Open No. Hei 6-2 7 9 3 10 describes that the antibacterial properties against skin pathogenic bacteria and cariogenic bacteria tend to increase by hydrolyzing lactoferrin with acid. . More specifically, various iron-binding ability products are obtained by treating ratatopherin derived from ushi with citrate and adjusting the pH to 3 or less and then treating at high temperature (95 ° C) for 5 minutes to 3 hours. It is described that a product having a lower iron-binding ability has a higher antibacterial effect against skin resident pathogenic bacteria and cariogenic bacteria. Disclosure of the invention

An object of the present invention is to provide an external preparation for skin having high antibacterial activity against skin resident bacteria.

The present invention provides a skin external preparation containing apolatatophorin, and in this skin external preparation,

The iron binding degree of the apolatatopherin is not more than 5%, and

When an aqueous solution containing the apolactophorin at a concentration of 1 w / v% is prepared, the total cation concentration in the aqueous solution is 5 mm o 1 Z L or less.

According to the present invention, a skin external preparation having high antibacterial properties against skin resident bacteria is provided. This topical skin preparation is safe and causes no side effects. Brief Description of Drawings

FIG. 1 is a graph showing the absorbance in an E. coli culture solution when apolactoferrin or lactoferrin obtained by each acid treatment is added at various concentrations.

FIG. 2 is a graph showing the absorbance in the E. coli culture solution when apolatatopherin was added together with various concentrations of sodium chloride.

FIG. 3 is a graph showing the absorbance in the culture medium of E. coli when various concentrations of apolatatophorin with different iron binding levels are added.

FIG. 4 is a graph showing the absorbance in the culture solution of E. coli when sample A, B, or C, which is apolactoferrin having various iron binding degrees and various total cation concentrations, is added.

FIG. 5 is a graph showing the absorbance in the culture solution of Staphylococcus epidermidis when sample A, B, or C, which is apolatatoferrin having various degrees of iron binding and various total cation concentrations, is added.

FIG. 6 is a graph showing the absorbance in the culture medium of Micrococcus luteu when various concentrations of apolatatoferin or lactoferrin are added. FIG. 7 is a graph showing the absorbance in Candida albicans culture medium when various concentrations of apolatatoferin or ratatopherin were added.

FIG. 8 is a graph showing the number of vaccinia colonies per dish when apolatatoferin or lactoferrin is added.

FIG. 9 is a graph showing the change in the dandruff score of the subject's scalp before and after the start of the test using ethanol water containing apolactoferrin or ethanol water containing no apolatatopherin.

Figure 10 shows the redness of the subject's scalp before and after the start of the test with ethanol water containing apolactoferrin or ethanol water without apolatatopherin. It is a graph which shows the change of a spot score. BEST MODE FOR CARRYING OUT THE INVENTION

(Aporactoferrin)

The external preparation for skin of the present invention contains apocyclic ferrin. Apolactoferrin is a glycoprotein molecule in which the iron bound in the lactoferrin molecule is released. Apolatopherin used in the present invention is not particularly limited as long as it has the following properties.

In the present invention, apolatatopherin has an iron bond degree in the molecule of 5% or less, preferably 4% or less, more preferably 3% or less. Here, the degree of iron binding refers to the ratio of the number of moles of iron to the number of moles of apolatatophorin. The degree of iron binding can be determined by measuring the absorbance of apolactoferrin by spectroscopic analysis, or by directly measuring the amount of iron in aporataferin by atomic absorption spectrometry or ICP spectroscopic analysis. . In the present invention, the degree of iron binding refers to a value obtained by dissolving an apo-ferferin powder in pure water to obtain an lw / v% solution, and measuring this with an absorbance of 470 ηm.

The apolactoferrin in the present invention has a total cation concentration of 5 mm o 1 ZL or less when an aqueous solution containing apolatatopherin at a concentration of 1 w / v% is prepared. To determine the total cation concentration, dissolve the apolactoferrin powder in 0.1 N hydrochloric acid to prepare a 0.1 lw / v% solution, and measure the amount of each cation by atomic absorption spectrophotometry. Obtain the concentration of each cation by adding the values. The total cation concentration may correspond to the salt (ion) contained as impurities in the apolatatopherin powder. This is because the above 0.1N hydrochloric acid can dissolve only the salt mixed in the powder, not the ions bound to lactoferrin. The total cation concentration is preferably 3. Ommo 1 / L or less, more preferably 1. Ommo 1 ZL or less. Apolatoferrin can be usually produced by adjusting the pH of an aqueous solution containing ratatopherin to the acidic side to dissociate the divalent iron ions of the ratatophorin molecule.

