Agent for improving or diversifying normal skin flora and composition containing same

Abstract

To provide agents or compositions for improving or variegating normal bacterial flora of skin, in particular, to provide agents or compositions for improving or variegating normal bacterial flora of skin which exhibit bacteriostatic or bactericidal activities only to harmful bacteria without damaging useful normal bacteria of human skin.SOLUTION: Disclosed is an agent for improving or variegating normal bacterial flora of skin which comprises lactoferrin or salt thereof as an effective ingredient. Also disclosed is a pharmaceutical composition or cosmetic composition comprising the agent for improving or variegating normal bacterial flora of skin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for improving or diversifying the normal flora of the skin and a composition for improving or diversifying the normal flora of the skin. [Background technology]

[0002] The skin is home to the second largest number of bacteria after the intestines, with approximately 1 trillion resident bacteria, equivalent to approximately 1,000 species, forming the resident skin flora. In recent years, it has been reported that individual differences in this resident skin flora are involved in the maintenance of skin homeostasis and disease. Healthy human skin is slightly acidic, and this pH regulation is achieved by Staphylococcus epidermidis and other bacteria present in various parts of the skin producing fatty acids with antibacterial properties, which suppress the growth of Staphylococcus aureus, which prefers a slightly alkaline environment.

[0003] It is known that Staphylococcus aureus is predominant in the affected areas of the skin of patients with atopic dermatitis, and that during the exacerbation phase, the diversity of the skin bacterial flora decreases and the proportion of Staphylococcus aureus increases. Myles et al. reported that when Roseomonas mucosa, a normal skin bacterium collected from a healthy individual, was transplanted into a mouse model of atopic dermatitis, the symptoms improved, but when Roseomonas mucosa was collected from the skin of atopic dermatitis patients, the symptoms remained unchanged or worsened (Non-Patent Document 1). They also reported that when Roseomonas mucosa collected from a healthy individual was applied to patients with atopic dermatitis, the severity was reduced by half, and no side effects or complications occurred (Non-Patent Document 2).

[0004] In this way, creating an appropriate environment on the skin, increasing beneficial bacteria, and improving bacterial diversity will suppress the numbers of harmful Staphylococcus aureus and Pseudomonas aeruginosa bacteria, and is expected to improve the skin flora and maintain healthy skin.

[0005] Currently, a wide variety of drugs and cosmetic ingredients have been reported that have bacteriostatic or bactericidal effects against Staphylococcus aureus, etc. However, for example, N-acylglutamic acid-containing cleansers and various plant extracts have bacteriostatic effects that are equal to or greater than those against beneficial skin flora such as Staphylococcus epidermidis, which are also resident skin bacteria belonging to the same genus Staphylococcus, and therefore have been inappropriate as agents for improving the resident skin flora.

[0006] Furthermore, an agent for adjusting the ecosystem balance of skin resident bacteria containing bittern and rosemary extract or licorice extract (Patent Document 1, JP 2005-139075 A) is insufficient in effectiveness, there is no disclosure of its bacteriostatic properties against Pseudomonas aeruginosa, and it is expensive and therefore not suitable for practical use. A skin resident flora improver containing cellobiose (Patent Document 2, JP 2008-50322 A) discloses only in vitro effects of its bacteriostatic action, and examples on humans only evaluate hair and moisturizing sensation, so its actual effect on improving skin resident flora is unknown.

[0007] Lactoferrin is a glycoprotein with a molecular weight of approximately 80,000 that is primarily found in mammalian milk, but is also found in neutrophils, tears, saliva, nasal secretions, bile, and semen. Lactoferrin's physiological activities include antibacterial, iron metabolism regulation, cell proliferation activation, hematopoietic, anti-inflammatory, antioxidant, phagocytosis enhancement, antiviral, bifidobacterial growth promotion, anticancer, anti-cancer metastasis, and translocation inhibition. Furthermore, recent studies have demonstrated that lactoferrin also has lipid metabolism-improving, analgesic, anti-stress, and anti-aging properties. Lactoferrin is a multifunctional, bioactive protein with diverse functions. It is expected to be used in pharmaceuticals and foods to restore or improve health, and lactoferrin-containing foods are already commercially available.

