JP6777794B1 - Composition for promoting collagen production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for promoting collagen production. SOLUTION: For promoting collagen production containing an extract of petals and / or buds of a plant belonging to the genus Rosa in the family Rosaceae and two types of tripeptides having sequences of Gly-Pro-Hyp and Gly-Pro-Ala as active ingredients. Composition. [Selection diagram] Fig. 1

Description

The present invention relates to a novel composition for promoting collagen production in a living body.

Collagen is a protein secreted extracellularly from fibroblasts. Collagen helps to give mechanical strength to various connective tissues in the living body, and the main component of the tendon is a neat arrangement of collagen fibers, which withstands a very strong force. In the skin, collagen is said to account for about 70% of the dry weight of the dermis, and plays an important role in maintaining the elasticity and flexibility of the skin. In addition, it is becoming clear that collagen also plays a role in transmitting information to give proliferation and differentiation signals to cells in contact with it. It is known that there are various types of collagen, and more than 30 types have been reported. Among them, the basic type that constitutes the fibrosis of collagen is type I collagen, which is a major constituent of the living body. Collagen, which is the most abundant in vertebrates at 85% to 90%, is contained in a large amount in bones and functions to make bones elastic. It is also very abundant in the dermis of the skin, and has the function of producing the strength of the skin by the thick fibers of type I collagen.
In recent years, collagen and collagen hydrolysates have been orally ingested for the purpose of improving skin elasticity and wrinkles, and collagen and collagen hydrolyzate under the names such as "beauty drink" and "skin-beautifying drink". Beverages containing decomposition products are commercially available. It is said that it is effective to ingest 900 to 10,000 mg of these beverages in terms of collagen at one time.
The detailed mechanism of the mechanism of action of orally ingested collagen and collagen peptide to improve skin elasticity and wrinkles has not been clarified, but some collagen peptides are used when collagen is ingested orally. It is known to appear in blood. It is also known that the collagen peptide promotes the production of type I collagen in fibroblasts.

In Patent Document 1, a tripeptide (collagen tripeptide) having a Gly-Pro-Hyp structure obtained when collagen is enzymatically decomposed with collagenase has a biological collagen synthesis promoting action, and a collagen hydrolyzate containing the same. Is described as a technique for utilizing as a collagen synthesis promoter.
Patent Document 2 describes that the above-mentioned collagen tripeptide and silymarin and apple extract, which are one of flavonoids, can be used for improving wrinkles by contracting a collagen gel containing collagen and fibroblasts.
In Patent Document 3, a collagen-derived peptide having a structure of Gly-Pro-Hyp, Gly-His-Lys, Lys-Thr-Thr-Lys-Ser, or Gly-Glu-Pro-Arg synergistically with silybin. It has been described to promote the production of type I collagen.
In Patent Document 4, a plurality of tripeptides or dipeptides appearing in blood immediately after oral ingestion of collagen are synthesized to synthesize Ala-Hyp-Gly, Pro-Hyp-Gly, Pro-Hyp, Leu-Hyp, Ala-. It has been confirmed that a Hyp dipeptide or tripeptide promotes collagen synthesis in cultured mouse fibroblasts, and based on the results, it is proposed to use it as a skin collagen production promoter.
Patent Document 5 describes a collagen production promoter containing a dipeptide having a sequence of any one of Gly-Pro, Pro-Gly, Pro-Hyp, Hyp-Gly, and Gly-Hyp as an active ingredient.
As described above, from the research and development so far, it has been clarified that many substances such as collagen protein hydrolyzate, collagen peptide, and amino acid have a collagen production promoting action, and the search for useful substances is continued. ing.