Lactoferrin, the raw material for Apolata tofrin, is separated and purified from mammalian secretions such as milk (eg, milk) or processed milk products such as skim milk and whey (eg, whey) (for example, adsorbed on cation exchange resin) And then desorbing with a high-concentration salt solution, separation method by electrophoresis, separation method using affinity chromatography, etc.). Furthermore, various cells obtained by gene recombination (including microorganisms, plant cells, animal cells, insect cells, etc.), plants, animals and the like may be used. Ratatopherin may be commercially available as a pharmaceutical, a reagent, or the like. Ratatofurin is preferably derived from natural products. Preferably, it is derived from whey. Whey obtained as a by-product generated when producing a dairy product (eg, cheese, casein, etc.) from cow's milk or skim milk can be suitably used as a source of ratatopherin.

Apo-actoferrin can be preferably produced, for example, by adding an acid to the liquid when ultrafiltration of the ratatopherin-containing liquid is carried out to dissociate iron ions bound to ratatofurin. . Examples of the acid that can be used here include citrate, hydrochloric acid, phosphoric acid, malic acid, or oxalic acid (0.4 M or more), and citrate is preferred. Alternatively, apolatatoferin is composed of, for example, bipolar membranes and force thione exchange membranes, which are composite ion exchange membranes having a structure in which a cation exchange membrane and an anion exchange membrane are bonded to each other. The use of an electrodialyzer having a partitioned acid chamber and a base chamber can also be suitably produced. In this case, hydrochloric acid produced during the manufacturing process using an electrodialyzer is used as the acid.

In the production of aporophylline in the present invention, the acidic side to be regulated The pH is preferably 0.5 to 3.0, more preferably 1.5 to 2.5. When the pH is near neutral (eg 5.5), the antibacterial activity of the resulting apolatatoferin may be weak. As a pH adjuster for an aqueous solution containing ratatopherin, not only the above-mentioned acids but also phthalic acid, glycine and the like can be used. These pH adjusters are added to an aqueous solution containing ratatoferrin in an amount appropriate for adjusting its pH to the above value.

The temperature at which the pH of the aqueous solution containing ratatopherin is adjusted to the acidic side is preferably not high in consideration of protein denaturation. Usually, it is 5 to 60 °, more preferably 15 ° to 35 ° C., and even more preferably room temperature. The specific production of apolatatopherin in the present invention will be described in detail in the following preparation examples, but the production method of aporaltophorin is not limited thereto. The apoloratoferrin in the present invention can also be obtained by modifying a commercially available apolactoferrin so as to have the above iron binding degree and total cation concentration.

In the production of Apolata topherin, Apolata topherin can usually be obtained in the form of an aqueous solution. When the external preparation for skin of the present invention is prepared using apolar lactin, it may be used in the form of an aqueous solution or in a powdered form after removing the solvent. (External preparation for skin)

The amount of apolatatopherin contained in the external preparation for skin of the present invention is not particularly limited because it varies depending on the dosage form. Preferably 0. 0 1% by mass to 20 ^_0/0 or 0. 0 lw / v% ~2 Ow / v%, more preferably 0.1 1 mass% to 1 0 wt ^_0/0 or 0. It is 1 w / v% to 10 w / v%, and even more preferably 1% to 5% by mass or 1 w / v% to 5 w / v%.

The external preparation for skin of the present invention further improves the appearance, feeling of use and storage stability. Therefore, if necessary, it may contain bases and additives usually used by those skilled in the art for external preparations for skin. Furthermore, various auxiliary agents can be added for the purpose of further enhancing or supplementing the function of the external preparation of the present invention.

Examples of the base include glycerol, ethanol, paraben, or butylene glycol. Examples of the additive include excipients (such as dextrins), fragrances, pigments, preservatives (such as parabens), and thickeners. (Silicon polymers, acrylic polymers, carboxyvinyl polymers, etc.), chelating agents (EDTA, etc.), sweeteners (sucralose, etc.), refreshing agents (menthol, etc.), antiseptic / antifungal agents (phenoxyethanol, etc.) Etc.

Examples of the auxiliary agents include other medicinal ingredients and other oils (unsaturated fatty acids such as linoleic acid, linolenic acid, palmitic acid, DHA, EPA, and derivatives thereof, linseed oil, coconut oil, jojoba oil, olive oil, etc. Oil, squalene, squalene, horse oil, rice oil, castor oil and other oils and derivatives thereof, moisturizers (collagen or its degradation products, collagen-like peptides contained in force lot extract, etc.) , Soy peptides, amino acids, mucopolysaccharides such as hyaluronic acid, amino sugars such as chondroitin, saccharides such as trehalose, seaweeds, water-soluble dietary fibers such as alginic acid, dalcomannan, pectin, phospholipids, etc.), surfactants (Lecithin, fatty acid ester, amino acid conductor, etc.), UV absorber (zinc oxide or And the like) and absorption enhancers.