[0008] However, although in vitro evaluation of lactoferrin's effects on normal skin bacteria has reported that it also has antibacterial activity against Staphylococcus epidermidis, it was not known what kind of antibacterial effect it actually has on the skin. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-139075 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-50322 [Non-patent literature]

[0010] [Non-Patent Document 1] JCI Insight. 2018 May 3;3(9). pii: 120608. doi: 10.1172 / jci.insight.120608. “First-in-human topical microbiome transplantation with Roseomonas mucosa for atopic dermatitis.” Myles, et al. [Non-patent document 2] JCI Insight. 2016 Jul 7;1(10). pii: e86955. “Transplantation of human skin microbiota in models of atopic dermatitis.” Myles, et al. Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide an agent and a composition for improving or diversifying the normal skin flora.A further object of the present invention is to provide an agent and a composition for improving or diversifying the normal skin flora that have a bacteriostatic or bactericidal effect only on generally harmful bacteria, without particularly damaging the normal skin flora that are beneficial to human skin. [Means for solving the problem]

[0012] The present inventors conducted an intensive search for a substance that has bacteriostatic or bactericidal activity against generally harmful bacteria among the normal skin flora but does not harm the beneficial bacteria on human skin. As a result, they surprisingly found that lactoferrin, although it exhibits bacteriostatic or bactericidal activity against beneficial bacteria in vitro, affects only harmful bacteria on human skin, increasing the diversity of normal skin bacteria and improving the normal skin flora, and thus completed the present invention.

[0013] That is, according to the present invention, [1] An agent for improving or diversifying the normal skin flora, containing lactoferrin or its salt as an active ingredient; [2] A composition comprising the agent for improving or diversifying the normal skin flora according to [1] above; [3] a pharmaceutical composition comprising the agent for improving or diversifying the normal skin flora described in [1]; [4] A cosmetic composition comprising the agent for improving or diversifying the skin's resident bacterial flora described in [1] above; [5] The pharmaceutical composition according to the above [3], which is an external preparation for skin containing lactoferrin or a salt thereof in an amount of 0.001 to 50% (W / W); [6] The cosmetic composition according to [4] above, containing lactoferrin or a salt thereof in an amount of 0.00001 to 50% (W / W); [7] The cosmetic composition according to [6] above, which is a bath additive containing lactoferrin or a salt thereof in an amount of 0.00001 to 10% (W / W). is provided. [Effects of the Invention]

[0014] The agent or composition of the present invention does not exhibit bactericidal activity against Staphylococcus epidermidis, which is beneficial to the skin, but has bactericidal or growth-inhibitory activity against harmful Staphylococcus aureus, which can promote the diversification of the normal skin flora. Furthermore, since lactoferrin is a component of milk, mucus, and neutrophils, it is very safe whether administered orally or used as an external preparation, with no reported side effects, and can be used in combination with various ingredients without impairing the biological activity of other drugs. Therefore, the agent or composition of the present invention is also very safe and can be used in combination with other ingredients without any problems. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows calibration curves prepared for Staphylococcus epidermidis (left) and Propionibacterium acnes (right). [Figure 2] FIG. 2 is a diagram showing a comparison of the normal skin bacterial flora before and after 4 weeks of application of the composition of the present invention. [Figure 3] FIG. 3 is a diagram showing a comparison of the Shannon diversity index of the indigenous skin flora before and after 4 weeks of application of the composition of the present invention (per individual). [Figure 4] FIG. 4 is a graph showing a comparison of the Shannon diversity index of the indigenous skin flora before and after 4 weeks of application of the composition of the present invention (average). [Figure 5] FIG. 5 is a graph showing the average rate of change in normal skin bacteria (good bacteria: Staphylococcus epidermidis, bad bacteria: Staphylococcus aureus, opportunistic bacteria: Propionibacterium acnes) after applying the composition of the present invention for 4 weeks. [Figure 6] FIG. 6 is a photograph showing a comparison of the keratinous skin before and after 4 weeks of application of the composition of the present invention. [Figure 7] FIG. 7 is a photograph showing the keratin grade. [Figure 8] FIG. 8 shows a comparison of the keratin grade (Panel A), nucleus count (Panel B), and multilayer peeling area (Panel C) before and after 4 weeks of application of the composition of the present invention. [Figure 9]FIG. 9 shows the results of a questionnaire using a visual analogue scale (VAS) given to subjects after applying the composition of the present invention for 4 weeks. DETAILED DESCRIPTION OF THE INVENTION

[0016] An agent for improving or diversifying resident skin flora in one embodiment of the present invention contains lactoferrin or a salt thereof as an active ingredient. Another embodiment of the present invention is a composition for improving or diversifying resident skin flora, which contains the agent for improving or diversifying resident skin flora of the present invention, which contains lactoferrin or a salt thereof as an active ingredient, together with one or more other ingredients.

[0017] The "lactoferrin" used in the present invention is not limited to lactoferrin derived from mammalian milk or other sources, but may be any lactoferrin that exhibits the biological activity of lactoferrin, particularly the improvement or diversification of skin flora. Examples include natural lactoferrin (e.g., bovine lactoferrin contained in bovine milk) obtained from various mammals, including humans (e.g., cows, horses, pigs, sheep, goats, camels, etc.), apolactoferrin (iron-free form) obtained by removing iron from lactoferrin using conventional methods, metal-saturated or unsaturated lactoferrin obtained by chelation of apolactoferrin with metal ions (e.g., iron, copper, zinc, manganese, etc.), recombinant lactoferrin produced by genetic engineering, and lactoferrins bound to polyethylene glycol chains. Recombinant lactoferrin includes recombinant lactoferrin produced based on modified lactoferrin genes, as well as lactoferrin produced by transgenic animals and functional equivalents such as active fragments of lactoferrin.