On the other hand, various actions have been found in the extracts of petals or buds of plants belonging to the genus Rosa in the family Rosaceae. It is also known that this can be incorporated into cosmetics and health foods. Patent Document 6 describes the buds or petals of at least one Rosaceae plant of the Rosaceae genus selected from dog rose, apotecary rose, sweet-brier, damask rose, rose and rose, water, hydrophilic organic solvent or these. The antibacterial action composition and food and drink containing the extract obtained by the extraction treatment with the mixed solution of the above as an active ingredient are described.
Patent Document 7 is an extract similar to Patent Document 6, and is a bud or petal of at least one Rosaceae plant selected from dog rose, apotecary rose, sweet briar, damask rose, rose rose and hamanashi. Is described as an extract obtained by extracting the mixture with water, a hydrophilic organic solvent or a mixture thereof. Then, an anti-influenza virus action is found in this extract, and an invention of an anti-influenza virus agent containing the same as an active ingredient and a food or drink containing the same is described.

Japanese Patent No. 38027221 Japanese Patent No. 5571406 Japanese Patent No. 40338777 Japanese Patent No. 4995155 JP-A-2016-169199 Japanese Patent No. 4633226 JP-A-2002-145790

The present inventors have focused on enhancing the collagen production promoting function of collagen peptides. It has already been confirmed that the collagen-producing ability of collagen peptide is improved by using rose tsubomi extract in combination with collagen peptide.
The present invention relates to the tripeptides Gly-Pro-Hyp (hereinafter "GPO") and Gly-Pro-Ala (hereinafter "GPA") contained in collagen peptide, and the petals and / or petals of plants belonging to the genus Rosaceae. It was made because it was found that when the bud extract is used in combination, it exerts an extremely strong collagen production promoting effect.
That is, it is an object of the present invention to provide a novel composition containing an extract of petals and / or buds of a plant belonging to the genus Rosa in the family Rosaceae and tripeptides GPO and GPA.

The main configuration of the present invention is as follows.
(1) A composition for promoting collagen production containing an extract of petals and / or buds of a plant belonging to the genus Rosa in the family Rosaceae and two types of tripeptides having the sequences of Gly-Pro-Hyp and Gly-Pro-Ala as active ingredients. Stuff.
(2) 1 to 200 parts by mass of two types of tripeptides having the sequences of Gly-Pro-Hyp and Gly-Pro-Ala per 1 part by mass of the extract of petals and / or buds of plants of the genus Rosaceae. The composition according to (1).
(3) The composition according to (1) or (2), wherein the plant of the genus Rosa in the family Rosaceae is one or more species selected from Galica rose, dog rose, apotecary rose, sweet briar, damask rose, rose rose and hamanashi. Stuff.
(4) Any of (1) to (3) obtained by extracting the petal and / or bud extract of a plant belonging to the genus Rosa in the family Rosaceae with water, a hydrophilic organic solvent or a mixture thereof. The composition according to.

The composition of the present invention has an action of promoting the production of biological collagen. The composition of the present invention is composed of two types of tripeptides having a collagen production promoting action exhibited by an extract of petals and / or buds of a plant belonging to the genus Rosaceae, and a sequence of Gly-Pro-Hyp and Gly-Pro-Ala. The collagen production promoting action shown in the above acts synergistically, and exerts a high collagen production promoting action far exceeding the collagen production promoting action shown by both.
In addition, since it also has antibacterial and antiviral effects of the petals and / or bud extracts of plants belonging to the genus Rosaceae, it can be used as a food or drink for protection against infectious diseases and recovery of prognosis and physical strength. Furthermore, the composition of the present invention can be orally ingested as a food or drink because the ingredients used as raw materials have a long eating experience and their safety has been confirmed.

It is a graph which shows the result of the type I collagen production promotion test by the rose bud extract, Gly-Pro-Hyp (GPO), Gly-Pro-Ala (GPA) 1: 1 mixture. It is a graph which shows the result of the type I collagen production promotion test by the rose bud extract Gly-Pro-Hyp (GPO), Gly-Pro-Ala (GPA) 2: 1 mixture.

The present invention relates to a collagen production promoting composition containing an extract of petals and / or buds of a plant belonging to the genus Rosaceae and a tripeptide.
The composition of the present invention will be described.
The petals and / or buds of plants belonging to the genus Rosa in the Rosaceae family may be collected from any variety, but those cultivated in large quantities as horticultural species are industrially preferable. These include, in particular, Rosa gallica, dog rose (Rosa canina), apotecalis rose (Rosa gallica officinalis), sweet briginosa, damask rose (Rosa dasai rose), damask rose (Rosa dasai). And hamanashi (Rosa rugosarubra) are preferred.

