JP2008074747A - Collagen production promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a collagen production promoter for promoting the production of collagen, especially Type III collagen, which is a constituent of dermis. <P>SOLUTION: This collagen production promoter containing, as an active ingredient, eugeniin represented by formula (1) which is contained in hydrolysable tannin, especially in an extract from a Duabanga grandiflora, in a high content. An external preparation for skins, containing the collagen production promoter exhibits excellent effects for preventing/improving skin aged symptoms such as the wrinkles, flabbiness, tension and the like of the skin. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a collagen production promoter, particularly a type III collagen production promoter.

  Skin aging symptoms such as wrinkles caused by external stress such as aging and ultraviolet rays, generation of spots, decreased skin elasticity, decreased function of skin dermal fibroblasts, degeneration and decrease of collagen, mucos including hyaluronic acid The decrease of polysaccharides, the formation and deposition of melanin by ultraviolet rays, and the oxidation of cells are important factors. However, as with conventional cosmetics, it is clear that skin aging symptoms cannot be sufficiently prevented simply by trying to retain moisture by combining biochemical products such as mucopolysaccharides and collagen and synthetic polymer products. It has become. Therefore, in order to obtain a skin external preparation having an anti-aging and improving action on the skin, any of cell activation, collagen production promoting action, melanin production inhibiting action, hyaluronic acid production promoting action and antioxidant action, etc. Of these, attempts have been made to search for and combine ingredients having a plurality of actions, but none are sufficient from the viewpoints of stability, side effects and effects.

  As collagen existing in the dermis tissue, type I and type III are known. Type I collagen has a function of maintaining elasticity and protecting, and type III collagen is said to have a function of maintaining freshness and supporting flexibility. It is said that the ratio of type III and type I collagen is 1: 4 in infancy, but 1:10 in adults, and type III collagen decreases rapidly with age. In order to maintain and recover the elasticity of the skin, not only type I collagen but also type III collagen plays an important role.

  Concerning hydrolyzable tannins, astringent action, antioxidant action, etc. have been known for a long time. Regarding Eugenin, which is one of hydrolyzable tannins, antiviral action (see Patent Document 1), DNA synthesis inhibitory action (see Patent Document 2), IgE-IgR receptor binding inhibitory action (see Patent Document 3), glucosidase inhibition An action (see Patent Document 4), a free radical scavenging action (see Patent Document 5), and the like are known. However, it has not been known so far that hydrolyzable tannin promotes the production of collagen.

JP-A-57-91922 Japanese Patent Laid-Open No. 10-195095 Japanese Patent Laid-Open No. 11-269075 Japanese Patent Laid-Open No. 2000-72682 JP 2001-335503 A

  An object of the present invention is to provide a collagen production promoter that promotes the production of collagen, particularly type III collagen, which is a constituent component of the dermis.

  The present invention is a collagen production promoter, particularly a type III collagen production promoter comprising hydrolyzable tannin as an active ingredient.

  The hydrolyzable tannin, which is an active ingredient of the collagen production promoter of the present invention, and sometimes the type III collagen production promoter, has the chemical formula (1)

(In Formula 1,
R1 is a hydroxyl group or a residue represented by chemical formula (2), or a residue represented by chemical formula (3) in combination with R2,
R2 is a hydroxyl group or a residue represented by the chemical formula (2), or a residue represented by the chemical formula (3) together with R1,
R3 is a hydroxyl group or a residue represented by the chemical formula (2),
R4 is a hydroxyl group or a residue represented by the chemical formula (2),
R5 is a hydroxyl group or a residue represented by the chemical formula (2),
R6 may be the same or different and is a hydrogen group or a group represented by the chemical formula (4), the chemical formula (5) or the chemical formula (6);
R1 to R5 are not all hydroxyl groups)

  The hydrolyzable tannin shown by can be used.

  In the present invention, as the hydrolyzable tannin, eugeniin represented by the chemical formula (7) can be used.

  In the present invention, as the hydrolyzable tannin, a compound represented by the chemical formula (8) can be used.

(R7 in formula 8 may be the same or different, and is a hydrogen group, chemical formula (4), chemical formula (5) or chemical formula (6),
R8 is a hydrogen group or a compound represented by chemical formula (9) or chemical formula (10))

  ADVANTAGE OF THE INVENTION According to this invention, the collagen production promoter excellent in the collagen dermal fibroblast, especially the III type collagen production promotion effect can be provided.

  The present invention relates to a collagen production promoter, particularly a type III collagen production promoter, comprising hydrolyzable tannin as an active ingredient.

