JP2018168082A - Skin repairing function promoting composition - Google Patents

Abstract

To provide a skin repairing function promoting composition that can effectively promote a skin repairing function, has no side effects and is safe, and is easy to take and ingest, and food and drink prepared therewith.SOLUTION: A skin repairing function promoting composition contains polysaccharides produced by Lactococcus lactis subsp. cremoris FC, FERM AP-20185, as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a composition for promoting the repair function of damaged skin.

  A living body has various functions against internal and external stimuli in order to maintain homeostasis. One of them is a skin repair function. Skin repair is said to consist of a blood coagulation / fibrinolytic system and a tissue repair system. When skin tissue is damaged by chemicals, physical stimuli, ultraviolet rays, etc., blood vessels around the tissue bleed, various blood coagulation factors are involved, blood coagulation reaction occurs, and a scab is formed to stop the bleeding. The damaged tissue is then repaired and the fibrinolytic system functions to resume blood flow and drop the scab.

  On the other hand, the stratum corneum in the surface layer of the epidermis has a function of protecting the subcutaneous tissue from stimulation from the outside world. In order for the stratum corneum to exert a protective function, it is necessary that the cytoplasm of the stratum corneum is filled with filaggrin, keratin, and their degradation products. Since filaggrin is the main protein that fills the cytoplasm of keratinocytes, and filaggrin degradation products also act as moisturizing factors in the granule layer, filaggrin plays an important role not only in protecting functions but also in maintaining moisture in the skin. Plays.

  Therefore, if this stratum corneum is excessively damaged by chemicals, physical stimuli, ultraviolet rays, etc., the repair cannot catch up, and if the synthetic power of filaggrin is insufficient, the moisturizing power decreases, the stratum corneum becomes hard, cracks occur and the skin becomes rough The condition of the skin worsens, such as dryness, inflammation, suppuration and bleeding.

  By the way, an adrenal cortex hormone is known to have an anti-inflammatory action for suppressing inflammation of damaged skin, a hemostatic action for preventing bleeding by tightly sealing blood vessels. Cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone propionate have been developed as chemical drugs that chemically synthesize the effects of such corticosteroids.

   On the other hand, some compositions and food and drink for the purpose of improving and treating the skin contain lactic acid bacteria and cultures thereof. As prior arts concerning these compositions and foods and drinks, Lactococcus lactis subsp. Cremoris FC, FERM AP-20185, cultures using the same, and polysaccharides produced thereby In connection with this, a stress-derived skin blood flow reduction improving composition (Patent Document 1), an anti-allergic skin inflammation-inhibiting action via an immunoregulatory function, and the like (Non-Patent Document 1) have been reported.

JP 2006-069940 A

Abstracts of Annual Meeting 2014 Agricultural Chemical Society of Japan p1485

  However, although the above-mentioned chemicals are relatively safe, they have habits and side effects, so it is not preferable to take them frequently for general skin function repair in daily life. If you stop using it later, rebounds may occur and even the symptoms worsen, which is not ideal.

  Moreover, in the said prior art, the relationship between lactic acid bacteria, its fermented product, and a product and a skin repair function was not examined.

  And it is not known that the lactic acid bacteria, fermented products, and products thereof promote damage repair function through the regulation of gene expression related to blood coagulation, keratinization, and fibrinolysis in skin tissue. There have been no reports of attempts to use it to improve skin repair and keep skin healthy.

  The present invention has been made in view of such circumstances, can effectively prevent and treat skin diseases and inflammation, lacerations and deterioration thereof, and is safe without causing allergies and side effects, and The object is to provide a composition having a function of promoting skin repair, which is easy to take and ingest.

  As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that a special strain [Lactococcus lactis subsp. Cremoris FC] deposited by the present applicant as a kind of lactic acid bacteria. Promotes blood coagulation, keratinogenesis, fibrinolytic (scab detachment) action, and blood coagulation action is fibrinogen gene expression suppression action, FGF-9 gene expression promotion action and thrombopoietin gene Facilitated by expression promoting action, keratinizing action promoted by filaggrin gene expression promoting action, and fibrinolytic action promoted by MMP-9 gene expression promoting action and PAI-1 gene expression promoting action In addition, these comprehensive actions can significantly promote the skin repair function. Heading, which resulted in the completion of the present invention.