The external preparation for skin of the present invention can take any form applicable to the skin. The dosage form of the external preparation for skin of the present invention can be, for example, a lotion, emulsion, gel, cream, ointment, powder, granule, tablet or the like. The external preparation of the present invention can be used as cosmetics, pharmaceuticals, quasi drugs, toiletries and the like. These contain components usually used by those skilled in the art, if necessary, and can be prepared according to methods usually used by those skilled in the art. For cosmetics, for example, makeup for cleaning It can also be used as cosmetics, basic cosmetics, finishing cosmetics, and hair cosmetics. Examples of cleansing cosmetics include facial cleansers, stone trials, and body shampoos. Examples of basic cosmetics include lotions, cosmetic creams, emulsions, and packs. Examples of finishing cosmetics include foundation, white powder, lipstick, lip gloss, scarlet, and eye shadow. Examples of hair cosmetics include chamboo, hair rinse, treatment, and styling agent.得る It can also be used for shaving agents, hair removal agents, depigmenting agents, etc. It can also be included in a sheet for wiping (for example, a wet tissue or a type of wiping paper). Examples of pharmaceuticals include hair restorers, aqueous ointments, oily ointments, ships, and gels.

Apolata topherin can be added to, added to, or incorporated into a topical skin preparation according to procedures commonly used by those skilled in the art. For example, after pre-dissolving in a solvent such as water or alcohol, mixing with other ingredients, or stirring and mixing with other ingredients as a powder, add, compound or include in a skin external preparation. obtain.

The external preparation for skin of the present invention is in the form as described above, and can be administered or applied to an individual according to the means usually used for administration or application in that form (for example, application, rubbing, spraying).

The external preparation for skin of the present invention can be effective against skin resident bacteria. Such as fungus, eg; J "3: Staphylococcus 晨 (Staphylococcus epidermidis, Staphylococcus aureus, etc.), Micro coccus _i / 禺 (Micrococcus luteu, etc.), Streptococcus 牌, Examples include Pityrosporumfe Candida genus (Candida albicans etc.), Propionibacteriumfe (Propionibacterium acnes etc.), Malassezia genus (Malassezia furfur etc.), Echerichia coli (Escherichia coli) etc.

The topical skin preparation of the present invention reduces unpleasant odor (deodorant action) It may have effects such as improvement (including anti-dandruff), prevention of acne, alleviation of atopic dermatitis, prevention of dermatitis (including pyoderma).

Further, the external preparation for skin of the present invention is excellent in skin moisture retention, and is suitable for daily use as a product such as cosmetics and toy retail products.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these exemplifications. Example

(Preparation Example 1: Production of apolactoferrin by various acid treatments)

LOV (hollow fiber module: membrane inner diameter 0.8 mm, effective membrane area 4 lm ² , membrane material: polyacrylonitrile) manufactured by Microza UF Lab Testing Machine (LX-22001; Asahi Kasei Chemicals Corporation)) Using an ultrafiltration apparatus incorporating a nominal molecular weight cut-off: 50,000), apolactoferrin was produced as follows.

10 kg of a 5 Omg / mL ratatopherin (Fontera; iron binding degree of about 20%) solution was used. In the production process of apolata topherin, lactoferrin was treated with any of the following acids: 0.1 M citrate, 0.1 M lactic acid, 0.1 M hydrochloric acid, 0.1 M malic acid, 0.1 M acetic acid, or 0. 4M acetic acid. First, the rato-ferrin solution was put into the supply tank of the apparatus, circulated for 10 minutes, and then circulated in the reverse direction for 5 seconds to concentrate the solution. At this time, the pressure at the inlet and outlet of the UF membrane and the circulating fluid flow rate were set to 0.12 Mpa, 0.08 Mpa, and 15 LZ, respectively. This operation was repeated until the non-permeate concentrate was halved (this is one round). Next, instead of the lactoferrin solution, the citrate solution was put into the tank, and the same operation as above was performed for two rounds. Next, 8 Ω · cm or more of pure water was put into the tank, and the above operation was performed for 5 rounds to remove the acid remaining in the non-permeated concentrate. The temperature of the circulating fluid is Throughout the manufacturing process, it was in the range of 10-28 ° C and the pH was 2-3. A concentrated solution of 4 O kg was obtained by the above production process. The concentrated solution was then freeze-dried to obtain 9.5 g of white powder.

