JP2013151442A - Antimicrobial peptide secretion promoter that mammalian secretes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition that promotes the secretion of anti-bacterium peptides secreted from epidermis in a cavity such as of β-defensin and CAP-18.SOLUTION: A mammalian antimicrobial peptide secretion promoter includes epigallocatechin gallate alone, or includes: the epigallocatechin gallate; and at least one chosen from the group consisting of vitamin C, quercetin, and a glycoside of the quercetin as active ingredients. In the mammalian antimicrobial peptide secretion promoter, the content ratio of the vitamin C or the quercetin mixed with the epigallocatechin gallate is 1.0:0.1 to 0.1:1.0, respectively.

Description

  The present invention relates to a secretagogue for antimicrobial peptides secreted by mammals.

  β-Defensin is a cationic peptide having a wide range of antibacterial activities. Among them, HBD-1 and HBD-2 were discovered in 1995 and 1997, and are representative examples of human β-defensins. Recent studies have confirmed the expression of HBD-1 and HBD-2 mRNA in mucosal cells such as salivary glands, tongue, gums and cheeks, and β-defensins in saliva. HBD-1 is a constitutive peptide that is always secreted in a constant amount, and HBD-2 is induced by stimuli such as IL1β and lipopolysaccharide. (For example, Mathews et al., Infection and Immunity, 1999, 67: 2740-2745). Thus, human β-defensins play an important role in preventing and preventing infection of microorganisms on the oral and mucosal surfaces. Human 18-kd (kilodalton) cationic antibacterial protein (CAP-18) is also secreted from the epithelial cells of the gums, which is also important for preventing infection from microbial bacteria, just like β-defensins (For example, Lehreretal., Curr Opin Hematol. 2002 Jan; 9 (1): 18-22).

Recently, it has been found that these antibacterial peptides are closely related to skin immunity, for example, healing of diseases such as atopic dermatitis, psoriasis and dry eye, which are said to be skin autoimmune diseases. It is reported that these antibacterial peptides are related.
Recently, an invention using an organic acid such as fumaric acid as an active ingredient has been disclosed as a secretory promoter for human β-defensin (Patent Document 1: International Publication Pamphlet WO2005 / 027893). Also disclosed is a pharmaceutical composition that directly or indirectly promotes secretion of type 2 human β-defensin. In the present invention, quinoa extract, Bordeaux extract, jasmonic acid and the like are disclosed as plant-derived active ingredients. (Patent Document 2: JP 2008-19264). Furthermore, bifidobacteria and lactic acid bacteria have been disclosed as materials for dramatically increasing the expression level of β-defensin (Patent Document 3: International Publication Pamphlet WO2007 / 020884).

International Publication Pamphlet WO2005 / 027893 JP 2008-19264 International publication pamphlet WO2007 / 020884

Infection and Immunity, 1999, 67: 2740-2745 Curr Opin Hematol. 2002Jan; 9 (1): 18-22

  The problem to be solved is the provision of a novel drug that promotes the secretion of antimicrobial peptides.

In the present invention, natural epigallocatechin gallate promotes secretion of antibacterial peptides, β-defensins (eg, HBD-1, HDB-2, and HBD-3) and CAP-18 secreted from epidermal cells in the oral cavity. Based on this novel finding, it is based on the finding that when some natural substances are combined with epigallocatechin gallate, the promoting effect is dramatically increased and a so-called unexpected synergistic effect is obtained. The present invention provides an application as a skin and oral cleanser containing this combination and its carrier. For example, the carrier may be a carrier suitable for delivery to a target site on the skin or oral cavity (for example, colloidal silica, water, alcohol or hydrous alcohol). Epigallocatechin gallate can be used in a content of 0.05 wt% to 50 wt%, 0.25 wt% to 20 wt%, or 0.5 wt% to 15 wt%. Substances that show a synergistic effect when mixed with epigallocatechin gallate are vitamin C and quercetin. These structures are used for oral cleansing of the skin in the form of toothpaste, gargle, gum, gel, paste, etc. in addition to the oral preparation. It can also be used to treat skin and oral diseases caused by bacteria. Creams, gels, and pastes are particularly suitable for application to the target area of the oral cavity.
Another aspect of the present invention is that the present invention provides a method for promoting secretion of β-defensin and CAP-18. This method can be achieved by bringing epigallocatechin gallate or a combination thereof into contact with a cell surface capable of purifying and secreting β-defensin and CAP-18.
As a further aspect, the present invention provides a method for treating periodontal disease, gingivitis, caries, gastritis and candida disease caused by bacterial infection. This method includes a method of delivering a therapeutically effective concentration of epigallocatechin gallate or a combination thereof to a disease site.