Specifically, the gist of the present invention is the following composition for promoting skin repair function (1) to (10).
(1) A composition for promoting a skin repair function comprising a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC, FERM AP-20185 as an active ingredient.
(2) Blood coagulation, keratinogenesis and fibrinolysis, containing polysaccharides produced by Lactococcus lactis subsp. Cremoris FC, FERM AP-20185 as active ingredients A composition for promoting a skin repair function.
(3) Suppression of fibrinogen gene expression, fibroblast growth factor, containing polysaccharide produced by Lactococcus lactis subsp. Cremoris FC, FERM AP-20185 as an active ingredient 9 (FGF-9) gene expression promotion and thrombopoietin gene expression promotion blood coagulation action, filaggrin gene expression promotion keratinization action and matrix metalloproteinase-9 (MMP-9) gene expression promotion and plasminogen activator A composition for promoting a skin repair function having a fibrinolytic action by promoting expression of an inhibitor 1 (PAI-1) gene.
(4) Promoting the skin repair function according to the above (1) to (3), wherein the polysaccharide contains rhamnose, galactose and glucose in a molar ratio of 1: 1: 3 and 3 to 7% by mass of phosphoric acid. Composition.
(5) The composition for promoting skin repair function (1) to (4), wherein the composition is a food or drink.
(6) A composition for promoting skin repair function, wherein the composition is yogurt.
(7) A composition for promoting blood coagulation, comprising a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC (FERM AP-20185) as an active ingredient .
(8) A composition for promoting keratinogenesis, comprising a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC (FERM AP-20185) as an active ingredient. .
(9) A composition for promoting fibrinolysis, comprising a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC (FERM AP-20185) as an active ingredient. .
(10) A yogurt for promoting skin repair function, comprising a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC, FERM AP-20185 as an active ingredient.

  The composition containing a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC as an active ingredient according to the present invention has no side effects and habits unlike conventional chemicals. In addition, the blood coagulation action, the keratinization action and the fibrinolytic action are promoted, and the skin repair function can be promoted by these comprehensive actions.

  In particular, when the polysaccharide contains rhamnose, galactose, and glucose in a molar ratio of 1: 1: 3 and contains 3 to 7% by mass of phosphoric acid, various functions as the product of the present invention are further improved. Significantly expressed.

It is a figure which shows the result of having isolate | separated EPS into acidic polysaccharide and neutral polysaccharide by anion exchange resin TOYOPEARL DEAR-650M. It is a figure which shows the sugar composition of EPS by gas chromatograph GC-17A. It is a figure which shows the coupling | bonding mode of the constituent sugar of EPS by the GC-MS apparatus equipped with DB-225 column and EI detector.

  Hereinafter, modes for carrying out the present invention will be described. The present invention is not limited to the following description.

  The composition for promoting skin repair of the present invention is a culture containing a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC, or a polysaccharide purified from the culture. Is contained as an active ingredient.

  In the present invention, skin repair means blood coagulation, stratum corneum formation, and scab detachment by fibrinolysis that occurs when skin tissue is damaged by chemicals, physical stimulation, ultraviolet rays, or the like. And it can be set as the parameter | index of a repair promotion effect | action together with the measurement result of the thickness of an auricular tissue. Such skin tissue damage can be caused by application of a drug such as trinitrochlorobenzene (TNCB), sodium lauryl sulfate (SLS), acetone ether mixture (AEW), dinitrofluorobenzene (DNFB), or the like.

  In the present invention, the blood coagulation action promotes hemostasis in damaged skin tissue by the fibrinogen gene expression suppressing action, the FGF-9 gene expression action, and the thrombopoietin gene expression action.

  In the present invention, the formation of keratin promotes the generation of a moisturizing component precursor of the epidermal stratum corneum in damaged skin tissue by the expression of filaggrin gene.

  In the present invention, fibrinolysis promotes detachment of the scab in damaged skin tissue by the MMP-9 gene expression promoting action and the PAI-1 gene expression promoting action.

  In the present invention, Lactococcus lactis sub-species Cremolis FC is a lactic acid belonging to the genus Lactococcus that Fujicco Corporation, the applicant of the present application, has deposited with the National Institute of Advanced Industrial Science and Technology Patent Biodeposition Center. It refers to a cocci having a production capacity, Lactococcus lactis subsp. Cremoris FC, FERM AP-20185.