  The hydrolyzable tannin used in the present invention is a compound having a structure in which an aromatic compound such as gallic acid or ellagic acid and a sugar such as glucose form an ester bond, and is universally present in the plant kingdom. In the present invention, among such hydrolyzable tannins, the general formula (1)

(In Formula 1,
R1 is a hydroxyl group or a residue represented by chemical formula (2), or a residue represented by chemical formula (3) in combination with R2,
R2 is a hydroxyl group or a residue represented by the chemical formula (2), or a residue represented by the chemical formula (3) together with R1,
R3 is a hydroxyl group or a residue represented by the chemical formula (2),
R4 is a hydroxyl group or a residue represented by the chemical formula (2),
R5 is a hydroxyl group or a residue represented by the chemical formula (2),
R6 may be the same or different and is a hydrogen group or a group represented by the chemical formula (4), the chemical formula (5) or the chemical formula (6);
R1 to R5 are not all hydroxyl groups)
The hydrolyzable tannin shown by can be used.

  In the present invention, as the hydrolyzable tannin, eugeniin represented by the chemical formula (7) can also be used.

  In the present invention, as the hydrolyzable tannin, a compound represented by the chemical formula (8) can be used.

(R7 in formula 8 may be the same or different, and is a hydrogen group, chemical formula (4), chemical formula (5) or chemical formula (6),
R8 is a hydrogen group or a compound represented by chemical formula (9) or chemical formula (10))

  The hydrolyzable tannin which is an active ingredient of the present invention may be a commercially available hydrolyzable tannin or one extracted from a plant.

  Eugenin is a hydrolyzable tannin contained in clove, chiyu, genokosho, rose and the like. In the present invention, eugeniin separated from these plants by a known method can be used, but an extract of a plant containing these eugeniins can also be used. Also, commercially available eugenin (for example, eigenin manufactured by Nagara Science Co., Ltd.) can be used.

  The present inventors have now found that Eugeniin is contained at a high content in the extract of the genus Duavanga. Table 1 shows the contents of duabanga, clove and rose eugenin.

  As shown in Table 1, it was shown that duabanga leaves contained eugeniin at a higher content than roses and cloves known to contain eugeniin.

  Eugeniin, for example, is subjected to a purification operation effective for concentrating hydrolyzable tannin, such as liquid-liquid partition extraction, various chromatographies, membrane separation, and the like, after being subjected to octadecylsilylated silica gel (ODS). ) Etc. can be obtained by performing liquid chromatography.

Specifically, eugeniin can be obtained by the following steps (a) to (c).
Step (a)
Step (a) is a step of extracting Duabanga plants with water, a hydrophilic organic solvent, or a mixed solvent thereof.

  The part of the plant belonging to the genus Duabanga used as the extraction raw material is not particularly limited, and for example, the above-mentioned parts such as leaves, branches, trunks, and the like can be used as the extraction raw material. In particular, it is preferable to use a leaf or the like as an extraction raw material.

  After the extraction raw material is dried, it is pulverized as it is or using a crusher, and is subjected to extraction with an extraction solvent. At this time, the extraction raw material may be dried in the sun or using a commonly used dryer.

  As the extraction solvent, it is preferable to use a polar solvent, and it is particularly preferable to use water, a hydrophilic organic solvent, or a mixture thereof at room temperature or a temperature not higher than the boiling point of the solvent.

  Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as water that has been subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. Accordingly, water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  Examples of the hydrophilic organic solvent that can be used as the extraction solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol C2-5 polyhydric alcohols such as propylene glycol and glycerin.

  When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when using the liquid mixture of water and a lower aliphatic alcohol, the mixing ratio of water and a lower aliphatic alcohol can be set to 1: 99-99: 1 (volume ratio).

  The extraction method when extracting Eugenin from a plant of the genus Duavanga is not particularly limited, and any apparatus can be used at room temperature or under reflux heating. For example, the extraction raw material is put into a treatment tank filled with an extraction solvent, and after standing for 1 to 48 hours with occasional stirring as necessary to elute soluble components, the solids are removed by filtration to obtain The extract is obtained by evaporating the extraction solvent from the extract and drying. The amount of extraction solvent is usually 1 to 100 times (weight ratio) of the extraction raw material, and the extraction temperature is usually 20 to 90 ° C.

Step (b)
Step (b) is a step in which the extract obtained in step (a) is adsorbed on an adsorbent and then eluted with water, a water-soluble solvent or a mixed solvent thereof.
The step (b) is a step of increasing the hydrolyzable tannin content of the extract obtained in the step (a) and purifying it for the purpose of decolorization, deodorization, activity improvement and the like.