[0018] The lactoferrin salts that can be used in the present invention are any physiologically acceptable salts of lactoferrin as described above, such as sodium salts, potassium salts, sulfate salts, phosphate salts, and the like.

[0019] The agent or composition for improving or diversifying the normal skin flora of the present invention may contain only one type of lactoferrin as described above, or may contain two or more types. Since lactoferrin is a known substance, commercially available products can be used. Alternatively, lactoferrin isolated or purified from lactoferrin-containing milk or other sources by known methods, such as a method for purifying lactoferrin using a sulfonated carrier (Japanese Patent Laid-Open Publication No. 3-109400), can be used. Furthermore, depending on the application, fractions from milk or other sources containing high concentrations of lactoferrin (for example, fractions obtained by removing sugars from milk) can also be used.

[0020] The agent for improving or diversifying resident skin flora of the present invention contains lactoferrin as the only essential ingredient, but may contain other active ingredients. Furthermore, if desired, it may contain various ingredients and additives known in the pharmaceutical, food, cosmetics, and other industries. The composition for improving or diversifying resident skin flora of the present invention contains one or more of the above-mentioned ingredients together with the agent for improving or diversifying resident skin flora of the present invention. The composition of the present invention may be a pharmaceutical composition, a food or beverage composition, a cosmetic composition, or the like, and their specific form is not particularly limited. Furthermore, the route of administration (or ingestion or application) of the agent or composition for improving or diversifying resident skin flora of the present invention can be appropriately selected from, for example, oral, transdermal, enteral, and rectal routes.

[0021] The pharmaceutical composition according to one embodiment of the present invention is preferably in the form of an external preparation for skin (ointment, cream, lotion, etc.).

[0022] In the context of the present invention, the term "cosmetic composition" refers to any composition that is used by being applied or stuck to the skin or the like and is not classified as a pharmaceutical composition, and includes so-called basic cosmetics, makeup cosmetics, medicated cosmetics, perfumes and colognes, finishing cosmetics, skin cosmetics, hair cosmetics such as hair tonics, special-purpose cosmetics, shampoos, conditioners, soaps, bath additives, and the like.

[0023] Examples of additives that may be contained in the composition of the present invention include excipients, disintegrants, lubricants, binders, surfactants, flow enhancers, colorants, solvents, thickeners, dispersants, pH adjusters, moisturizers, stabilizers, preservatives, fragrances, etc. that are routinely used in the pharmaceutical, food, cosmetic, etc. industries. These additives are appropriately selected depending on the desired dosage form, etc.

[0024] For example, when a composition such as the pharmaceutical composition of the present invention is in the form of a powder, granules, tablets, capsules, etc., excipients to be used include monosaccharides or disaccharides such as lactose, sucrose, glucose, sorbitol, lactitol, etc., starches such as corn starch and potato starch, crystalline cellulose, and inorganic substances such as light silica gel, synthetic aluminum silicate, magnesium aluminometasilicate, calcium hydrogen phosphate, silicon dioxide, etc. In addition to the excipients, binders, disintegrants, surfactants, lubricants, flow enhancers, colorants, flavors, etc. may also be used as needed.

[0025] Disintegrants include starches, carboxymethylcellulose (CMC), hydroxypropylcellulose (HPC), carboxymethylcellulose sodium salt, and polyvinylpyrrolidone. Examples of binders include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, carboxymethylcellulose sodium salt, methylcellulose, crystalline cellulose, ethylcellulose, and polyvinylpyrrolidone.

[0026] Examples of surfactants include soybean lecithin and sucrose fatty acid esters; examples of lubricants include talc, wax, sucrose fatty acid esters, hydrogenated vegetable oil, calcium stearate, and magnesium stearate; and examples of flow enhancers include anhydrous silicic acid, dried aluminum hydroxide, and magnesium silicate.

[0027] Pharmaceutical or cosmetic compositions for topical administration, such as topical skin preparations, can be formulated into various dosage forms, such as liquids, solids, and semisolids, by blending them with commonly used and known ingredients depending on the intended use. Preferred compositions include ointments, gels, creams, sprays, patches, lotions, emulsions, solutions, suspensions, and powders. For example, a pharmaceutical or cosmetic composition for topical skin preparations can be prepared by mixing the agent for improving or diversifying the normal skin flora of the present invention with one or more ingredients, such as hydrocarbons (e.g., petrolatum), higher fatty acid lower alkyl esters (e.g., stearyl alcohol and isopropyl myristate), animal fats and oils (e.g., lanolin), polyhydric alcohols (e.g., glycerin), surfactants (e.g., glycerin fatty acid esters and polyethylene glycol monostearate), thickeners (e.g., carboxymethylcellulose, hydroxypropylcellulose, and xanthan gum), humectants (e.g., sodium hyaluronate and sodium pyrrolidonecarboxylate), inorganic salts, organic salts, waxes, resins, water, and preservatives (e.g., phenoxyethanol and methyl parahydroxybenzoate).