  The polysaccharide produced by Lactococcus lactis subspecies Cremolis FC is a polysaccharide produced by the bacterium by culturing the bacterium according to a conventional method of culturing lactic acid bacteria in advance. , So-called exopolysaccharide. In addition, since the exopolysaccharide comes to be stably produced by containing milk in the medium used for the culture, the effect of the present invention is more exhibited, which is preferable. .

  In addition, as the milk, for example, animal products such as cow's milk (which may be skim milk powder) or vegetable products such as soy milk are used. These may be used alone or in combination of two or more. The soy milk can be obtained by processing, for example, whole soybeans, moulted soybeans, flake soybeans, etc. containing fats and oils as raw materials.

  In addition, when using only soy milk as said milk, since fermentation is hard to be promoted as it is, it is preferable to add glucose and lactose in the ratio of about 1 to 5 weight%. Moreover, even if it is not the case where only soy milk is used as said milk, saccharides, such as glucose, lactose, and fructose, may be suitably added to the said milk as needed.

  Examples of the form of the composition in the present invention include fermented milk such as yogurt, beverages and the like prepared by the above-described culture method and containing polysaccharides in foods. It can be used as a food, a nutritional supplement, or a food displaying a skin repair promoting action.

  In addition, polysaccharides extracted and purified by conventional methods from the culture obtained by the above culture method can be added to existing foods, or food hygienically acceptable formulations such as stabilizers and preservatives. Add ingredients such as colorants, fragrances, and vitamins as appropriate, mix them, and prepare foods such as tablets, granules, granules, powders, capsules, liquids, jellies, creams, beverages, etc. by conventional methods. You can also.

  Since ingestion of the food containing the composition for promoting skin repair in such a form promotes skin repair, the food and drink obtained in the present invention is skin damaged by chemicals, physical stimuli, ultraviolet rays, etc. It can contribute to tissue repair, prevention of atopic dermatitis and ichthyosis vulgaris, and prevention of progression.

  Hereinafter, examples will be shown together with comparative examples, and the details of the present invention will be specifically described. However, the present invention is not limited thereto.

First, prior to Examples and Comparative Examples, a polysaccharide (hereinafter referred to as EPS) produced by Lactococcus lactis subspecies Cremolis FC shown below was prepared.
1. Cultivation of lactic acid bacteria 3% of Lactococcus lactis subspecies Cremolis FC was inoculated into milk, followed by stationary culture at 30 ° C. for 24 hours.
2. EPS fractionation
Mix 20% milk fermented product with 4% 100% trichloroacetic acid and centrifuge protein (18890 xg, 4 ℃, 20min), then add 4% 100% trichloroacetic acid to the collected supernatant. The protein was removed again by centrifugation (18890 xg, 4 ° C, 20 min). After adding 1.5 times the amount of cold EtOH to the obtained supernatant, the precipitate was collected with a spatula, dissolved in 10% trichloroacetic acid, and the protein was removed by centrifugation (18890 × g, 4 ° C., 20 min).
After dialysis against MilliQ water and lyophilization at 4 ° C for 3 days, the column was dissolved in 50 mM Tris-HCl (pH 8.5) and anion exchange resin TOYOPEARL DEAR-650M (Tosoh) was used (φ1cm × 10cm) Was used to separate neutral and acidic polysaccharides (FIG. 1). Neutral polysaccharides were collected as a flow-through fraction, and acidic polysaccharides were collected by elution with a 0-1M ionic strength gradient using NaCl.
3. Analysis of EPS composition 3-1) Analysis of constituent sugars
90% formic acid was added to 0.25 mg of EPS (0.2 w / v%) and hydrolyzed (100 ° C., 6 hours). After removing the formic acid by drying under reduced pressure with an evaporator, 2M TFA was added (0.2 w / v%) to perform hydrolysis (100 ° C., 6 hours). TFA was removed by drying under reduced pressure with an evaporator. The obtained hydrolyzate was reduced with sodium borohydride, and acetylated with acetic anhydride and pyridine. This was analyzed with a DB-225 column (J & W Scientific, 0.25 mm × 30 m). The apparatus used was a gas chromatograph GC-17A (Shimadzu Corporation) equipped with an FID detector.
3-2) Quantification of phosphorus Using the above hydrolyzed EPS, the phosphorus content was quantified by the Fiske and Subbarow method. The weight% of phosphoric acid was calculated in terms of (H2PO3).
3-3), methylation analysis
5 mg of EPS was suspended in 0.5 mL of DMSO, and dissolved by repeating heating (130 ° C., 30 min) and sonication (30 min) on an oil bath. After adding 50 mg of sodium hydroxide and 0.25 mL of methyl iodide and replacing with nitrogen gas, methylation was performed by heating (110 ° C., 2 hours). The methylated product was recovered by chloroform extraction. After repeating this process twice, gel filtration chromatography was performed using a Sephadex LH-20 (Pharmacia) column (eluent: chloroform-methanol = 2: 1) to obtain a polysaccharide fraction. The resulting methylated product was hydrolyzed in the same manner as described above, reduced with sodium borohydride, acetylated by adding 0.2 mL of pyridine and 0.2 mL of acetic anhydride and heating (110 ° C., 2 hours). did. The obtained partially methylated alditol acetate was dissolved in chloroform, 1 L was introduced into a DB-225 column (J & W Scientific, 0.25  mid x 30 m), and a GC-MS device equipped with an EI detector ( GCMS-QP2010, Shimadzu Corporation). The carrier gas uses helium, the column inlet pressure is 100kPa (total flow rate 34mL / min), the column temperature is raised from 170 ℃ (1min) to 210 ℃ at 3 ℃ / min, vaporization chamber temperature is 230 ℃, detector The temperature was analyzed at 230 ° C.