BIOXYTECH (registered trademark) Lac. To f EIA ™ (OXIS International Inc. USA / Oregon) after the powder was dissolved in pure water, the powder obtained by each acid treatment was apolactoferrin and the purity of apolata toferin It was determined by performing antibody quantification using. The purity of apollatoferrin was as follows: 93% for 0.1 M taenoic acid, 94% for 0.1 M lactic acid, 96% for 0.1M hydrochloric acid, 9 for 0.1M malic acid. 1%, 9 1% with 0.1M acetic acid, 91% with 0.4M acetic acid.

Furthermore, the iron binding degree of the powder obtained by each acid treatment was dissolved in pure water so as to have a concentration of lw / v%, and then the amount of iron bound to apolitoferrin was determined as the absorbance at 470 nm. It was determined by measuring with. Table 1 shows the degree of iron binding of the obtained aporatopherin. Here, the degree of iron binding was calculated by the formula: degree of iron binding (%) = (number of moles of iron in lw / v% solution Zlw / v% number of moles of apolatatof phosphorus in solution) × 100. table 1

(Preparation Example 2: Production of apolactoferrin by various treatments with citrate) Except for increasing or decreasing the number of treatments with citrate, an apolatatopherin powder was produced according to the procedure described in Preparation Example 1 above, and the degree of iron binding was measured. Table 2 shows the iron binding degree of the obtained apoptophylline. Table 2

(Example 1: Apolatoferin antibacterial test using E. coli)

The antibacterial properties of the apolatatopherin powders prepared in Preparation Example 1 and treated with various acids were examined. As a control, lactoferrin powder (manufactured by Fontera) was used.

E. coli (NBRC 3972) purchased from the Biotechnology Headquarters, Biotechnology Headquarters, Biotechnology Headquarters, National Institute of Technology and Evaluation, E. coli (NBRC 3972) is subcultured in 5 mL of S CD bouillon (Nissui Pharmaceutical Co., Ltd.) Stored in culture (liquid). 50 μL of this stored E. coli solution was inoculated into 5 mL of SCD broth and cultured for 16 hours at 30 ° C. in a shaking water bath (preculture). The pre-cultured bacterial solution was diluted with sterile water to prepare 10-fold serial dilutions up to 10 ⁷ -fold. In each well of a 96-well flat-bottom microplate (BD Falcon), 50 μL of a series obtained by dissolving apo-actoferrin or ratatofurin powder in sterile water and doubling the dilution, 100 μL of SCD bouillon at 2 times concentration, And 1 of the dilutions prepared above The bacterial suspension of 0 ⁵ dilution was added 5 0 mu L. The microplate was cultured at 35 ° C for 24 hours. After culturing, the microplate was measured for turbidity (absorbance) at a wavelength of 630 nm using a microplate reader (Multiscan JX: Thermo Labsystems) to confirm the antibacterial action.

FIG. 1 is a graph showing the absorbance in an E. coli culture solution when apolatatophorin or ratatopherin obtained by each acid treatment was added at various concentrations. The horizontal axis indicates the concentration of apolatatoferin or ratatopherin (%) (w / v) in the culture medium. The vertical axis represents the absorbance, and the lower the absorbance, the smaller the number of viable bacteria, that is, the higher the antibacterial activity. As is clear from Fig. 1, antibacterial action was not seen with ratatofurin, whereas apolatatophorin showed antibacterial action with increasing concentration. In particular, the antibacterial effect of apolactoferrin obtained by treatment with citrate was most excellent.

(Example 2: Effect of iron binding degree in antibacterial test of apolactoferrin using E. coli)

Using the apolactoferrin obtained in Preparation Example 2 above, the commercially available lactoferrin (manufactured by Fontera), and the commercially available apolactoferrin (manufactured by Taza Daily) in the same manner as in Example 1 above, Antibacterial activity against was investigated. Here, the degree of iron binding of commercially available ratatopherin was 19.8%, and the degree of iron binding of commercially available apoactoferrin was 4.39%.

FIG. 2 is a graph showing the absorbance in the culture solution of E. coli when various concentrations of apolactophorin with different iron binding levels are added. The horizontal axis indicates the concentration of apolactoferrin (%) (w / v), and the vertical axis indicates the absorbance. The lower the absorbance, the smaller the number of viable bacteria, that is, the higher the antibacterial activity. From FIG. 2, lactoferrin, whose iron binding degree was 19.8%, did not show any decrease in absorbance and no antibacterial action was observed. For apolatatophorin with an iron binding level of 5% or less, The absorbance decreased with the increase, and an excellent antibacterial effect was observed. In addition, apolata toporin with an iron bond degree of about 4% or less (iron bond degree 4.09%, iron bond degree 2.95%, iron bond degree 2.2%, iron bond degree 1.66%, and iron At a binding degree of 1.0 4%), sufficient antibacterial activity was observed even in an aqueous solution containing apolatapherin at a concentration of 5 w / v%.