[0028] The agent or composition of the present invention may be administered or used alone or in combination with other drugs. When the agent or composition of the present invention is used in combination with other drugs, the two may be used simultaneously or in succession.

[0029] The daily administration (or ingestion or application) amount of the agent or composition of the present invention effective for improving or diversifying the normal skin flora varies depending on the formulation, method and route of administration, the age and body weight of the subject, etc. For example, in the case of oral administration, the active ingredient lactoferrin is generally administered or ingested in humans at a dose of about 0.001 g to about 10 g / kg / day, preferably about 0.001 g to about 2 g / kg / day, and most preferably about 0.01 g to about 1 g / kg / day, all at once or in divided doses. For external use, for example, a composition containing the active ingredient lactoferrin or a salt thereof in an amount of 0.001 to 50% (w / w), or in the case of a cosmetic composition, 0.00001 to 50% (w / w), can be applied once to several times a day. For bath additive compositions, for example, a composition containing lactoferrin or a salt thereof in an amount of 0.00001 to 10% (W / W) can be prepared and dissolved in water or warm water so that the lactoferrin concentration at the time of use is 0.000002 to 0.2% (W / V).

[0030] The agent or composition of the present invention can be administered (or ingested or applied) to humans and non-human animals, preferably mammals. Examples of non-human animals include livestock such as cows, horses, pigs, and sheep, and companion animals such as dogs and cats. The dosage of the topical agent can be the same as that described above.

[0031] Whether or not the skin bacterial flora has been improved or normalized by the agent or composition of the present invention can be determined by calculating the diversity index of the normal skin bacterial flora using a bacterial flora analysis method based on amplification of the 16S rRNA gene using samples collected from the skin of a subject at two or more time points before, during, or after administration (or ingestion or application) of the agent or composition of the present invention, or after administration (or ingestion or application), and comparing the values ​​of multiple samples collected at different time points.

[0032] The specimen can be any sample collected from the skin that contains skin bacteria, and the collection method is not limited. For example, commercially available tape for collecting skin resident bacteria can be used conveniently.

[0033] The combination of time points for collecting samples is not particularly limited, and may be any time point before, during, or after administration (or ingestion or application) of the agent or composition for improving or diversifying skin resident flora of the present invention, as long as there is an interval of one day or more. Samples taken at two or more time points preferably include at least one sample taken during or after administration (or ingestion or application) of the agent or composition for improving or diversifying skin resident flora of the present invention. For example, when samples are taken at two time points, the samples are preferably taken before, during, or after administration (or ingestion or application) of the agent or composition of the present invention; at two different time points during administration (or ingestion or application); during and after administration (or ingestion or application); or at two different time points after administration (or ingestion or application).

[0034] The bacterial flora analysis techniques used in methods for assessing the effectiveness of bacterial flora improvement or diversification generally include the following steps: (1) extracting genomic DNA from a sample collected from a subject and amplifying a portion or the entire DNA sequence of the bacterial 16S rRNA gene; (2) creating a library by attaching adapter sequences to the DNA amplification products and sequencing them using a next-generation sequencer; (3) combining the obtained sequence reads with data analysis software to estimate the origin of the bacteria and their occupancy in the sample.

[0035] Examples of bacterial flora analysis methods based on amplification of the 16S rRNA gene include 16S amplicon sequencing, shotgun sequencing, 16S full-length amplicon sequencing, and equivalent bacterial flora analysis methods.

[0036] In the present invention, an improved state of the resident skin flora refers to a state in which, when samples collected from the skin at two time points are measured and compared using the above-described bacterial flora analysis method based on amplification of the 16S rRNA gene, the Shannon Diversity Index in the sample collected later is increased.

[0037] The effects of the present invention can also be determined by evaluating the condition of the skin or keratin. For example, skin cell turnover becomes insufficient with age, resulting in an increase in the area of ​​keratinocytes peeled off by tape. The more appropriate the turnover, the smaller the peeled area, indicating healthy skin. Therefore, the keratin of a sample obtained from the skin can be evaluated to determine which of the predetermined keratin grades corresponds to, and the improved state of the skin can be determined.

