JP6903306B1 - Oral composition for improving the barrier function of human keratinized epithelial cells in the oral cavity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent effective for improving an epithelial cell barrier function and safe for humans, an oral composition and a method, and a food composition and a beverage composition. SOLUTION: An epithelial cell barrier function improving agent containing a hot water extract of eleuthero root as an active ingredient, a composition for gargle, application, toothpaste or other oral use containing an epithelial cell barrier function improving agent, eleuthero Functional foods containing root hot water extract as an active ingredient, dietary supplements, supplements, various processed foods, seasonings, food compositions for improving epithelial cell barrier function such as chewing gum, and epithelial cell barrier function such as soft drinks. A method for improving the epithelial cell barrier function by using a beverage composition for improvement and a hot water extract of Siberian ginseng root. [Selection diagram] None

Description

The present invention relates to agents, oral compositions and methods effective for improving epithelial cell barrier function, and food compositions and beverage compositions for improving epithelial cell barrier function.

Prevention of oral infections, including periodontal disease, is an important issue for modern people, especially for the protection of the health of the elderly.

As one of the preventive measures, it is considered to enhance the barrier function of the oral mucosal epithelium that functions as a physical barrier against bacterial infection.

Tight junction proteins, which are intercellular ligation devices, are present in the mucosal epithelium and function as a physical barrier against bacterial infection.

Japanese Unexamined Patent Publication No. 2014-136692 discloses an epithelial barrier function enhancer containing yeast (Saccharomyces cerevisiae) or an extract thereof, but it is effective in improving the epithelial cell barrier function of oral mucosal epithelial cells and the like. There are many further demands for agents, compositions and methods that are safe for humans.

Japanese Unexamined Patent Publication No. 2014-136692

An object of the present invention is to provide an oral composition that is effective in improving the barrier function of human keratinized epithelial cells in the oral cavity and is safe for humans.

The present invention can be expressed, for example, as follows.

(1) An epithelial cell barrier function improving agent containing an extract of Siberian ginseng root as an active ingredient.

(2) The agent according to (1) above, wherein the epithelial cell barrier function is a keratinized epithelial cell barrier function.

(3) The agent according to (1) or (2) above, wherein the barrier function is a barrier function due to a tight junction.

(4) The agent according to (3) above, in which the transepithelial electrical resistance of epithelial cells increases as the barrier function of epithelial cells improves.

(5) The agent according to (3) above, which increases the expression of CLDN-4 in epithelial cells and improves the barrier function of epithelial cells.

(6) The agent according to any one of (1) to (5) above, wherein the epithelial cell barrier function is a human epithelial cell barrier function.

(7) A composition for gargling, application, dentifrice or other oral cavity containing the epithelial cell barrier function improving agent according to (6) above.

(8) A food composition for improving epithelial cell barrier function containing an extract of Siberian ginseng root as an active ingredient.

(9) A beverage composition for improving epithelial cell barrier function containing an extract of Siberian ginseng root as an active ingredient.

(10) A method for improving the epithelial cell barrier function using an extract of Siberian ginseng root.

The oral composition of the present invention is effective in improving the human keratinized epithelial cell barrier function in the oral cavity.

The measurement result of transepithelial electrical resistance (TER) is shown. The measurement result of CLDN-4 by the quantitative RT-PCR method is shown. It is a photographed image which shows CLDN-4 by immunofluorescent tissue staining.

Embodiments of the present invention will be described.

(1) The epithelial cell barrier function improving agent of the present invention contains an extract of Siberian ginseng root, which is highly safe for humans, as an active ingredient. The oral composition of the present invention also contains the epithelial cell barrier function improving agent of the present invention. In addition, the method of the present invention uses an extract of Siberian ginseng root to improve the epithelial cell barrier function. Further, the food composition and the beverage composition for improving the epithelial cell barrier function of the present invention contain an extract of Siberian ginseng root as an active ingredient.

(2) For the extract of the root of Siberian ginseng (scientific name: Acanthopanax senticosus), for example, the root of Siberian ginseng is crushed into fine pieces, hot water extraction is performed on the fine pieces, and the extract is concentrated under reduced pressure. Further, the ethanol insoluble matter is removed, and the obtained concentrated extract is spray-dried with a spray dryer to obtain a dry powder of a hot water extract. The dry powder of the hot water extract of Siberian ginseng root thus obtained contains isofraxisdin, eleutheroside B, B1 and E, chlorogenic acid and the like as main components.