(Example 3: Effect of salt in antibacterial test for apolatatopherin using Escherichia coli)

In the antibacterial test of Escherichia coli of Example 1 above, the influence of salt was examined using citrate-treated apolatatophorin. The antibacterial activity was examined according to the same procedure as in Example 1 except that sodium chloride was added to the lw / v% citrate-treated apolactoferrin solution so as to have various ion concentrations.

FIG. 3 is a graph showing the absorbance in an E. coli culture solution containing various concentrations of sodium chloride and 2. Ow / v% apolar lactin. The horizontal axis indicates sodium chloride concentration (mmo 1 / L), and the vertical axis indicates absorbance. The lower the absorbance, the smaller the viable count, that is, the higher the antibacterial activity. In Fig. 3, the antibacterial action decreased as the salt concentration increased. Therefore, it is understood that the amount of salt that can be mixed as impurities in the apopherophine powder affects the antibacterial properties. Furthermore, it was found that the amount of such salt is desirably 5 mm o 1 / L or less for all ions when 1% solution of apolatatopherin is used.

Therefore, the total cation concentration of apolatatophorin having an iron binding degree of 2.95% obtained in Preparation Example 2 was determined. By adding 0.1N hydrochloric acid to lyophilized powder of apolactoferrin to prepare 0.1.lw / v% apolactoferrin solution and measuring Na, K :, Ca, Mg, and Cu by atomic absorption spectrophotometry. The concentration of each of these cations was determined, and the total was obtained as the total cation concentration. For comparison, the total cation concentration of commercial ratatofurin and apolatatophorin was also determined. The results are shown in Table 3. Table 3

Apolatofurin with a degree of iron binding of 2.95% obtained in Preparation Example 2 had a total cation concentration of 5 mm o 1ZL or less. In Example 2, in the case of commercially available apolata topherin (manufactured by Tazza Dairy), the absorbance decreased to a certain level or more even though the iron binding degree was 5% or less. As shown in this example, the aporal ferrin has a total cation concentration exceeding 5 mm o 1ZL. Therefore, it was considered that both iron binding degree and total cation concentration of apolatatopherin powder affect antibacterial properties.

(Example 4: Antibacterial test of apolactoferrin having a predetermined degree of iron binding and total cation concentration)

Considering the results of Example 3 above, the antibacterial activity of apolatatopherin having an iron binding degree of 5% or less and a total cation concentration of 5 mmo 1 / L or less was further examined. The following three types of apo-lactoferrin were used: Sample A (Aporatatophorin commercially available: 4.39% iron binding, 14.7 m total cation concentration) mo l / L); Sample B (Sample A was retreated at a temperature of 25 ± 1 ° C and pH 2.6 according to the method of Preparation Example 1: iron binding degree 3.89%, total cation concentration 4.5 m mo l / L); and Sample C (apolatatophorin obtained in Preparation Example 2 above: degree of iron binding 4.56%, total cation concentration 4.4 mmol ZL).

Antibacterial activity was tested using Escherichia coli and Staphylococcus epidermidis (NBRC 3972 and NBRC 13889, respectively, obtained from the National Institute for Biotechnology, Biotechnology Headquarters, NBRC 3972 and NBRC 13889, respectively) The procedure was the same as in Example 1 except that. Staphylococcus epidermidis is a skin-resident bacterium that causes odor. The final concentration of apolatatophorin was 2 OmgZmL.

Figures 4 and 5 show the graphs showing the absorbance in the culture solution of Escherichia coli and Staphylococcus epidermidis when samples A, B, and C are added, respectively. The vertical axis represents the absorbance, and the lower the absorbance, the smaller the viable count, that is, the higher the antibacterial activity. Sample A, which has an iron binding degree of 5% or less but a total cation concentration of more than 5 mm o 1 / L, showed little difference in absorbance compared to the culture medium without addition of apolatatoferrin. In contrast, Samples B and C, which have an iron binding degree of 5% or less and a total cation concentration of 5 mm o 1 / L, are compared with the culture medium without the addition of apolactoferrin. The absorbance was low and it showed good antibacterial properties.

(Example 5: Antibacterial test of Apolata topherin against Micrococcus spp.)