[0038] Furthermore, normal skin turnover occurs on a 28-day cycle, but various factors are known to disrupt this cycle, resulting in incomplete keratinization. Epidermal cells normally become denucleated and turn into keratinocytes as they approach the outermost layer, but in the state of incomplete keratinization, nucleated cells with nuclei appear on the epidermis. Nucleated cells are immature keratinocytes, and their moisturizing and barrier functions are impaired, which accelerates skin aging. Therefore, the condition of the skin can be determined by evaluating the number of nucleated cells (nucleated count) in cells from a sample obtained from the skin.

[0039] When keratinocytes are peeled off with tape or other tools, the stratum corneum peels off in layers, which is called multilayer peeling. Skin with a lot of multilayer peeling has a reduced barrier function and is in a state of moisture retention and inflammation, so measuring the amount of multilayer peeling can also be used to evaluate the condition of the skin. [Example]

[0040] The present invention will be described in more detail below by way of examples. The examples shown below, particularly the formulation examples, are typical or general examples and are not intended to limit the present invention.

[0041] [Test Example 1] Antibacterial activity of lactoferrin in vitro The antibacterial activity of lactoferrin against Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes was investigated using liquid culture and solid culture methods (modified cup method).

[0042] 1) Examination of culture conditions for test bacteria Staphylococcus aureus IFO12732 (hereafter referred to as SA) and Staphylococcus epidermidis NBRC 100911 (hereafter referred to as SE) were cultured in Mueller-Hinton medium (BD), and Cutibacterium acnes NBRC 10765 (hereafter referred to as CA) was cultured in diluted GAM medium (Nissui) (1 / 2, 1 / 5, 1 / 10) at 37°C (liquid culture, solid culture). Growth was observed after 20 hours. Because CA is an anaerobic bacterium, an anaerobic culture was performed using an anaerobic jar and Anaeropack (model number A-110, Mitsubishi Gas Chemical Company, Inc.).

[0043] As a result, growth of all bacteria was confirmed after 20 hours at 37°C in all diluted media (liquid and solid media), so it was decided to carry out subsequent experiments using 1 / 10 medium.

[0044] 2) Preparation of a calibration curve SE was cultured in 1 / 10 MH medium at 37°C with shaking at 120 rpm for 22 hours, then serially diluted (four times) and measured for turbidity (OD660). After appropriate dilutions, the samples were plated on 1 / 10 MH agar plates in triplicate and cultured at 37°C for 44 hours. The number of colonies was counted to create a calibration curve.

[0045] For CA, colonies were scraped from the petri dish, suspended in 1 / 10 GAM agar medium at 37°C for 22 hours under anaerobism, and then serially diluted (four times) to measure turbidity (OD660). After appropriate dilutions, these were plated on 1 / 10 GAM agar medium in triplicate and incubated at 37°C for 70 hours. The number of colonies was counted to create a calibration curve.

[0046] The results are shown in Figure 1. In Figure 1, the vertical axis represents OD660 and the horizontal axis represents cfu / ml (x10 7 )

[0047] 3) Verification of antibacterial activity by liquid culture method 1 The minimum inhibitory concentration (MIC) of lactoferrin against major skin flora was measured using a liquid culture method. Lactoferrin (milk-derived purified lactoferrin powder, manufactured by Tatura or Morinaga Milk Industry, also used in other examples; hereafter referred to as LF) and Bacto Peptone (manufactured by BD; hereafter referred to as BP), used as a negative control, were adjusted to a final concentration of 100 mg / ml in 1 / 10 medium, and chlortetracycline hydrochloride (hereafter referred to as CTC), used as a positive control, was adjusted to a final concentration of 10 μg / ml in 1 / 10 medium, and then each was filter-sterilized before use.

[0048] Each sample was serially diluted 20 times in a 96-well plate and used in the assay. For SA and SE, 1 / 10 MH medium was used and cultured at 37°C and 120 rpm for 20 hours with shaking. The OD660 of the bacterial solution was measured, and the final concentration of 1 x 10 was determined from the calibration curve obtained above. 4 The CA was prepared so that the final concentration was 1 × 10 cfu / ml. After static anaerobic culture at 37°C for 20 hours using 1 / 10 GAM agar medium, the bacteria were scraped off the petri dish and suspended in 1 / 10 GAM medium. The OD660 of the suspension was measured, and the final concentration was determined from the calibration curve obtained above. 4 The concentration was adjusted to cfu / ml.

[0049] These bacterial solutions were added to a 96-well plate at 10% (V / V) concentration (final concentration 1 × 10 3cfu / ml), and after shaking and stirring for about 1 minute using a plate mixer, static culture was carried out at 37°C (static anaerobic culture for CA).

[0050] Observations after 22 and 27 hours of culture showed that SA showed poor growth in the control, but strong antibacterial activity in LF. At this point, no antibacterial activity was observed in BP, and weak antibacterial activity was observed in CTC. SE showed almost no growth in the control, LF, or CTC, but BP clearly promoted growth. CA showed no growth at all, including in the control.