(3) Examples of epithelial cells for improving the barrier function include human epithelial cells, keratinized epithelial cells, mucosal epithelial cells, keratinized mucosal epithelial cells, oral mucosal epithelial cells, keratinized oral mucosal epithelial cells, etc. However, it is not limited to these.

(4) The barrier function of epithelial cells may be due to tight junctions, but is not limited to this.

Evaluation of the improvement of the barrier function of epithelial cells by tight junctions can be performed by various means.

For example, the improvement of the barrier function of epithelial cells by tight junctions generally limits the permeation of ions between the upper layer side and the basement membrane side of epithelial cells, and an increase in transepithelial electrical resistance (TER) is observed. This TER measurement is widely used as a method for evaluating the barrier function due to tight junctions.

Also, for example, the adhesion molecule Claudin (also called "CLDN") is involved in the basic structure of tight junctions, and has a physical barrier function by bringing adjacent cells into close contact with each other. Evaluation of the improvement of epithelial cell barrier function by tight junctions can be performed by confirming the increased expression of Claudin (CLDN) 4 in epithelial cells.

(5) The elephant root extract (preferably hot water extract) should be used as an active ingredient of an epithelial cell barrier function improving agent, an epithelial cell barrier function improving food composition, or an epithelial cell barrier function improving beverage composition. Or, it can be used as a method for improving the epithelial cell barrier function. The epithelial cell barrier function improving agent can be used, for example, as an external preparation or by being contained in an external composition containing a composition for the oral cavity.

(5-1) When the epithelial cell barrier function improving agent is applied to humans as an external preparation or an external composition, the content of the dry powder of the hot water extract of eleuthero root as an active ingredient is, for example, 10 to 500 μg / g. It can be ml. Preferably, it is 50 to 500 μg / ml.

The amount of the epithelial cell barrier function improving agent applied to humans as an external preparation or an external composition is 1 for the content of the dry powder of the hot water extract of eleuthero root as an active ingredient, for example, 10 times or less per day. It can be 10 to 2000 μg per round. Preferably, it is 50 to 2000 μg at a time. The dose of the food composition for improving the epithelial cell barrier function or the beverage composition for improving the epithelial cell barrier function can be adjusted to a dose generally equivalent to the amount applied to humans as an external preparation or an external composition.

(5-2) Examples of external preparations or external compositions (including oral preparations or compositions) include gargling liquids, ointments or other coating compositions, patches, troches, and oral cavity. Examples thereof include disintegrating tablets, chewable tablets, paste-like, gel-like or liquid toothpaste compositions, cosmetics, medicated creams, and other skin preparations or compositions.

Examples of food compositions include functional foods, dietary supplements, supplements, various processed foods, seasonings, chewing gums, and various other foods, and examples of beverage compositions include soft beverages. Various beverages to be used can be mentioned.

(5-3) Further, the above-mentioned external preparations or external compositions, food compositions and beverage compositions are prepared with various excipients, binders, disintegrants, lubricants, coating agents, etc., as required. Colorants, flavoring agents, odorants, plasticizers and the like can be appropriately used.

Examples of excipients are sugars (lactose, sucrose, glucose, mannitol), starch (potassium, wheat, corn), inorganic substances (calcium carbonate, calcium sulfate, sodium hydrogen carbonate, sodium chloride), crystalline cellulose, plant powder (plant powder). Canzo powder, starch powder), etc. can be mentioned.

Examples of binders are starch paste, gum arabic, gelatin, sodium alginate, meth / recellulose (MC), ethylcellulose (EC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hydroxypropylcellulose (HPC). , Carpoxymethyl cellulose (CMC) and the like.

Examples of the disintegrant include starch, agar, gelatin powder, crystalline cellulose, CMC / Na, CMC / Ca, calcium carbonate, sodium hydrogencarbonate, sodium alginate and the like.

Examples of the lubricant include magnesium stearate, talc, hydrogenated vegetable oil, macrogol, silicone oil and the like.