The antibacterial activity against bacteria belonging to the genus Micrococcus was examined for the apolatatophorin having an iron binding degree of 2.95% prepared in Preparation Example 2 above. As a control, commercially available ratatopherin (manufactured by Fontera) was used. Micrococcus bacteria may be skin resident bacteria involved in the development of unpleasant odors. The test is Micrococcus The procedure was the same as in Example 1 except that luteu (NBRC 13867: Independent Administrative Institution · National Institute of Product Evaluation Technology Biotechnology Logistics Division (National Biogenetic Resource Division) (NBRC)) was used.

FIG. 6 is a graph showing the absorbance in the culture medium of Micrococcus luteu when various concentrations of apolatatophorin (Apo-Lfn) or lactoferrin (Lfn) are added. The horizontal axis shows the concentration of apolatatoferin or lactoferrin added (%) (w / v), and the vertical axis shows the absorbance. The lower the absorbance, the smaller the number of viable bacteria, that is, the higher the antibacterial activity. From Fig. 6, it was also observed that lactoferrin did not show much antibacterial effect in bacteria of the genus Micrococcus, whereas apo lactoferrin showed antibacterial action as the amount of addition increased.

(Example 6: Antibacterial test of apolatatopherin against Candida spp.) About apolactoferrin with a degree of iron binding of 2.95% prepared in Preparation Example 2 above, antibacterial against Candida spp. I examined the sex. Commercially available Ratatov ヱ Rin (manufactured by Fontera) was used as the contrast. Candida bacteria can contribute to dermatitis. The test is Candi da albicans (NBRC 1594: Independent Administrative Law, Biotechnology Division, Biotechnology Headquarters, Biotechnology Headquarters)

(Available from (NBRC)), and YM broth (Nippon Betaton Dickinson Co., Ltd.) was used as the growth medium for this bacterium, and the culture conditions for preculture and main culture were 24 hours at 24 ° C. Except for this, the same procedure as in Example 1 was performed.

FIG. 7 is a graph showing the absorbance in the Candida albicans culture medium when various concentrations of apolactoferrin (Apo-Lfn) or lactoferrin (Lfn) were added. The horizontal axis is the concentration of apolatatoferin or ratatopherin added

(%) (w / v), and the vertical axis represents absorbance. The lower the absorbance, the smaller the number of viable bacteria, that is, the higher the antibacterial activity. From Fig. 7, it can be seen that ratatopherin does not show much antibacterial activity even in Candida spp. In the case of kutoferin, the antibacterial effect was seen as the amount of addition increased.

(Example 7: Antibacterial test of apolatatoferrin against Propionibacterium acnes)

The antibacterial activity against acne bacteria was examined for the apolactoferrin having an iron binding degree of 2.95% prepared in Preparation Example 2 above. As a contrast, commercially available ratatofrin (manufactured by Fontera) was used. Acne is a skin-resident bacterium that causes acne. Propionibacterium acnes (manufactured by THE VAN Kampen Group, Inc.) was introduced, and a liquid medium S CD Bouillon (Nissui Pharmaceutical Co., Ltd.) was prepared and placed under anaerobic conditions (anaerobic container and anaerobic atmosphere). In an anaerobic glove box, apolactoferrin or ratatopherin was mixed with the liquid medium so that their concentration was 2 w / v%. The cells were cultured for 3 days under anaerobic conditions, and the bacteria were diluted 10 times and transferred to SCD agar medium (Nissui Pharmaceutical Co., Ltd.). Furthermore, the cells were cultured for 3 days under anaerobic conditions, and the number of colonies formed was counted. The number of colonies was determined by averaging 10 dishes.

FIG. 8 is a graph showing the number of Acne colonies per dish when apolatatoferin or ratatopherin is added. Compared with the addition of lactoferrin, which was the control group, the number of colonies was smaller when apolactoferrin was added, and the antibacterial activity of apolactoferrin was also confirmed against akune bacteria.

(Example 8: Antibacterial test of apolactoferrin against Malassezia furfur)

The anti-bacterial activity against Malassezia was investigated for 2.95% iron porphyrin prepared in Preparation Example 2 above. Malassezia is a skin resident yeast that causes seborrheic dermatitis that can cause dandruff hair loss.

Malassezia furfur (Independent Administrative Institution The YM liquid medium (manufactured by Difco) is adjusted to pH 5.6 with hydrochloric acid before adding olive oil, and the medium is adjusted to 10 OmL. Olive oil (10 g) was added. 1 platinum loop suspension of slant culture was suspended in 5 O mL of this medium to obtain a McFarland 2 suspension (about XI 0 ⁸ ). Using this suspension as a stock solution, 10-fold, 100-fold and 100-fold dilutions were prepared, and these were used as test bacterial solutions.