[0051] Since the control cultures did not grow sufficiently for any of the bacteria, extended culture was carried out, and after 48 hours of culture, OD650 was measured using a plate reader.

[0052] The results are shown in Table 1.

[0053] [Table 1]

[0054] As can be seen from Table 1, LF showed strong antibacterial activity against all bacteria, and the order of susceptibility to LF was SE > SA > CA. BP also showed weak antibacterial activity against CA.

[0055] After 144 hours of incubation, the MIC of LF did not change against any of the bacteria (bactericidal), but CTC completely grew the bacteria even at the highest concentration of 10 μg / ml (bacteriostatic).

[0056] 4) Verification of antibacterial activity by liquid culture method 2 As a result of Verification 1, the antibacterial activity of LF was stronger than expected, so LF alone was re-assayed using the standard method. That is, using a medium of normal concentration, the amount of inoculated bacteria was increased to a final concentration of 1 × 10 4 The concentration was returned to cfu / ml, and growth was observed 22 hours (SA, SE) and 48 hours (SA, SE, CA) after the start of culture.

[0057] After 22 hours, SA showed slight growth (about 10% of the control) at 100 mg / ml, but 100% growth inhibition at 50 to 12.5 mg / ml. SE showed growth (about 80% of the control) at 100 to 25 mg / ml, but 100% growth inhibition at 12.5 to 1.56 mg / ml. Since CA showed no growth at all even in the control and could not be measured, an extended culture was performed and the OD650 was measured after 48 hours.

[0058] The results are shown in Table 2.

[0059] [Table 2]

[0060] As shown in Table 2, LF had antibacterial activity against SA and SE, but no activity was confirmed against CA. The order of sensitivity was SE > SA > CA, as in Test 1.

[0061] 5) Verification of antibacterial activity using a solid culture method (modified cup method) LF was adjusted to a final concentration of 100 mg / ml in each 1 / 10 medium, sterilized by filter, and serially diluted (5 times).

[0062] Five sterilized penicillin cups were placed on each of the 1 / 10 agar plates (20 ml per dish) prepared in advance, and a final concentration of 1 x 10 4 5 ml of 1 / 10 agar medium containing a bacterial solution adjusted to cfu / ml was poured into the plate. After solidification, the cup was removed and 50 μl of the above LF solution was dispensed into the resulting depressions. Static culture (anaerobic culture for CA) was carried out at 37°C (two plates for each bacteria). After 20 hours, observation showed almost no growth of the bacteria, so the inhibition zone diameter (mm) was measured for the first time after 40 hours.

[0063] The results are shown in Table 3. Inhibition zones were observed in a concentration-dependent manner for both SA and SE, but in CA they were double circles, with the outer circle being opaque.

[0064] [Table 3]

[0065] After another 20 hours (60 hours of culture), the diameter (mm) of the inhibition zone was measured a second time. The results are shown in Table 4. Since bacteria were growing in the outer circle of CA, the inhibition zone was determined to be the inner transparent area.

[0066] [Table 4]

[0067] These results showed that LF exhibited antibacterial activity against all bacteria in the solid culture method (modified cup method), and the order of sensitivity was SE>SA>CA, as in the liquid culture method.

[0068] From the above, it was found that in vitro lactoferrin exhibited antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes in both liquid and solid culture methods (modified cup method), with the order of lactoferrin sensitivity being Staphylococcus epidermidis > Staphylococcus aureus > Propionibacterium acnes.

[0069] [Production Example 1] Production of a skin topical agent (cream) A lactoferrin-containing external skin preparation (cream) having the formulation shown in Table 5 was produced.

[0070] <Cream formulation example> [Table 5]

[0071] [Production Example 2] Production of lotion A lactoferrin-containing lotion was produced according to the formulation shown in Table 6.

[0072] <Example of lotion formulation> [Table 6]

[0073] [Production Example 3] Production of emulsion A lactoferrin-containing emulsion was produced according to the formulation shown in Table 7.

[0074] <Example of emulsion formulation> [Table 7]

[0075] [Production Example 4] Production of beauty serum A lactoferrin-containing beauty serum was prepared using the ingredients shown in Table 8.

[0076] <Example of serum formulation> [Table 8]

[0077] [Production Example 6] Production of gel A lactoferrin-containing gel was prepared with the ingredients shown in Table 9.

[0078] <Gel prescription example> [Table 9]

[0079] [Test Example 2] Examination of the effect of diversifying the normal skin flora in humans An open-label study was conducted to verify the usefulness of a lactoferrin-containing cream on the normal flora of the skin and the condition of the keratin. The study was conducted with approval from the ethics committee of NRL Pharma Co., Ltd.