The present invention comprises:
1. A mammalian antibacterial peptide secretion promoter comprising epigallocatechin gallate mixed with at least one selected from the group consisting of glycosides of vitamin C, quercetin and quercetin as an active ingredient.
2. In the antibacterial peptide secretion promoter comprising the epigallocatechin gallate described in the preceding item 1, at least one selected from the group consisting of glycosides of vitamin C, quercetin and quercetin as an active ingredient. 2. The mammalian antibacterial peptide secretion promoter according to the above item 1, wherein each comprises 0.25 to 80% by weight. In a mammalian antibacterial peptide secretagogue comprising the epigallocatechin gallate according to the preceding item 1, wherein at least one selected from the group consisting of glycosides of vitamin C, quercetin and quercetin is mixed as an active ingredient. 2. The mammalian antimicrobial peptide secretion promoter according to item 1 above, wherein the content ratio of vitamin C or quercetin mixed with catechin gallate is 1.0: 0.1 to 0.1: 1.0, respectively.
4). In the mammalian antibacterial peptide secretion promoter according to the preceding item 1, at least one selected from the group consisting of epigallocatechin gallate alone or epigallocatechin gallate and a glycoside of vitamin C, quercetin and quercetin is 0.5 to 50 wt. 2. The mammalian antimicrobial peptide secretion promoter according to 1 above, comprising%.
5. 2. The mammalian antimicrobial peptide secretagogue according to item 1, wherein the active ingredient is a combination of epigallocatechin gallate, vitamin C and quercetin.
6). The mammalian antibacterial peptide secretagogue according to item 1, wherein the weight ratio of epigallocatechin gallate, vitamin C and quercetin is 1: 2: 2.
7). The mammalian antimicrobial peptide secretion promoter according to any one of the preceding items 1 to 6, wherein the secreted antimicrobial peptide is β-defensin 2 and CAP-18.
8). The mammalian antimicrobial peptide secretion promoter according to any one of 1 to 7 above, which is used in combination with an antibacterial component derived from a natural plant or an oral cleansing component derived from a natural plant.
9. 9. The mammalian antimicrobial peptide secretion promoter according to 8 above, wherein the natural plant-derived antimicrobial component is derived from hops.
10. 10. The mammalian antibacterial peptide secretion promoter according to any one of items 1 to 9, which is used in skin or mucous membranes.
11. 10. The mammalian antibacterial peptide secretion promoter according to any one of 1 to 9 above, which is used in the lip or in the oral cavity.
12 A composition for external use for skin disease treatment for promoting mammalian antimicrobial peptide secretion, comprising the mammalian antimicrobial peptide secretion promoting agent according to any one of 1 to 9 above.
13. 10. A cosmetic composition for promoting mammalian antimicrobial peptide secretion, comprising the mammalian antimicrobial peptide secretion promoting agent according to any one of 1 to 9 above.
14 10. An internal composition for treatment of mucosal disease for promoting human antimicrobial peptide secretion, comprising the mammalian antimicrobial peptide secretion promoting agent according to any one of 1 to 9 above.
15. An external composition for treating eye diseases for promoting secretion of a mammalian antibacterial peptide comprising the mammalian antibacterial peptide secretion promoter according to any one of 1 to 9 above.
16. 10. A food product for promoting mammalian antimicrobial peptide secretion, comprising the mammalian antimicrobial peptide secretion promoter according to any one of 1 to 9 above.

