JP5577489B2 - Collagen production promoter - Google Patents

Description

  The present invention relates to a collagen production promoter, and a cosmetic composition, a cell culture composition and a wound healing pharmaceutical composition comprising the collagen production promoting action.

The skin epidermis and dermis are composed of extracellular matrix such as epidermis cells, fibroblasts, and collagen, and the skin's interaction with the skin tissue maintains its homeostasis, so that the skin's moisturizing function, flexibility, elasticity Sexuality, etc. are ensured, and a fresh skin state with tension and gloss is maintained. However, if the production of collagen, which is a major component of the extracellular matrix, decreases due to the influence of external factors such as ultraviolet irradiation and drying, or aging, the skin's moisturizing function and elasticity decrease, The tension and luster are lost, and skin aging symptoms such as roughening and wrinkles appear. For this reason, the development of cosmetics that increase the amount of collagen in the skin has been promoted for the purpose of preventing and / or improving skin aging, including the maintenance of skin tension and gloss and improvement of the skin moisturizing function. For example, Patent Documents 1 and 2 report those containing ascorbic acid or the like as a component having a dermal matrix component stabilizing effect and a collagen stimulating effect. However, many of the conventional cosmetics maintain the amount of collagen in the skin by suppressing the degradation of the amount of collagen.

On the other hand, Patent Document 3 discloses a collagen production improver comprising as an active ingredient a compound (eg, 1-acetoxy-1- (2,4-diacetoxyphenyl) -2-propene) contained in a ginger extract component. Has been. By using these compounds as cosmetic compositions, for example, collagen production in skin fibroblasts is remarkably promoted, and effects such as prevention of skin aging, firmness, and improvement in gloss are expected.
JP 2004-077564 A JP-A-11-246333 JP 2009-079004 A

However, although the above compound is excellent in enhancing collagen production in human normal fibroblasts, there is a problem that its stability in an aqueous solution is extremely low.

  The present invention solves the above-mentioned problems, is excellent in stability in an aqueous solution, is easy to synthesize, and has a collagen production promoter having an equivalent collagen production promoting effect, and useful including the production promoter. The main purpose is to provide a composition.

As a result of intensive studies aimed at solving the above-mentioned problems, the present inventor has not yet promoted collagen production even though the compound is completely decomposed by heat treatment after being dissolved in an aqueous solvent. As a result of finding that it is equivalent to the treatment and further studying it, the structure of the active degradation product was identified and this research was completed. That is, the present invention relates to the following collagen production promoter and composition.

（式中、R¹、R²、R⁴、R⁵ は同一又は異なって、水素又は水酸基又は炭素数1〜4のアルキル基又は−O−C(＝O)R⁶を示す。R⁶は炭素数1〜4のアルキル基を示す。R³は、水素、炭素数1〜4のアルキル基又は−C（＝O）R⁷を示す。R⁷は炭素数1〜8のアルキル基を示す。）で表される化合物を有効成分とするコラーゲン産生促進剤。
項２：項１に記載のコラーゲン産生促進剤を含有する化粧用組成物。
項３：項１に記載のコラーゲン産生促進剤を含有する細胞培養液用組成物。
項４：項１に記載のコラーゲン産生促進剤を含有する創傷治癒用医薬組成物。 Item 1: General formula (1)

(Where R ¹ , R ² , R ⁴ , R ⁵ Are the same or different and each represents hydrogen, a hydroxyl group, an alkyl group having 1 to 4 carbon atoms, or —O—C (═O) R ⁶ . R ⁶ represents an alkyl group having 1 to 4 carbon atoms. R ³ represents hydrogen, an alkyl group having 1 to 4 carbon atoms, or —C (═O) R ⁷ . R ⁷ represents an alkyl group having 1 to 8 carbon atoms. The collagen production promoter which uses the compound represented by this as an active ingredient.
Item 2: A cosmetic composition containing the collagen production promoter according to Item 1.
Item 3: A composition for cell culture medium containing the collagen production promoter according to Item 1.
Item 4: A pharmaceutical composition for wound healing containing the collagen production promoter according to Item 1.

Hereinafter, the present invention will be described in more detail.

1. Collagen production promoter The collagen production promoter of the present invention comprises a compound represented by the following general formula (1) as an active ingredient:
General formula (1)

In the general formula (1), R ¹ , R ² , R ⁴ , R ⁵ Are the same or different and each represents hydrogen, a hydroxyl group, an alkyl group having 1 to 4 carbon atoms, or —O—C (═O) R ⁶ . One preferred example is when R ¹ , R ₂ , R ⁴ , and R ⁵ are all hydrogen. Another example is the case where R ¹ is —O—C (═O) R ₆ and R ₂ , R ⁴ , and R ⁵ are hydrogen or —O—C (═O) R ⁷ . For example, R ¹ is —O—C (═O) R ₆ , and R ² , R ⁴ , and R ⁵ are hydrogen.

R ⁶ represents an alkyl group having 1 to 4 carbon atoms. Preferably a methyl group is shown.

R ⁷ represents an alkyl group having 1 to 8 carbon atoms. Preferably a methyl group is shown.