Examples of coating agents include sugar coating (white sugar, HPC, cellac), glue coating (gelatin, glycerin, sorbitol), film coating [hydroxypropyl methylcellulose (HPMC), EC, HPC, PVP], enteric coating [hydroxyprovyl]. Methyl cellulose phthalate (HPMCP), cellulose acetate phthalate (CAP)] and the like can be mentioned.

Examples of colorants include water-soluble food dyes, lake dyes) and the like. Examples of the flavoring agent include lactose, sucrose, glucose, mannitol) and the like. Examples of the odorant include aromatic essential oils), a light blocking agent (titanium oxide), and the like. Examples of the plasticizer include phthalates, vegetable oils, polyethylene glycol) and the like.

1. 1. Manufacture of test substance

A dry powder of a hot water extract of the roots of Siberian ginseng (Acanthopanax senticosus) was produced as follows.

From 19.9 parts by weight of the root portion of Siberian ginseng, 7.1 parts by weight of a concentrated extract was obtained by hot water extraction, concentration under reduced pressure, and removal of ethanol insoluble matter. This concentrated extract was spray-dried with a spray dryer to obtain 1 part by weight of a dry powder (hereinafter, also simply referred to as “Siberian ginseng root extract”) of a hot water extract of Siberian ginseng root having a water content of 4% by weight or less.

2. Measurement of transepithelial electrical resistance (TER)

(1) Preparation of cell culture medium

1% penicillin-streptomycin (EmbryoMax PEN./STREP. 100 ×: Millipore) and 10% fetal bovine serum (FBS: Gibco) were added to Dulbecco's modified Eagle medium (DMEM: SlGMA). The obtained culture medium was used.

(2) Cultured cells

A human keratinized epithelial cell line (HaCaT) [manufactured by Cell line service] was used.

(3) TER measurement

Using a cell culture insert (manufactured by BD Falcon) as an _{incubator, incubate in a 5% CO 2} incubator at 37 ° C until HaCaT becomes confluent, and then Millicell ERS-2 before adding the eleuthero root extract (0 hr). (Millipore, Millicell is a registered trademark) was used to measure ^{TER (Ω / cm 2).}

After that, the solvent DMSO (dimethyl sulfoxide) was used in the control culture medium, and the final concentrations of Siberian ginseng root extract were 1, 10, 50, 100, 250, and 500 (μg / μg /) in the culture media of the six samples, respectively. The eleuthero root extract dissolved in DMSO was added so as to be in mL), and the TER was measured after 24 hours (24 hrs), 48 hours (48 hrs) and 72 hours (72 hrs), respectively. The measurement results are shown in FIG.

(4) Result

Before the addition of the eleuthero root extract (0 hr), no significant difference was observed in the TER measured in the control and each sample, but the concentration of the added eleuthero root extract was 50 24 hours after the addition of the eleuthero root extract. , 100, 250, and 500 (μg / mL) showed a significant increase in TER. 48 hours after the addition of the eleuthero root extract, the concentration of the added eleuthero root extract is 100 μg / mL, and 72 hours after the addition, the concentration of the added eleuthero root extract is 10, 50, 100, 250 and 500 (μg / mL). ), A significant increase in TER was observed. For statistical analysis, after one-way ANOVA, multiple comparison test was performed, and a risk rate of 0.05 or less was considered to be significant (the same applies below).

From the above results, it was confirmed that the eleuthero root extract enhances the physical barrier function of epithelial cells.

3. 3. Changes in CLDN-4 expression

(1) Of the same culture media used in the above transepithelial electrical resistance (TER) measurement, 100 μg / mL culture medium recognized as the optimum concentration for the barrier function of epithelial cells as the elephant root extract concentration was used. Using, changes in the expression of CLDN-4 in human keratinized epithelial cells (HaCaT) [manufactured by Cell line service] were analyzed by real-time PCR method and immune tissue fluorescent staining.

(2) real-time PCR method

(2-1) RNA was extracted from HaCaT cultured in the culture medium by TRIzol (manufactured by Ambion) and reversed using Oligo (dt) 12-18 (manufactured by Invitrogen) and SuperScript II (manufactured by Invitrogen). CDNA was prepared by photoreaction.