  The present invention relates to a cleansing agent for mucous membranes such as skin, oral cavity, nose and eyes by epigallocatechin gallate alone or a combination of epigallocatechin gallate and a specific substance. Epigallocatechin gallate is one of six typical catechins having a flavan skeleton in green tea, but although not similar in structure, not all catechins have this effect. Surprisingly, epigallocatechin gallate is the only one in this catechin that promotes secretion of human antimicrobial peptides of the present invention, β-defensins (eg, HBD-1, HDB-2, and HBD-3) and CAP-18 Promote. Furthermore, in order to further enhance the promoting action of epigallocatechin gallate, a combination with various food additives was tested, and it showed a synergistic effect in at least one mixture with vitamin C, quercetin and quercetin glycoside. It was revealed.

  Epigallocatechin gallate, vitamin C, and quercetin are known compounds and are widely available on the market and can be used. The purity is generally acceptable as long as it is acceptable in the medical field, edible field, or cosmetic field. Generally, the purity is 70% or more, preferably 80% or more, and more preferably 90%. % Or more is sufficient. In the present invention, the term quercetin glycoside means a sugar derivative having a quercetin skeleton. Specifically, quercetin-3-O-glucoside, quercetin-5-O-glucoside, quercetin-7-O-glucoside, quercetin-9-O-, which means rutin, isoquercetin, and sugar derivatives of quercetin glucoside, quercetin-3-O-rutinoside, quercetin-3-O- [a-rhamnosyl- (1 ® 2) -a-rhamnosyl- (1 ® 6)]-b-glucoside, quercetin-3-O-galactoside, quercetin-7-O-galactoside, quercetin-3-O-rhamnoside, and quercetin-7-O-galactoside.

  In the antibacterial peptide secretion promoter containing at least one selected from the group consisting of epigallocatechin gallate of the present invention alone or epigallocatechin gallate and a glycoside of vitamin C, quercetin and quercetin as an active ingredient, The compounding amount of each component is 0.25 to 80% by weight, or 0.5 to 50% by weight, and the preferable compounding amount can be easily changed by the effect of promoting secretion of mammalian antimicrobial peptide.

  In the mammalian antibacterial peptide secretion promoter containing as an active ingredient at least one selected from the group consisting of epigallocatechin gallate according to the present invention and vitamin C, quercetin and a glycoside of quercetin, the mixing ratio of these components is 1.0. : 0.1 to 0.1: 1.0, and the preferred compounding ratio can be easily changed by the effect of promoting secretion of mammalian antimicrobial peptide.

  In the mammalian antibacterial peptide secretion promoter containing epigallocatechin gallate of the present invention and at least one selected from the group consisting of glycosides of vitamin C, quercetin and quercetin as active ingredients, It is 0.25 to 80% by weight, or 0.5 to 50% by weight, and is a combination of epigallocatechin gallate and vitamin C and quercetin or quercetin glycoside. The blending weight ratio is, for example, 1: 2: 2. This blending ratio can be changed within a range of ± 0.5, if desired. A combination of at least one selected from the group consisting of vitamin C, quercetin and quercetin glycosides, and a combination of vitamin C and quercetin or quercetin glycoside rather than epigallocatechin gallate alone The antibacterial peptide secretion promoting effect is significantly increased.

The preparation of the present invention can be produced by a known method. For example, a mouthwash can be prepared by dissolving 50 g of a mixture containing 10% epigallocatechin gallate, 20% vitamin C and 10% quercetin in 170 mL of ethanol and diluting with 780 mL of distilled water. In use, the patient gargles with approximately 14 mL of the gargle.
As another application, epigallocatechin gallate, at least one selected from the group consisting of vitamin C, quercetin and quercetin glycoside, and a predetermined toothpaste substance are combined and used as a toothpaste component. be able to.
In addition, these combinations are applicable to various skin disease preventive treatments, mucous membranes, oral cleansing products, and bad breath prevention products. The formulation can be prepared by blending at least one selected from the group consisting of epigallocatechin gallate and vitamin C, quercetin and quercetin glycosides into the external preparation by the formulation of external preparations known per se. Moreover, it is also possible to mix other antibacterial agents (for example, quaternary salt and triclosan) in these combinations. They can also contain aromatic components such as spearmint oil, peppermint oil, sassafras oil, clove oil and winter green oil. If necessary, sweeteners such as aspartame, stevian, saccharin, sodium cyclamate, dextrose, xylitol, and levulose may be mixed. Furthermore, it can be combined with natural plant-derived antibacterial materials to enhance the effect. A preparation comprising epigallocatechin gallate according to the present invention and at least one selected from the group consisting of glycosides of vitamin C, quercetin and quercetin is a skin disease, periodontal disease, caries fungus, candida disease, gastroenteritis It is used for the treatment and prevention of oral diseases caused by such bacterial infections. Plant-derived antibacterial materials include hinokitiol (hinoki), xanthohumol (hop), capsaicin (hot pepper), rosemariic acid (rosemari), artemisin (propolis), arteviline (propolis), oregano oil (oregano), peryl Examples thereof include aldehyde (seed), carvacrol (seed), cinnamaldehyde (cinnamon), and lauryl aldehyde (dokudomi).