A 10% (w / v, lOOmg / mL) solution is prepared by dissolving the iron binding degree 2.95% prepared in Preparation Example 2 above in distilled water, and then a 0.45 / zm solution. Sterile filtered with a filter. Then, it was diluted to 4% and 2% (both w), and these were used as sample solutions.

The sample solution 500 μL and the bacterial cell suspension 500 ju ju L were added to the 24-well plate tool. Sterile water was used as a blank. For this reason, the concentration of apolatatophorin in each test group is diluted 2-fold to 5%, 2%, and 1% (all are w-8) as shown in the table below. The culture was allowed to stand at 30 ° C for 3 days, and the growth of the bacteria was visually confirmed. The test was performed under the condition of n = 3. The results are shown in Table 4 below. In the table, + indicates “fungus growth” and 1 indicates “fungus growth could not be detected”. Table 4

As shown in Table 4 above, Malassezia furfur grows in the water. In contrast, no growth of Malassezia furfur was observed in any of the concentrations in the Apollataferin Test Zone.

(Example 9: Effect of apolatatopherin on scalp)

Using the iron binding degree of 2.95% prepared in Preparation Example 2 above, the effect on the scalp was examined in a double-blind study using volunteers.

Prepare a solution (also referred to as apolitatopherin-containing ethanol water) containing 1% of the iron binding degree 2.9 5% prepared in Preparation Example 2 above in 5 _v / v% ethanol water and bottled. did. With 5 v ^_0/0 ethanol free water Aporata Tofuwerin as control (also referred to as Aporata Tofuerin-free aqueous ethanol). All volunteers (10 people) had their scalp and hair washed one week before the start of the study using the same commercially available shampoo and pre-sales conditioner. From the start of the test, 5 volunteers from each of these volunteers should apply either apolactofurin-containing ethanol water or apolatatof-phosphorus-free ethanol water to the ground contact skin after washing the scalp and hair as described above. It was. This application test was conducted for 4 weeks. The scalp was diagnosed by a dermatologist before and after the study. The volunteer's head is divided into the right frontal head, left frontal head, upper head, right back head, and left back head, so that the light hits the judgment area vertically, and the amount of dandruff is determined by the dermatologist. Scoring and scalp erythema determination were performed.

(Standard for scoring the amount of dandruff)

1 No dandruff

2 Extremely dandruff

3 Dandruff that anyone can check

4 Medium amount of dandruff 5 Large amounts of dandruff

(Criteria for scalp erythema)

1 No redness

2 Some are slightly red

3 Overall slightly red

4 Entirely red

Figure 5 and Figure 10 show the results of dandruff scoring and scalp erythema determination, respectively. In Fig. 9, the vertical axis shows the dandruff score shown above, and the horizontal axis shows the course before and after the test. In Fig. 9, the black circles represent the results for ethanol water containing apolatatopherin, and the white circles represent the results for ethanol water not containing apolatatopherin. In Fig. 10, the vertical axis shows the score of erythema shown above, and the horizontal axis shows the course before and after the test. Similarly, in FIG. 10, black circles represent the results of ethanol water containing apolactoferrin, and white circles represent the results of ethanol water not containing apolatapherin.

As shown in Fig. 9, in ethanol water containing apolactoferrin, dandruff score decreased after the test and the amount of dandruff decreased. In addition, as shown in Fig. 10, it was found that with ethanol solution containing apolactoferrin, the redness score decreased after the test and the scalp condition could be improved.

(Example 10: Moisturizing effect of apollatatophosphorus)

In order to measure the skin moisturizing effect, 125 women (average age 21.3 ± 6.5 years old) were moisturized (skin moisture in the forehead, eyelids, heels, and forearms). The amount ^_0/0) and the pH Moisture Checker (MY- 7 0 7 S; Sato Shoji Ltd., Japan) and Skin ρΗ meter (Skincheck-1; Sato Shoj i Ltd., was measured using a Japan).

After measuring the moisture retention and pH of the bare skin where the subject's makeup was sufficiently removed, whey water (made by Ohm Dairies) was applied to the right and right arms of the face with cotton. Also, on the left and left arms of the face, use a solution of 102.5 mg / ml of apolata topherin with iron binding degree 2.95% prepared in Preparation Example 2 in whey water (Ohm Milk Industry Co., Ltd.). It was applied with. Both application sites were air-dried for 5 minutes and then the moisture retention and pH were measured. These results are shown in Table 5 and Table 6 below. Table 5

(The result is the water content%) Table 6

(The result is the pH value) From the results shown in Tables 5 and 6, it was found that the skin moisturizing effect was retained when apolactofurin was contained.