[0080] Ten Japanese women aged 30 to 49 (average age 33.5 years) who were aware of dry skin were selected as subjects (Table 10).

[0081] [Table 10]

[0082] When selecting subjects, we excluded those with atopic dermatitis, those taking health foods containing lactoferrin, pregnant and breastfeeding women, those with drug or food allergies (especially milk allergies), those deemed unsuitable for this study, and those whose family members worked for cosmetics, pharmaceuticals, or hygiene product companies.

[0083] The test method was as follows. Subjects applied an appropriate amount (approximately 0.5 g) of lactoferrin-containing cream (prepared in Preparation Example 1; containing 0.5% (w / w) lactoferrin) to their entire faces twice daily, morning and evening, during face wash and skin care. This was continued for 4 weeks. Furthermore, to examine the resident skin flora and keratinocytes as primary endpoints, subjects used sampling tapes (skin flora collection: MySkin® sample collection sticker, TAK-Circulator Inc.; keratinocyte collection: Keratin Checker (trade name) PRO type, PROMOTOOL Inc.) to collect resident skin flora (by pressing one tape against the forehead and peeling it off) and keratinocytes (by pressing one tape against the right cheek and peeling it off) before and after 4 weeks of application. The samples were then stored in a home refrigerator. Secondary endpoints included subjective evaluation of skin condition via self-administered questionnaires before and after 4 weeks of application. Each subject continued to use the same commercially available cosmetics up until the day before the start of application and throughout the test period.

[0084] 1) Analysis of resident skin flora DNA was extracted from the skin flora collection tape collected as described above. The extraction and analysis of the bacterial flora were performed by TAK-Circulator using MySkin (registered trademark). DNA extraction can also be performed using the method of Morita et al. (MicrobesEnviron. Vol. 22, No. 3, pp. 214-222, 2007).

[0085] Using the obtained DNA solution, the 16S ribosomal RNA gene contained in the solution was amplified by PCR, and then DNA analysis was performed using a sequencer (NGS) (manufactured by Miseq Illumina).

[0086] The results obtained in this manner were analyzed using the pipeline analysis tool Qiime, and the relative expression level of each bacterium was evaluated, with the total expression level of 16S ribosomal RNA of 577 bacterial species set as 1. Furthermore, the bacteria were classified into the following phyla: Thermi, Acidobacteria, Acinetobacteria, Armatimonadetes, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres, Firmicutes, Fusobacteria, Gemmatimonas, and others. The bacteria were classified into 23 types: monadetes, GN02, Planctomycetes, Proteobacteria, Spirochaetes, SR1, Synergistetes, Tenericutes, TM6, TM7, Verrucomicrobia, and "others." The diversity index for each phylum of the subjects' skin resident bacteria was calculated using the Shannon-Wiener index h', and the diversity index H' was calculated as the sum of the 23 types. The diversity index was then calculated before and after 4 weeks of use. Formula: h' = - Relative expression level of phylum / Total expression level of all phyla × log2 Relative expression level of phylum / Total expression level of all phyla H' = sum of h'

[0087]

number

[0088] The results are shown in Figures 2 to 5. Figure 2 shows the results of an analysis of the normal skin flora of each subject. In each subject, changes in the normal skin flora were observed following application of the lactoferrin-containing topical skin preparation of one embodiment of the present invention.

[0089] Figure 3 shows the diversity index of the resident skin flora for each subject before and after 4 weeks, calculated using the Shannon-Weaver index. With the exception of a few subjects, an increase in the diversity index was observed, indicating an improvement in the diversity of resident skin bacteria.

[0090] Figure 4 shows the average diversity index of the resident skin flora before and after 4 weeks for all subjects shown in Figure 3. Overall, a trend toward improvement in diversity was observed.

[0091] Figure 5 shows the rate of change in the major resident skin bacteria (three types of bacteria evaluated in vitro) for all subjects (calculation formula: % after 4 weeks / % before starting). As the diversity index increased, the number of beneficial bacteria, Staphylococcus epidermidis, more than doubled, while the number of harmful bacteria, Staphylococcus aureus, decreased. This increase was also significant compared to the opportunistic bacteria, Propionibacterium acnes (p<0.05). This suggests that the resident skin flora improved, which in turn improved the skin condition (see below).

[0092] As described above, after 4 weeks of use, the diversity index increased and the resident skin flora tended to improve compared to before application of the lactoferrin-containing topical skin preparation of one embodiment of the present invention.

[0093] 2) Analysis of keratinocytes The keratin checker was immersed in the staining solution and stained for approximately 10 minutes. The staining solution was made by dissolving gentian violet (for cytoplasmic staining) at 1.0 (w / v)% and brilliant green (for nuclear staining) at 0.5 (w / v)% in distilled water. The keratin checker was immersed in water for approximately 10 minutes, and excess staining solution was washed away. Care was taken during washing to avoid peeling off the keratinocytes.