  Treatment means administering the components of the general invention including an effective amount to a human who is symptomatic of skin, oral cavity or mucosal disease due to bacteria or the like, or who is expected to prevent it. Administration means the delivery of the components of the invention to the diseased part of the mucous membranes such as the skin, oral cavity, eyes and nose. Effective amount means the amount of ingredients necessary to treat or prevent a disease. This effective amount depends on the type of disease being treated. It can also be used in combination with other therapeutic agents. The optimal amount can be determined by relevant experiments in vitro or in vivo. In addition, the scope of the present invention is that β-defensin and CAP can be obtained by contacting the general components of the present invention with cells capable of secreting antimicrobial peptides such as β-defensin and CAP-18 in vitro or in vivo. Vitro or vivo methods that promote the secretion or production of antimicrobial peptides such as -18 are included.

  Some examples are shown below, but the present invention is not limited thereto.

(Example 1) Gargle effect by composition A Epigallocatechin gallate (80% purity), vitamin C and quercetin were purchased from Sigma Sigma, St. Louis, MI. Composition A was prepared as follows. 1 g of a mixture of epigallocatechin gallate, vitamin C and quercetin 1: 2: 2 (weight ratio) was dissolved in 99 mL of 27 w / v% aqueous ethanol. (Solution of mixture 10 mg / mL (1 w / v%)). A commercially available mouthwash, Listerin (registered trademark) (containing 27 w / v% ethanol) and 27 w / v% ethanol were used as controls.
Nine oral healthy adults (7 males, 2 females: age 16 to 40) were used for the study. Saliva samples (approximately 500-750 μL) were first collected from subjects prior to testing. Thereafter, the subject gargled the oral cavity with 14 mL of Composition A, water, Listerin (registered trademark), and 27 w / v% aqueous ethanol for about 2 minutes. After 1 hour, about 500 to 750 μL of saliva samples were collected again from each person. Each saliva sample was centrifuged and then diluted to 0.5% solution with phosphate buffer (PBS). 60 μL of bacterial solution was added to each solution, and cultured in a sheep blood agar medium at 37 ° C. for 48 hours under both anaerobic and aerobic conditions. Thereafter, the bacterial colonies generated in each medium were counted. The inhibitory effect was judged by the difference in the number of bacterial colonies in the sample prepared with a diluted saliva sample collected before and after gargling. The results are shown in Table 1. The percentage of bacteria in the saliva after gargling when the value before gargling is 100 is expressed in%. According to this, it can be seen that gargle in Listerin (registered trademark) and composition A has a significantly reduced number of bacteria than water and 22 w / v% ethanol. Furthermore, it can be seen that gargle by composition A is superior to Listerin (registered trademark) containing benzoic acid as a main component in 22 w / v% ethanol as an organic acid.