The constituent sugars of EPS were rhamnose, galactose, and glucose, and the constituent sugars were estimated to be rhamnose: galactose: glucose = 1: 1: 3. (Figure 2). As a result of quantifying EPS phosphorus, it was found that phosphoric acid (H2PO3) contained about 3 to 7% phosphoric acid by weight (Table 1).

  <Confirmation test of filaggrin gene expression by oral administration to mice using EPS>

[Example 1] EPS administration group
Application of TNCB and oral administration of EPS 0.5 mg / kg were performed. (N = 7)

[Comparative Example 1] Control group
Only application of TNCB was performed, and oral administration of EPS was not performed. Instead, 0.2 ml of PBS was administered (n = 8).

[Reference Example 1] Positive Control group
Application of TNCB and oral administration of prednisolone having a function of promoting skin repair, 3 mg / kg (n = 7) were performed.
EPS and prednisolone were each dissolved in 0.2 ml of sterile PBS and used for administration.
[Control group] Sham group Trinitrochlorobenzene (TNCB) application and oral administration of EPS were not performed, and instead, 0.2 ml of phosphate buffer (PBS) was administered. (N = 7)

Experiment schedule
After 29 Balb / c mice (male, 6-week-old, Claire Japan) were brought in, they were acclimated for 7 days using AIN-93 purified feed and divided into 4 groups so that the average body weight was uniform. . For 4 days from the start of the test, the above-mentioned administration was continuously administered orally with a sonde. On the 4th day from the start of the test, 20 μl of 0.3% TNCB was applied to the right ear to induce ear inflammation. In addition, 20 μl of acetone was applied to the left ear as a target. Oral administration was carried out every other day after the 6th day, and TNCB application was carried out from the 8th day to every other day until the 22nd day. On day 23, dissection was performed and auricular tissue was collected. In addition, during the test period, the thickness of the auricle was measured with calipers before applying TNCB to evaluate the repaired state of the skin.

Measurement of auricular filaggrin mRNA expression level by real-time PCR After the collected auricular tissue was immersed in RNA later (Ambion), RNA was recovered with TRIzol reagent (Life Technologies). From 0.5 μg of the recovered RNA, cDNA was synthesized by ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo).
Using this cDNA as a template, real-time PCR analysis was performed using the primers shown in Table 2. Equipment is Light Cycler (Roche), PCR enzyme is THUNDERBIRD SYBR qPCR Mix (Toyobo), cDNA 2.0μl, Enzyme 5.0μl, 10μM Primer Fw, Rv 0.4μL each, 10μl Reaction System It carried out in. The results were corrected by the expression level of the GAPDH gene, and expressed as a relative ratio with the expression level of the control group as 1.0.