[0094] After air-drying, the samples were observed under a microscope. Images of the stained stratum corneum were taken using a 5-megapixel CMOS camera (microscope adapter 3R-DKMC01, 3R Corporation) and Anyty Microscope software (Figure 6). The stratum corneum grade was evaluated on a six-point scale, with Grade 0 representing the smallest peeled area and Grade 5 representing the best condition, according to the criteria shown in Figure 7. The amount of peeled stratum corneum area, the number of nucleated cells, and the stratum corneum layer peeling area ratio (layer peeling area ratio (%) = layer peeling area / stratum corneum peeling area × 100) were calculated visually, and the results before and after 4 weeks were compared.

[0095] The results are shown in Figure 8. Panel A shows the keratin grades of each subject in Figure 6, evaluated before and after four weeks, based on the keratin grade photographs in Figure 7. Overall, a decrease in the keratin grade values ​​was observed after four weeks, and the average values ​​also improved after four weeks. Student's t-test showed a significant improvement with p=0.00458 when evaluating the keratin grades before and after four weeks.

[0096] Panel B shows the number of nucleated cells counted for each subject in Figure 6 before and four weeks after treatment. Subjects who had a high number of nucleated cells before treatment showed a significant decrease in the number after four weeks, indicating improvement, while subjects who had a low number of nucleated cells showed no change. The average value also improved after four weeks. Student's t-test was used to evaluate the number of nucleated cells before and four weeks after treatment, indicating a significant improvement with p=0.0174.

[0097] Panel C shows the area ratio of lamellar detachment before and after 4 weeks for each subject in Figure 6. Although the lamellar detachment area worsened after 4 weeks in some subjects, there was an overall tendency for it to decrease, and the average value also showed a tendency for improvement. This demonstrates that use of an embodiment of the lactoferrin-containing topical skin preparation of the present invention diversifies the normal skin flora and improves skin condition.

[0098] 3) Secondary endpoints The subjects were asked to complete a questionnaire survey using a visual analogue scale (VAS) (0 to 10, 0 being greatly improved, 10 being greatly worsened, and 5 being no change) regarding their impressions of using the topical skin preparation of Preparation Example 1 (dryness, dullness, redness, age spots / freckles, acne, pimples, wrinkles on the forehead / between the eyes, wrinkles at the corners of the eyes, sagging around the mouth, smoothness, firmness, luster, texture, makeup application, makeup staying power, and overall evaluation of skin condition), to determine the degree of improvement due to the lactoferrin-containing topical skin preparation of one embodiment of the present invention.

[0099] The results are shown in Table 11 and FIG.

[0100] [Table 11]

[0101] Improvements were seen in all items, and t-tests showed statistically significant differences in dryness, dullness, redness, wrinkles on the forehead and between the eyebrows, wrinkles at the corners of the eyes, sagging around the eyes, sagging around the mouth, smoothness, firmness, luster, texture, makeup application, makeup staying power, and overall skin condition.

[0102] [Production Example 7] Production of bath additives Lactoferrin-containing bath additives were prepared according to the formulation shown in Table 12. (When using, dissolve 10-60g of this bath additive in 150-300L of warm water.)

[0103] <Bath additive prescription example> [Table 12]

Claims

1. An agent for improving or diversifying the normal skin flora, the active ingredient of which is lactoferrin or a salt thereof, which promotes the growth of Staphylococcus epidermidis and kills or inhibits the growth of Staphylococcus aureus on human skin (however, this does not include agents for the purpose of improving dry skin or rough skin).

2. A composition for improving or diversifying the normal skin flora, which comprises the agent for improving or diversifying the normal skin flora according to claim 1 and which promotes the growth of Staphylococcus epidermidis and kills or inhibits the growth of Staphylococcus aureus on human skin (excluding compositions for improving dry skin or rough skin, and compositions containing mugwort extract, yeast extract, secretory immunoglobulin A and / or monk fruit extract).

3. A pharmaceutical composition for improving or diversifying the normal skin flora, which comprises the agent for improving or diversifying the normal skin flora according to claim 1 and which promotes the growth of Staphylococcus epidermidis and kills or inhibits the growth of Staphylococcus aureus on human skin (excluding compositions for improving dry skin or rough skin, and compositions containing mugwort extract, yeast extract, secretory immunoglobulin A and / or monk fruit extract).

4. A cosmetic composition for improving or diversifying the normal skin flora, which comprises the agent for improving or diversifying the normal skin flora according to claim 1 and which promotes the growth of Staphylococcus epidermidis and kills or inhibits the growth of Staphylococcus aureus on human skin (excluding compositions for improving dry skin or rough skin, and compositions containing mugwort extract, yeast extract, secretory immunoglobulin A and / or monk fruit extract).

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