Table 1: Reduction of bacteria in saliva 1 hour after mouth gargle

(Example 2) In vitro experiment of peptide secretion synergistic effect of epigallocatechin and other natural products
Three groups of spiders were immunized with human synthetic antimicrobial peptides HBD-1, HBD-2, and CAP-18 according to Midorikawa et al. (Midorikawa et al., Infection & Immunity. 2003, 71: 3730-3739). The IgG fraction obtained from each sputum serum was purified by a protein A column. Purified IgG showed strong affinity for each corresponding synthetic peptide, and it was confirmed by ELISA that there was no cross-reactivity between the three peptides. Purified IgG was labeled with biotin (EZ-Link Sulfo-NHS-Biotin, Pierce Biotechnology, Rockford, IL). A sandwich ELISA system was prepared by coating unlabeled rabbit IgG specific for each synthetic peptide. Specific IgG labeled with biotin was detected by Streptavidin-conjugated peroxidase (Roche Diagnostics Corp., Indianapolis, IN). The serial dilution method of HBD-1, HBD-2, and CAP-18 solution followed the standard method for ELISA detection. O-phenylenediamine was used as a substrate for peroxidase, and the color intensity was measured with a spectrometer at OD490nm.

  Samples of each concentration were prepared by dissolving in Dulbecco's modified Eagle's medium-10% fetalbovine serum. Human gingival epithelial cells (OBA-9) (see Saito et al., J. Dent. Res., 1997, 76: 441) are cultured in a 24-well plate in Keratinocyte-serum free medium until sub-confluent Allowed to grow. Next, after washing the cells, each test sample was added and cultured for various times. The supernatant was collected and stored at -70 ° C. until assayed by ELISA. The measurement of the produced antimicrobial peptide was repeated 3 times for each specimen. The peptide concentration was determined by standard titration. Data are basically expressed as purified peptide (ng / mL) ± SD. The results are shown in Tables 2-6.

Table 2 is an experiment of the synergistic effect of epigallocatechin on the promotion of secretion of human defensin 2. When quercetin is mixed with 10 μg of epigallocatechin, the effect is significantly increased in the range of 10 ⁻⁴ to 10 ⁻⁵ M (30 μg / mL to 3 μg / mL). On the other hand, rosmarinic acid and curcumin did not show the effect.

* これらの値はすべてコントロール（無添加）におけるヒトディフェンシン２の分泌量も同条件で測定し、それによって規格化した値である。 Table 2: Experiments on the synergistic effect of epigallocatechin and other natural products on the promotion of human defensin 2 secretion

* All of these values are values normalized by measuring the amount of secreted human defensin 2 in the control (no addition) under the same conditions.

Table 3 shows the results of a similar synergistic effect on the promotion of CAP-18 secretion. When quercetin that promoted the secretion of human defensin 2 was mixed with 10 μg of epigallocatechin gallate (in the range of 10 ⁻⁴ to 10 ⁻⁵ (30 μg / mL to 3 μg / mL), the effect was not significant.

* これらの値はすべてコントロール（無添加）におけるヒトディフェンシン２の分泌量も同条件で測定し、それによって規格化した値である。 Table 3: Experiments on the synergistic effect of epigallocatechin and other natural products on the promotion of CAP-18 secretion

* All of these values are values normalized by measuring the amount of secreted human defensin 2 in the control (no addition) under the same conditions.

  Table 4 is an experiment of synergistic effects with vitamin C on epigallocatechin promotion of defensin 2 secretion using human gum epithelial cells. It can be seen that when vitamin C is mixed with 10 g of epigallocatechin, the effect is significantly increased by adding 1 to 4 times the amount.

* これらの値はすべてコントロール（無添加）におけるヒトディフェンシン２の分泌量も同条件で測定し、それによって規格化した値である。 Table 4: Experiments on the synergistic effect of epigallocatechin and vitamin C on the promotion of defensin 2 secretion

* All of these values are values normalized by measuring the amount of secreted human defensin 2 in the control (no addition) under the same conditions.

  Table 5 is an experiment of the synergistic effect of vitamin C on the promotion of CAP-18 secretion by epigallocatechin. When vitamin C 3 that promoted the secretion of human defensin 2 was mixed with epigallocatechin, the effect significantly increased the secretion of CAP-18 by adding 2 to 4 times the amount of epigallocatechin gallate.

* これらの値はすべてコントロール（無添加）におけるヒトディフェンシン２の分泌量も同条件で測定し、それによって規格化した値である。 Table 5: Synergistic effect of epigallocatechin and vitamin C on the promotion of CAP-18 secretion

* All of these values are values normalized by measuring the amount of secreted human defensin 2 in the control (no addition) under the same conditions.