For example, it can be obtained by raw or drying the petals or buds of a plant belonging to the genus Rosa in the family Rosaceae, and then pulverizing the plants as they are or using a coarse crusher for solvent extraction. As the solvent used for extraction, it is preferable to use water or a hydrophilic organic solvent and a mixed solution thereof at a temperature of room temperature to a temperature equal to or lower than the boiling point of the solvent. Specifically, lower alcohols such as methanol and ethanol; lower aliphatic ketones such as acetone and methyl ethyl ketone; polyhydric alcohols having 2 to 4 carbon atoms such as 1,3-butylene glycol and propylene glycol, and these hydrophilic organic solvents. A mixed solvent of water and water can be used. When a mixed solvent of water and a hydrophilic organic solvent is used, the amount of lower alcohol is 1 to 90 parts by mass with respect to 10 parts by mass of water, and the amount of lower aliphatic ketone is 10 parts by mass of water. On the other hand, it is preferable to add 1 to 40 parts by mass, and in the case of polyhydric alcohol, 10 to 90 parts by mass with respect to 10 parts by mass of water.

In this case, the extraction process can be carried out at room temperature or under reflux heating using any device. For example, the buds (or petals) of plants belonging to the genus Rosaceae are put into a treatment tank filled with an extraction solvent, and the soluble components are eluted by allowing them to stand for 30 minutes to 2 hours with occasional stirring as needed. , The solid matter is removed by filtration, the extraction solvent is distilled off from the obtained extract, and the extract is dried to obtain a reddish brown rose extract. When water is used as the extraction solvent, the extraction conditions are usually about 30 minutes to 2 hours at 50 to 90 ° C. When a mixed solvent of water and ethanol is used as the extraction solvent, it is usually about 30 minutes to 2 hours at 40 to 80 ° C. The extract obtained by extracting with a solvent can be used as an active ingredient of the present invention by blending it as it is as long as the extraction solvent has high safety. Patent Document 6 discloses a specific production method, and an extract suitable for the present invention can be obtained according to this.

The extract of the Rosaceae plant of the Rosaceae family thus obtained has a preferable flavor derived from the raw material and can be used as it is in the present invention, but if necessary, it is purified for the purpose of decolorization or the like. It can be used as a solution of alcohol or other organic solvent or in the form of an aqueous solution, depending on the compounding application.

The extract of a plant belonging to the genus Rosaceae used in the present invention can be pulverized by a drying means such as spray drying by adding dextrin or cyclodextrin, if necessary.
As the extract of the plant of the genus Rosa in the family Rosaceae used in the present invention, a commercially available extract can be used. An example is “Rose Butts Extract Powder MF” manufactured by Maruzen Pharmaceuticals Co., Ltd., which is a spray-dried rose bud extract suitable for the present invention.
The plant extract of the genus Rosaceae used in the present invention contains 5% by mass or more of polyphenol, preferably 10 to 80% by mass, and particularly preferably 10 to 20% by mass.
The amount of the extract of a plant belonging to the genus Rosaceae used in the present invention refers to the amount equivalent to the solid content derived from the plant extract.

The tripeptide used in the present invention is preferably a tripeptide contained in a collagen peptide obtained by enzymatic hydrolysis of collagen, and any animal-derived tripeptide such as pig or cow, or fish-derived tripeptide can be used. Moreover, it may be a synthetic product.
The amino acid sequence of the tripeptide of the present invention contains both Gly-Pro-Hyp and Gly-Pro-Ala components.
Further, it is preferable that the ratio of GPO: GPA is 1: 1 to 1-3: 1, but this is not the case.

In the present invention, collagen production is promoted by the synergistic action of the petal and / or bud extract of a plant belonging to the genus Rosa in the family Rosaceae and a tripeptide. In order to obtain such a synergistic effect, the blending ratio of the petal and / or bud extract of the Rosaceae plant and the collagen tripeptide (total of GPO and GPA) should be adjusted to the petals and / or bud plant petals and / Or a composition containing 1 to 300 parts by mass, preferably 1 to 200 parts by mass, and particularly preferably 5 to 150 parts by mass with respect to 1 part by mass of the bud extract.