In addition, 70 women (average age 28 ± 7.6 years) were treated with whey water (Ohm Milk Industry Co., Ltd.) at 10 mg / ml, and the iron binding degree 2.9 5% aporatatopherin prepared in Preparation Example 2 above. The solution was dissolved and bottled, and used as a lotion for one month in the morning and evening. As a result, many respondents said that a moist feeling was obtained.

Therefore, Apolactoferrin is expected to have a skin moisturizing effect as well as its antibacterial properties.

(Example 11: Combination of aporactofylline with various reagents)

The effect of the iron binding degree 2.95% prepared in Preparation Example 2 above on antibacterial properties was examined in combination with various reagents. The combined reagents used are shown in Table 7 below.

The reagent was diluted in distilled water by a micro liquid dilution method using a microtiter, and the concentrations of the reagent were changed to prepare an apolatatophorin solution and a concomitant reagent solution. Both the apolatatopherin solution and the concomitant reagent solution were prepared so as to have a final concentration of 2 w / v%, and diluted sequentially. 10 ⁴ to 10 ⁵ cfu / m E. coli (NBRC 3972: Incorporated Administrative Agency · National Institute of Technology and Evaluation Technology Biotechnology) (Obtained from the Division of Biological and Genetic Resources (NBRC)) and incubated at room temperature for 24 hours. Next, the presence or absence of bacterial growth was determined by measuring the absorbance with a microplate reader, the minimum growth inhibitory concentration (MIC) was determined, and the fractional inhibitory concentration index (FI and index) was calculated according to my own equation. : FIC index

= Ap o solution at the time of combination MI C / Ap o solution at the time of single use Concomitant reagent at the time of combined use of MI C + MI CZ single-use reagent MIC From the obtained FIC index, ≤0.5 is synergistic, 0.5 <~ <2 The additive effect, =

Evaluation was made with 2 as unrelated and> 2 as an antagonistic relationship.

2 Casein peptide (HCP301) Tatua

3 Growth Factor Concentration (GFC) Ί atua Taza 'Japan Co., Ltd.

4 GLYCOMACROPEPTIDE (GMP) Tatua 7

5 Hydrolysed Casein Protein 105 (HCP 105) Tatua

6 Casein peptide (HCP 321) Tatua

7 Whey Peptide (HWP 202) Tatua

8 Whey peptide (HWP 205) Tatua

9 Whey peptide (HWP 214) Tatua

10 Whey Peptide (HWP 406) Tatua

1 1 Whey Peptide (HW-3) Snow Brand Taza 'Japan Co., Ltd. Snow Brand Milk Products Co., Ltd.

12 Nisin Avios SIGMA

13 Masahiro Ascorbate Co., Ltd. Nakarai Tesque Co., Ltd.

14 Poly-L-lysin SERVA Electrophoresis GmbH

15 L-Arginine Shogo Co., Ltd. Wako Pure Chemical Industries, Ltd.

16 Glycine Shogo Co., Ltd. Wako Pure Chemical Industries, Ltd.

17 Methyl p-hydroxybenzoate (methylparaben) Masami Co., Ltd. Wako Pure Chemical Industries, Ltd.

18 Cetylpyridinium chloride monohydrate (CPC) Shogo Co., Ltd. Wako Pure Chemical Industries, Ltd.

19 Polyphosphate Masami Co., Ltd. Wako Pure Chemical Industries, Ltd.

20 Potassium 2,4-hexagenate (sorbic acid K) Masami Co., Ltd. Wako Pure Chemical Industries, Ltd.

21 2-Phenoxyethanol (2-PE) Masami Co., Ltd. Wako Pure Chemical Industries, Ltd.

This results in a synergistic effect when the concomitant reagents are Nisin and Poly-L-lysin, and the concomitant reagents are citrate, whey peptide (HWP 202) Tatua, ascorbic acid, Glycine, methylparaben, cetylpyridinium chloride (CPC;) and 2,4-hexagenate potassium (sorbic acid K) showed an additive effect. Industrial applicability

The external preparation for skin of the present invention has high antibacterial properties against skin resident bacteria. It is safe and does not cause side effects. Therefore, the external preparation for skin of the present invention is used on a daily basis such as cosmetics and toiletries for the prevention of acne, atopic dermatitis, pyoderma, reduction of unpleasant odor, and improvement of the scalp condition. It can be used suitably also for the product used.

Claims

Billed

1. a topical skin preparation containing apolactoferrin,

The iron binding degree of the aporactophylline is 5% or less, and

When preparing an aqueous solution containing the apolatatopherin at a concentration of lw / v%, the total cation concentration in the aqueous solution is 5 mm o 1ZL or less.

Skin external preparation.