  In the above experimental results, vitamin C was effective in promoting secretion of human defensin 2 and CAP-18, and quercetin was effective in promoting secretion of human defensin 2 in combination with epigallocatechin gallate.

  In Table 6, composition A mixed with epigallocatechin gallate, vitamin C, and quercetin at a weight ratio (1: 2: 2), and epigallocatechin gallate and vitamin C with two components mixed at a ratio (1: 2) It is a comparative experiment of the promotion effect with the case. It can be seen that the promotion effect is further increased by mixing the three components.

* これらの値はすべてコントロール（無添加）におけるヒトディフェンシン２の分泌量も同条件で測定し、それによって規格化した値である。 Table 6: Results of three-component mixing experiment of epigallocatechin gallate, vitamin C and quercetin

* All of these values are values normalized by measuring the amount of secreted human defensin 2 in the control (no addition) under the same conditions.

Claims (16)

A mammalian antibacterial peptide secretion promoter comprising epigallocatechin gallate mixed with at least one selected from the group consisting of glycosides of vitamin C, quercetin and quercetin as an active ingredient. The epigallocatechin gallate according to claim 1, wherein at least one selected from the group consisting of glycosides of vitamin C, quercetin and quercetin is mixed as an active ingredient, and the mammalian antimicrobial peptide secretion promoter, The mammalian antibacterial peptide secretion promoter according to claim 1, wherein each component comprises 0.25 to 80% by weight. A mammalian antibacterial peptide secretagogue comprising the epigallocatechin gallate according to claim 1, wherein at least one selected from the group consisting of vitamin C, quercetin and a glycoside of quercetin is mixed as an active ingredient. The mammalian antimicrobial peptide secretion promoter according to claim 1, wherein the content ratio of vitamin C or quercetin mixed with gallocatechin gallate is 1.0: 0.1 to 0.1: 1.0, respectively. The mammalian antimicrobial peptide secretagogue according to claim 1, wherein at least one selected from the group consisting of epigallocatechin gallate alone or epigallocatechin gallate and a glycoside of vitamin C, quercetin and quercetin is 0.5 to 50, respectively. The mammalian antibacterial peptide secretion promoter according to claim 1, comprising% by weight. The mammalian antimicrobial peptide secretion promoter according to claim 1, wherein the active ingredient is a combination of epigallocatechin gallate, vitamin C and quercetin. The mammalian antimicrobial peptide secretion promoter according to claim 1, wherein the weight ratio of epigallocatechin gallate, vitamin C and quercetin is 1: 2: 2. The antimicrobial peptide secretion promoter according to any one of claims 1 to 6, wherein the antimicrobial peptide to be secreted is β-defensin 2 and CAP-18. The mammalian antimicrobial peptide secretion promoter according to any one of claims 1 to 7, which is used in combination with an antibacterial component derived from a natural plant or an oral cleansing component derived from a natural plant. The mammalian antimicrobial peptide secretion promoter according to claim 8, wherein the natural plant-derived antimicrobial component is derived from hops. The mammalian antibacterial peptide secretion promoter according to any one of claims 1 to 9, which is used in skin or mucosa. The mammalian antibacterial peptide secretion promoter according to any one of claims 1 to 9, which is used in the lip or in the oral cavity. A composition for external use for skin disease treatment for promoting mammalian antimicrobial peptide secretion, comprising the mammalian antimicrobial peptide secretion promoting agent according to any one of claims 1 to 9. A cosmetic composition for promoting mammalian antimicrobial peptide secretion, comprising the mammalian antimicrobial peptide secretion promoting agent according to any one of claims 1 to 9. An internal composition for treatment of mucosal diseases for promoting human antimicrobial peptide secretion, comprising the mammalian antimicrobial peptide secretion promoter according to any one of claims 1 to 9. An external composition for treating eye diseases for promoting secretion of a mammalian antibacterial peptide, comprising the mammalian antibacterial peptide secretion promoter according to claim 1. A food for promoting mammalian antimicrobial peptide secretion, comprising the mammalian antimicrobial peptide secretion promoting agent according to any one of claims 1 to 9.