  As a result, the expression of filaggrin gene increased in the Control group compared to the Sham group, and further increased in the EPS administration group and the Positive Control group. (Table 3). Furthermore, the thickness of the auricle increased significantly in the Control group compared to the Sham group, and the EPS group and the Positive Control group showed significantly lower values than the Control group. (Table 4)

<Confirmation of blood coagulation and fibrinolysis-promoting gene expression promoting action by oral administration to mice using polysaccharides>
[Example 2] EPS administration group 1
TNCB was applied and EPS was orally administered at 0.05 mg / kg. (N = 7)

[Example 3] EPS administration group 2
Application of TNCB and oral administration of EPS 0.5 mg / kg were performed. (N = 7)

[Comparative Example 2] Control group
Only TNCB was applied and oral administration of EPS was not performed. Instead, 0.2 ml of PBS was administered (n = 8)

[Reference Example 2] Positive Control group
Application of TNCB and oral administration of prednisolone 3 mg / kg (n = 7)
The EPS and prednisolone were each dissolved in 0.2 ml of sterile PBS and used for administration.

[Control group] Sham group Trinitrochlorobenzene (TNCB) application and oral administration of EPS were not performed, and instead, 0.2 ml of phosphate buffer (PBS) was administered. (N = 7)

Experiment schedule
After carrying in 36 Balb / c mice (male, 6 weeks old, CLEA Japan), they were habituated for 6 days using AIN-93 purified feed, and then experiments were conducted. The animals were divided into 5 groups so that the average body weight was uniform. For 4 days from the start of the test, the above-mentioned administration was continuously administered orally with a sonde. On the 4th day from the start of the test, 20 μl of 0.3% TNCB was applied to the right ear to induce ear inflammation. In addition, 20 μl of acetone was applied to the left ear as a target. Oral administration was carried out every other day after the 6th day, and TNCB application was carried out from the 8th day to every other day until the 14th day. On the 15th day, dissection was performed and the auricular tissue was collected. In addition, during the test period, the thickness of the auricle was measured with calipers before applying TNCB to evaluate the repaired state of the skin.

<Blood collection, biomarker test>
After anesthesia with diethyl ether, blood was collected from the inferior vena cava using a 1 ml syringe treated with heparin Na with a 26Gx1 / 2 injection needle. This was centrifuged at 10,000 rpm, 10 min, 4 ° C. to obtain plasma. The plasma was stored at −80 ° C., and a portion of the collected plasma was pooled for each group, and then subjected to a MAPs test at Charles River, fibrinogen gene, FGF-9 gene, thrombopoietin gene, MMP-9 gene, and The content of the PAI-1 gene was measured.

  As a result, there was a tendency for the fibrinogen gene expression to decrease in the EPS administration group 1 and the EPS administration group 2 relative to the Control group. Moreover, the FGF-9 gene, the thrombopoietin gene, the MMP-9 gene, and the PAI-1 gene tended to increase. (Table 5). Furthermore, the thickness of the auricle increased significantly in the Control group compared to the Sham group, and the EPS group and the Positive Control group showed significantly lower values than the Control group. (Table 6)

The composition of the present invention and foods and drinks using the composition are preferably used in the skin repair function promoting action.

Claims (10)

  A composition for promoting a skin repair function, comprising a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC (FERM AP-20185) as an active ingredient.   It contains polysaccharides produced by Lactococcus lactis subsp. Cremoris FC, FERM AP-20185 as an active ingredient, and has blood coagulation, keratinization, and fibrinolysis. A composition for promoting a skin repair function. Containing a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC, FERM AP-20185 as an active ingredient,
(1) Blood coagulation effect by suppressing fibrinogen gene expression, fibroblast growth factor 9 (FGF-9) gene expression, and thrombopoietin gene expression (2) Keratinogenesis effect by promoting filaggrin gene expression (3) Matrix metallo A composition for promoting a skin repair function, characterized by having a fibrinolytic action by promoting proteinase-9 (MMP-9) gene expression and promoting plasminogen activator inhibitor 1 (PAI-1) gene expression.   The said polysaccharide contains rhamnose, galactose and glucose in a molar ratio of 1: 1: 3, and contains 3 to 7% by mass of phosphoric acid, for promoting skin repair function according to claim 1-3 Composition.   The composition for promoting skin repair function according to claim 1, wherein the composition is a food or drink.   The composition for promoting a skin repair function according to claim 1, wherein the composition is yogurt.   A composition for promoting blood coagulation, comprising a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC (FERM AP-20185) as an active ingredient.   A composition for promoting keratinogenesis, comprising a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC (FERM AP-20185) as an active ingredient.   A composition for promoting fibrinolysis, comprising a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC (FERM AP-20185) as an active ingredient.   A yogurt for promoting skin repair function, comprising a polysaccharide produced by Lactococcus lactis subsp. Cremoris FC (FERM AP-20185) as an active ingredient.