When this composition is added to foods and drinks, the amount added is 0.01% by mass or more, preferably 0.5 to 95% by mass, and particularly preferably 5 to 90% by mass. In the case of a drug, the amount to be blended cannot be uniquely determined because it depends on the symptom, the target age, the body weight, and the dosage form to be administered.

As the food, in addition to ordinary foods, nutritional supplements, functional foods, health foods, foods for specified health uses and the like, for example, tablets, capsules, powders and the like are preferable. In addition, it can be added to beverages such as juice and jelly.

In pharmaceutical applications, the active ingredient is mixed with a solid or liquid medicinal non-toxic carrier suitable for administration methods such as rectal administration and injection in the case of oral administration or parenteral ingestion, and is used as a conventional pharmaceutical preparation. Can be administered in the form of. Examples of the form include solid preparations such as powders, powders, granules, tablets and capsules, liquid preparations such as solutions, suspensions and emulsions, and freeze-dried preparations. These formulations can be prepared by conventional means. Examples of the above-mentioned pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethylene starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, and water. , Physiological saline and the like. If necessary, additives such as stabilizers, wetting agents, emulsifiers, binders and isotonic agents can be added as appropriate.

Hereinafter, the present invention will be specifically described with reference to Examples of Collagen Production Promotion Test Examples and Comparative Test Examples Using the Composition of the Present Invention.
<1. Test to confirm the combined effect of a 1: 1 mixture of tripeptide GPO and GPA and rose bud extract>
(1) Test sample (sample)
Rose Butts Extract Powder MF sold by Maruzen Pharmaceuticals Co., Ltd. was used as an extract of petals and / or buds of plants belonging to the genus Rosa in the family Rosaceae. Further, as the tripeptide, Gly-Pro-Hyp (GPO) SEQ ID NO: 1 and Gly-Pro-Ala (GPA) SEQ ID NO: 2 were used. As GPO and GPA, those having reagent purity of chemically synthesized products were used.

(2) Test method 1) Cells used Normal human skin fibroblasts (NHDF: Lonza Japan Co., Ltd.)

2) Cell culture The cells were cultured under the conditions of 37 ° C., 5% carbon dioxide and 95% air. As the medium, Dulbecco's modified Eagle's medium (high glucose content, life technologies) supplemented with 10% of deactivated fetal bovine serum (FBS) (Hyclone laboratories) and 1% of penicillin-Streptomycin (Sigma-Aldrich) was used. Hereinafter, this medium will be referred to as "basal medium". A 10 cm or 15 cm dish was used for normal culture, and 0.05% Trypsin-EDTA (Sigma Aldrich) was used for cell detachment during passage.

(3) Preparation of test sample The test sample was dissolved in the medium. A basal medium was used before UVA irradiation, and an FBS-free basal medium was used after UVA irradiation.
Final Concentration of Test Sample The test was carried out at the concentration shown in Table 1 below.

(4) Cell morphology observation A morphology observation was performed to confirm the effect of the addition. The morphology was observed using an inverted system microscope (OLYMPUS IX70).