  The hydrophilic organic solvent contained in the extract is distilled off as necessary before adsorbing to the adsorbent. The extract obtained by distilling off the hydrophilic organic solvent is dissolved or suspended in, for example, water, a water-soluble solvent or a mixed solvent thereof, and then adsorbed on an adsorbent.

  As the water-soluble solvent used for dissolution, suspension or elution, the above-mentioned hydrophilic organic solvents can be used, and the above-mentioned lower alcohols having 1 to 5 carbon atoms, particularly methanol, ethanol, etc. are used. Is preferred. When a mixed solvent of water and a water-soluble solvent is used, the mixing ratio can be adjusted as appropriate. For example, when a mixed solvent of water and ethanol is used, the mixing ratio of water and ethanol is 90:10 to 1:99 (volume ratio), preferably 80:20 to 20:80 (volume ratio). it can.

  The adsorbent is not particularly limited, but known adsorbents such as ion exchange resin, synthetic adsorption resin, activated carbon, chelate resin, silica gel, alumina gel type adsorbent, and porous glass are used alone or in combination. be able to. Preferably, column chromatography using a porous synthetic adsorbent such as Diaion HP-20 and Sepabead SP-207 (both manufactured by Mitsubishi Chemical Corporation), which are porous synthetic adsorption resins, and octadecylsilylated silica gel ( ODS) and chromatolex ODS, DM1020T (manufactured by Fuji Silysia Chemical Co., Ltd.) and the like are used in combination with reverse phase column chromatography using a filler.

Step (c)
Step (c) is a step of fractionating the eluate obtained in step (b) by liquid chromatography using water, a water-soluble solvent or a mixed solvent thereof as a mobile phase.

  The fractionation method by liquid chromatography is not particularly limited, and can be performed by a usual method. For example, as the stationary phase, known carriers such as silica gel, ODS, and ion exchange resin can be used alone or in combination.

  As the water-soluble solvent, the above-described hydrophilic organic solvent can be used, and it is preferable to use the above-described lower alcohol having 1 to 5 carbon atoms, particularly methanol, ethanol, acetonitrile and the like. When a mixed solvent of water and a water-soluble solvent is used, the mixing ratio can be adjusted as appropriate. For example, when a mixed solvent of water and methanol is used, the mixing ratio of water and ethanol can be set to 2: 8 to 6: 4 (volume ratio), preferably 3: 7 (volume ratio).

  By fractionating a fraction containing eugenin using liquid chromatography, purified eugeniin can be obtained.

  The collagen production promoter of the present invention is not only externally applied to the skin, but can also be taken orally, and can be applied to foods, beverages, pharmaceuticals, and the like.

  In addition, by incorporating the collagen production promoter of the present invention into a skin external preparation, a skin external preparation that exhibits an excellent effect in preventing and improving skin aging symptoms such as skin wrinkles, tarmi, and firmness can be obtained. . Furthermore, the collagen production promoter of the invention can also be used in foods and beverages for the purpose of beauty, health maintenance or nutritional supplementation.

  The blending amount when blending the collagen production promoter of the present invention into the external preparation for skin can be adjusted according to the type and intended use of the external preparation for skin, but from the viewpoint of effect and stability, the total amount 0.0001-50.0 mass% is preferable, More preferably, it is 0.001-25.0 mass%.

  The dosage form of the external preparation for skin containing the collagen production promoter of the present invention is arbitrary, and can be provided, for example, as a solubilizing system such as lotion, an emulsifying system such as cream or emulsion, or a dispersing system such as calamine lotion. . Furthermore, it can also be provided in various dosage forms such as aerosols, ointments, powders and granules filled with a propellant.

  The details of the present invention will be described below with reference to examples, but the technical scope of the present invention is not limited thereto.

[Production Example 1] Preparation of hydrolyzable tannin-containing plant extract 100 g of dried dry ground powder of Duabanga grandiflora was dispersed in 1 L of 50 vol% ethanol aqueous solution and stirred at 70 ° C for 2 hours. Extracted. The extraction supernatant was filtered off. The filtrate was concentrated under reduced pressure and lyophilized to obtain 19.1 g of Duabanga extract.

[Production Example 2] Preparation of hydrolyzable tannin-containing plant extract fraction A porous resin (Diaion HP-20) filled with an open column was prepared by adding 50 mL of water to 17.19 g of the resulting Duabanga extract and suspending it. : Made by Mitsubishi Chemical Co., Ltd.), and water: ethanol solvent is sequentially eluted in the order of 100: 0, 80:20, 60:40, 40:60, 20:80, 0.5: 99.5, The eluate was concentrated to dryness to obtain elution fractions 0, 2, 4, 6, 8, and 10.