In addition, as a control, cDNA was prepared in the same manner using a culture medium supplemented with DMSO instead of Siberian ginseng root extract.

Use TaqMan probe (manufactured by Applied Biosystems, TaqMan is a registered trademark) of the same type as CLDN-4 specific primer to each cDNA, add TaqMan Universal PCR Master Mix (manufactured by Applied Biosystems), and generate Gene Amp 7500 Real-Time. A quantitative RT-PCR method was carried out by carrying out a reaction of 40 cycles at 95 ° C. for 15 seconds and 60 ° C. for 1 minute using a PCR System (manufactured by Applied Biosystems). The measurement result is shown in FIG.

The primers and TaqMan probe used were CLDN-4: HsOO533616_s1 and GAPDH: Hs99999905_m1.

(2-2) As shown in FIG. 2, a significant increase in the expression of CLDN-4 was observed by using a culture medium having a concentration of 100 μg / mL of eleuthero root extract, and the eleuthero root extract physically increased the epithelial cells. It was found that the barrier function was improved.

(3) Immunofluorescent tissue staining

(3-1) Using the same culture medium (before addition of Ezokogi root extract) used in the above transepithelial electrical resistance (TER) measurement, use Lab-Tek II Chamber Slide (manufactured by Nalge Nunc). Using, human keratinized epithelial cells (HaCaT) [manufactured by Cell line service] were _{cultured in a 5% CO 2} incubator at 37 ° C until 100% confluent, and then 100 μg of Ezokogi root extract dissolved in DMSO. The cells were added to the culture medium to a concentration of / mL and cultured for 24 hours. For control, DMSO used as a solvent for Siberian ginseng root extract was added and cultured for 24 hours.

After washing each of these cultured cells with PBS, the cells were fixed with 70% ethanol, and mouse monoclonal anti-CLDN-4 antibody (32-9400, manufactured by Invitrogen) was diluted 200-fold with PBS containing 1% BSA as the primary antibody. The reaction was carried out at 4 ° C. for 16 hours.

Next, those cells were washed 3 times with PBS containing 0.1% BSA and then diluted 200-fold with PBS containing 1% BSA Alexa Fluor 488 goat anti-mouse IgG (manufactured by Invitrogen, Alexa Fluor is a registered trademark). After reacting at room temperature for 1 hour under shading, washing with PBS containing 0.1% Tween 20 3 times, encapsulating and drying with Dapi-Fluoromount-G tissue specimen encapsulant (manufactured by Southern Biotech), and then confocal Observation and imaging were performed using a confocal laser scanning microscope DIGITAL ECLIPSE C1 (manufactured by Nikon).

The photographed image of CLDN-4 is shown in FIG.

(3-2) CLDN-4 did not show a clear stained image under the control to which DMSO was added, but when it was cultured in a culture medium having a concentration of 100 μg / mL of eleuthero root extract, it was linear in the intercellular spaces. Many positive images were observed.

It was found that the eleuthero root extract improves the physical barrier function of epithelial cells.

4. Based on the above results, an agent containing eleuthero root extract as an active ingredient, an oral composition containing the agent, a method for improving epithelial cell barrier function using eleuthero root extract, and an epithelial cell barrier function containing eleuthero root extract as an active ingredient. The effectiveness of the food composition and the beverage composition for improvement in improving the epithelial cell barrier function was confirmed.

Claims (4)

A composition containing an extract of Siberian ginseng root as an active ingredient.
Except for those containing at least one selected from water and / or organic solvent extracts of Quercus salicina, and water and / or organic solvent extracts of choy sum and hinokitiol.
Oral composition for improving the barrier function of human keratinized epithelial cells in the oral cavity . The composition according to claim 1, wherein the improvement of the barrier function of the human keratinized epithelial cells in the oral cavity is the improvement of the barrier function due to the tight junction associated with the increased expression of CLDN-4. The composition according to claim 1 or 2, which is for gargling, application or toothpaste. The composition according to any one of claims 1 to 3, wherein the transepithelial electrical resistance of the human keratinized epithelial cells increases as the barrier function of the human keratinized epithelial cells in the oral cavity is improved.

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