(5) Confirmation test of collagen production The cells were seeded on a 24-well plate at 1 × 10 ⁵ cells / 500 μL / well. After culturing one day before, the cells were replaced with 500 μL of the basal medium containing the evaluation sample and cultured for one day. Then, it was replaced with 500 μL of Hanks balanced salt solution (HBSS, calcium / magnesium-containing, phenol red-free), and the dishes with the lid removed were placed in a row under the UVA irradiation lamp and irradiated with 12 J / cm ² (about 132 minutes). ). After irradiation, HBSS was removed, replaced with 500 μL of FBS-free basal medium containing a sample, and cultured for 2 days. Medium was collected and stored at -80 ° C. After collecting the medium, the dish was washed 3 times with 500 μL of phosphate buffered saline, suspended in 200 μL of 0.1 N sodium hydroxide solution, and the cells were recovered.
The collected medium was centrifuged at 4 ° C. and 10,000 × g for 10 minutes, and the amount of type I collagen in the supernatant was measured using Collagen Type I, Human, and ELISA Kit (Acel Co., Ltd.). The cell suspension was centrifuged at 4 ° C. and 10,000 × g for 10 minutes, and the amount of protein in the supernatant was measured using a Pierce BCA Protein Assay kit (Thermo Fisher SCIENTIFIC). The amount of type I collagen in the medium was corrected by the amount of cellular protein. As for the measurement result, the amount of increase with respect to the amount of collagen produced by the control (additive-free) cells was obtained, and the rate of increase was obtained. The results are shown by the average value of the collagen production increase rate as a result of the three tests.

(6) Test results The test results are shown in Table 1 and FIG.

From Table 1 and FIG. 1, it was clarified that the combined use of rose bud extract, GPO and GPA 1: 1 mixed tripeptide significantly promotes collagen production.

The evaluation of the synergistic effect of type I collagen production by the combined use of GPO and GPA 1: 1 mixed tripeptide and rose bud extract was judged according to the following criteria.

Evaluation of synergistic effect: The case where the theoretical value of the additive effect <the measured value was judged as having the synergistic effect.
Theoretical value of additive effect = (A / D-1) x 100 + (B / D-1) x 100
Measured value = (C / D-1) x 100

A: Type I collagen production by adding rose bud extract alone B: Type I collagen production by adding tripeptide mixture C: Type I collagen production by using rose bud extract and tripeptide mixture together D: Control group (sample without addition) ) I type collagen production amount The judgment table of the combined effect is shown in Table 2 below.

It was confirmed that the combined use of the rose bud extract, the tripeptide GPO, and the GPA mixture synergistically increased the amount of type I collagen produced.

<2. Test to confirm the combined effect of a 2: 1 mixture of tripeptide GPO and GPA and rose bud extract>
(1) Test sample (sample)
Rose Butts Extract Powder MF sold by Maruzen Pharmaceuticals Co., Ltd. was used as an extract of petals and / or buds of plants belonging to the genus Rosa in the family Rosaceae. As the tripeptide, GPO and GPA were used as in Test 1.

(2) Test method 1) Cells used Normal human skin fibroblasts (NHDF: Lonza Japan Co., Ltd.)

2) Cell culture The cells were cultured under the conditions of 37 ° C., 5% carbon dioxide and 95% air. As the medium, Dulbecco's modified Eagle's medium (high glucose content, life technologies) supplemented with 10% of deactivated fetal bovine serum (FBS) (Hyclone laboratories) and 1% of penicillin-Streptomycin (Sigma-Aldrich) was used. A 10 cm or 15 cm dish was used for normal culture, and 0.05% Trypsin-EDTA (Sigma Aldrich) was used for cell detachment during passage.

(3) Preparation of test sample The test sample was dissolved in the medium. Use basal medium before UVA irradiation and FBS after UVA irradiation
A basal-free medium was used.
Final Concentration of Test Sample The test was carried out at the concentrations shown in Table 3 below.

(4) Cell morphology observation A morphology observation was performed to confirm the effect of the addition. The morphology was observed using an inverted system microscope (OLYMPUS IX70).

(5) Confirmation test of collagen production The cells were seeded on a 24-well plate at 1 × 10 ⁵ cells / 500 μL / well. After culturing one day before, the cells were replaced with 500 μL of basal medium containing a sample and cultured for one day. Then, it was replaced with 500 μL of Hanks balanced salt solution (HBSS, calcium / magnesium-containing, phenol red-free), and the dishes with the lid removed were placed in a row under the UVA irradiation lamp and irradiated with 12 J / cm ² (about 132 minutes). ). After irradiation, HBSS was removed, replaced with 500 μL of FBS-free basal medium containing a sample, and cultured for 2 days. Medium was collected and stored at -80 ° C. After collecting the medium, the dish was washed 3 times with 500 μL of phosphate buffered saline, suspended in 200 μL of 0.1 N sodium hydroxide solution, and the cells were recovered.