[Production Example 3]
Dissolve the eluted fraction 4 in a 40% by volume methanol aqueous solution, collect the ultraviolet absorption fraction with LC-MS, concentrate and dry to obtain the Duabanga A phase (molecular weight: 938) and Duabanga B phase ( Molecular weight: 1106) was isolated. The yields were 4.06% and 8.51%, respectively.
[Liquid chromatography conditions]
Stationary phase: Inertsil ODS-3 (GL Sciences)
Column diameter: 4.6 mm
Column length: 150mm
Mobile phase: 30% by volume methanol aqueous solution Mobile phase flow rate: 1 mL / min
Detection: UV

¹ H-NMR and ¹³ C-NMR analyzes were performed on the isolated eugenin and the compound represented by the chemical formula (7). The results are shown in FIGS. As a result of molecular weight and ¹ H-NMR and ¹³ C-NMR analyses, it was confirmed that the Duavanga A phase was eugeniin represented by the chemical formula (6).

[Promoting action of type III collagen production]
Normal human dermal fibroblasts were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells per well. As the seeding medium, Dulbecco's modified Eagle medium (DMEM) to which 5% by mass of fetal bovine serum (FBS) was added was used. After 24 hours, the culture medium was replaced with a sample culture solution adjusted to each concentration in a DMEM medium supplemented with 0.5 mass% FBS, and further cultured for 24 hours.
The amount of type III collagen secreted into the culture supernatant was determined by ELISA, and finally 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) against labeled peroxidase. And after adding hydrogen peroxide and making it react, the light absorbency of 405 nm was measured with the microplate reader. In the evaluation, 0.5% by mass FBS-added DMEM medium was used as a negative control in addition to the sample culture solution. The amount of protein was measured with BCA PROTEIN ASSAY Kit manufactured by PIERCE, and the amount of collagen produced per unit protein was determined.
The evaluation was performed by obtaining a relative value when the collagen production amount per unit of the negative control was 100. Moreover, the significant difference test (t test) with control was performed, and it showed with * and ** in the table | surface. A significance probability of less than 5% (P <0.05) was represented by *, and a significance probability of less than 1% (P <0.01) was represented by **.

  The evaluation results are shown in Tables 2 to 10 as relative values with the type III collagen production action in the control with no sample added as 100. Moreover, the significant difference test (t test) with control was performed, and it showed with * and ** in the table | surface. A significance probability of less than 5% (P <0.05) was represented by *, and a significance probability of less than 1% (P <0.01) was represented by **.

  As apparent from Tables 3 to 10, the plant extract containing the hydrolyzable tannin of the present invention and the hydrolyzable tannin exhibited a high type III collagen production promoting effect.

Duabanga A phase 13C-NMR spectrum Duabanga A phase 1H-NMR spectrum Duabanga B phase 13C-NMR spectrum Duabanga B phase 1H-NMR spectrum

Claims (5)

Collagen production promoter containing hydrolyzable tannin as an active ingredient A type III collagen production promoter comprising hydrolyzable tannin as an active ingredient. The following chemical formula (1)

(In Formula 1,
R1 is a hydroxyl group or a residue represented by chemical formula (2), or a residue represented by chemical formula (3) in combination with R2,
R2 is a hydroxyl group or a residue represented by the chemical formula (2), or a residue represented by the chemical formula (3) together with R1,
R3 is a hydroxyl group or a residue represented by the chemical formula (2),
R4 is a hydroxyl group or a residue represented by the chemical formula (2),
R5 is a hydroxyl group or a residue represented by the chemical formula (2),
R6 may be the same or different and is a hydrogen group or a group represented by the chemical formula (4), the chemical formula (5) or the chemical formula (6);
R1 to R5 are not all hydroxyl groups)
The collagen production promoter of Claim 1 or Claim 2 which uses the hydrolyzable tannin shown by this as an active ingredient.

Hydrolyzable tannin has the chemical formula (7)

The collagen production promoter of Claim 1 or Claim 2 which is eugeniin shown by these. Hydrolyzable tannin has the chemical formula (8)

(R7 in formula 8 may be the same or different, and is a hydrogen group, chemical formula (4), chemical formula (5) or chemical formula (6),
R8 represents a hydrogen group, chemical formula (9), or chemical formula (10).

The collagen production promoter according to claim 1 or 2, which is a compound represented by (1).

Images