The collected medium was centrifuged at 4 ° C. and 10,000 × g for 10 minutes, and the amount of type I collagen in the supernatant was measured using Collagen Type I, Human, and ELISA Kit (Acel Co., Ltd.). The cell suspension was centrifuged at 4 ° C. and 10,000 × g for 10 minutes, and the amount of protein in the supernatant was measured using a Pierce BCA Protein Assay kit (Thermo Fisher SCIENTIFIC). The amount of type I collagen in the medium was corrected by the amount of cellular protein. As for the measurement result, the amount of increase with respect to the amount of collagen produced by the control (additive-free) cells was obtained, and the rate of increase was obtained. The results are shown by the average value of the collagen production increase rate as a result of the three tests.

(6) Test results The test results are shown in Table 3 and FIG.

From Table 3 and FIG. 2, it was clarified that the combined use of rose bud extract, GPO and GPA 2: 1 mixed tripeptide promotes collagen production.

The evaluation of the synergistic effect of type I collagen production by the combined use of GPO and GPA 2: 1 mixed tripeptide and rose bud extract was judged according to the following criteria.

Evaluation of synergistic effect: The case where the theoretical value of the additive effect <the measured value was judged as having the synergistic effect.
Theoretical value of additive effect = (A / D-1) x 100 + (B / D-1) x 100
Measured value = (C / D-1) x 100

A: Type I collagen production by adding rose bud extract alone B: Type I collagen production by adding tripeptide mixture C: Type I collagen production by using rose bud extract and tripeptide mixture together D: Control group (sample without addition) ) I type collagen production amount The judgment table of the combined effect is shown in Table 4 below.

It was confirmed that the combined use of the rose bud extract, the tripeptide GPO, and the GPA mixture synergistically increased the amount of type I collagen produced.

<3. Test result summary>
The following points were clarified from the above tests 1 and 2.
(1) When a mixture of rose bud extract and GPO and GPA is used in combination, collagen production in a living body is synergistically promoted.
(2) The mixing ratio of tripeptide GPO and GPA was mixed and tested so that the ratio of GPO: GPA was 1: 1 or 2: 1. In either case, it should be used in combination with the rose bud extract. Collagen production increased synergistically.

Reference manufacturing example 1
Example of production of bud extract of a plant belonging to the genus Rosa of the Rosaceae family Add 2000 mL of 50% by mass ethanol to 300 g of buds (Rosa gallica officinalis), attach a reflux cooler, and extract at 80 ° C. for 1 hour. , The extract 1 was obtained by filtering with a filter paper. Further, 1500 mL of 50 wt% ethanol was added to the extraction residue, and the mixture was similarly attached with a reflux condenser, extracted at 80 ° C. for 1 hour, and then filtered through a filter paper to obtain an extract 2. The obtained extracts 1 and 2 were combined, concentrated under reduced pressure, and dried to obtain 100 g of a powder extract (yield 33.3%).

Reference manufacturing example 2
Example of production of extract of buds of plants belonging to the genus Rosa in the family Rosaceae Add 2000 mL of water to 300 g of buds of roses (Rosa centifolia), extract at 90 ° C. for 1 hour, and then filter with filter paper to obtain an extract. It was. The obtained extract was concentrated under reduced pressure and dried to obtain 100 g of the powder extract of Production Example 2 (yield 33.3%).

Claims (2)

And it rose species petals and / or buds of the plant extracts, met Gly-Pro-Hyp and Gly-Pro-Ala 2 types of collagen production promoting composition containing tripeptide as an active ingredient having the sequence of hand,
Collagen production containing 1 to 200 parts by mass of two types of tripeptides having the sequences of Gly-Pro-Hyp and Gly-Pro-Ala per 1 part by mass of the extract of petals and / or buds of plants of the genus Rosaceae. Acceleration composition . The composition according to claim 1, wherein the plant of the genus Rosa in the family Rosaceae is at least one selected from Galica rose, dog rose, apotecary rose, sweet-brier, damask rose, rose rose and hamanashi.