Specifically, the compound represented by the general formula (1) includes
4-((E) -3-Hydroxy-1-propenyl) phenyl acetate (4-((E) -3-Hydroxyprop-1-enyl) phenyl
acetate or p-acetoxy cinnamic alcohol (hereinafter also referred to as HPA)),
2-Acetoxy-4-((E) -3-hydroxy-1-propenyl) phenyl acetate (2-Acetoxy-4-((E) -3-hydroxyprop-1-enyl) phenyl
acetate, hereinafter also referred to as 2,4-HPA),
2-Hydroxy-4-((E) -3-hydroxy-1-propenyl) phenyl acetate (2-Hydroxy-4-((E) -3-hydroxyprop-1-enyl) phenyl
acetate, hereinafter also referred to as 2H, 4-HPA),
2-Acetoxy-4-((E) -3-hydroxy-1-propenyl) phenol (2-Acetoxy-4-((E) -3-hydroxyprop-1-enyl) phenol, hereinafter referred to as 2,4H-HPA) )
4-((E) -3-hydroxy-1-propenyl) -2-methylphenyl acetate (4-((E) -3-hydroxyprop-1-enyl) -2-methylphenyl
acetate, hereinafter also referred to as 2M, 4-HPA),
3-Acetoxy-4-((E) -3-hydroxy-1-propenyl) phenyl acetate (3-Acetoxy-4-((E) -3-hydroxyprop-1-enyl) phenyl
acetate (hereinafter also referred to as 3,4-HPA)
3-Hydroxy-4-((E) -3-hydroxy-1-propenyl) phenyl acetate (3-Hydroxy-4-((E) -3-hydroxyprop-1-enyl) phenyl
acetate, hereinafter also referred to as 3H, 4-HPA),
3-Acetoxy-4-((E) -3-hydroxy-1-propenyl) phenol (3-Acetoxy-4-((E) -3-hydroxyprop-1-enyl) phenol, hereinafter referred to as 3,4H-HPA) )
4-((E) -3-hydroxy-1-propenyl) -3-methylphenyl acetate (4-((E) -3-hydroxyprop-1-enyl) -3-methylphenyl
acetate, hereinafter also referred to as 3M, 4-HPA).

  The above compound is produced according to a known method, or 1′-acetoxychavicol acetate (hereinafter also referred to as “ACA”) or an analog thereof is dissolved in an aqueous solvent and heat-treated. can do.

  For example, using 4-iodophenol as a raw material and acetylating a hydroxyl group, an allyl alcohol moiety is introduced by an organometallic reaction to obtain the desired compound. Preferably, it can be obtained according to the method described in the Examples.

The collagen production promoter of the present invention contains the compound represented by the general formula (1) as an active ingredient. The compound represented by the general formula (1) has an action of promoting the same amount of collagen production as that of a conventionally used compound.

Furthermore, the collagen production promoter of the present invention is excellent in the sustainability of the collagen production promoting action.

The target cells are not particularly limited as long as the effects of the present invention are exerted, but remarkably promote collagen production of dermal fibroblasts in particular.

The collagen production promoter of the present invention may be composed of the compound itself represented by the above general formula (1), or a pharmaceutically or sanitary acceptable carrier or additive comprising the compound as an active ingredient. It may further comprise other components. The kind and blending amount of such a carrier or additive can be appropriately set as long as the effects of the present invention are not impaired.

The form of the collagen production promoter is not particularly limited, and can be set according to the dosage form, form, use, etc. of the applied product. For example, it can be prepared into a liquid, emulsion, cream, powder, granule, pill, ointment and the like.

  The application site of the collagen production promoter is not particularly limited, and can be set according to the purpose. For example, it can be applied to a living body and used to improve skin condition and promote wound healing. Moreover, it can add to a culture medium and a culture solution, and can use for promotion of formation of collagen containing tissue, such as an artificial tissue and a scaffold material. Moreover, it can utilize as a form of the following compositions.

2. Cosmetic composition The cosmetic composition of the present invention is characterized by containing the collagen production promoter.

The cosmetic composition of the present invention can be prepared according to a conventional method. For example, it can be produced by preparing the collagen production promoter together with an appropriate medium or carrier.

The compounding ratio of the compound represented by the general formula (1) in the cosmetic composition of the present invention varies depending on the type of compound, the application site, the age and characteristics of the application target, the form of the cosmetic composition, etc., and is uniform. For example, in the case of HPA, it is about 0.1 to 1.0 mM, preferably about 0.1 to 0.5 mM with respect to the entire composition. For example, in the case of 2,4-HPA, it is about 0.1 to 2.0 mM, preferably about 0.1 to 0.5 mM, with respect to the entire composition.

The ratio as described above is relatively low as the component concentration, but the compound exhibits an excellent collagen production promoting action while being at a low concentration.

The cosmetic composition of the present invention can be blended with other components as long as the effects of the present invention are not impaired. Examples of other components include diethylenetriaminepentaacetic acid (hereinafter also referred to as “DTPA”), tetrakis (2-pyridylmethyl) ethylenediamine,
Hereinafter, compounds that can improve collagen production ability such as ATP) and ACA, or compounds that enhance collagen production such as vitamin C or its derivatives can also be added. The types and amounts of these other components can be appropriately set within a range that does not impair the effects of the present invention.

Further, the cosmetic composition of the present invention can be appropriately prepared in an appropriate form, for example, liquid, emulsion, cream, powder, granule or the like as desired.

In addition, using the cosmetic composition of the present invention, various cosmetics and cosmetics such as lotion, lotion, tonic, emulsion, cream, ointment, pack, lipstick, bath preparation, hair styling, etc. are produced according to conventional methods. You can also.

  The application site of the cosmetic composition is not particularly limited as long as the effect of the present invention is achieved, and can be used for various sites such as for the face, for the body, and for the hair.

The cosmetic composition of the present invention has the effect of remarkably promoting collagen production in skin fibroblasts, and is used for preventing and / or improving skin aging, skin firmness, gloss maintenance and skin moisturizing. It can be used to maintain and improve functions.

3. Composition for cell culture The composition for cell culture of the present invention is characterized by containing the collagen production promoter.

The ratio of the compound represented by the above general formula (1) in the composition for cell culture medium varies depending on the type of cell, culture medium, culture medium, etc. and cannot be specified uniformly. For example, HPA or 2,4- In the case of HPA, it is about 0.1-2.0 mM with respect to the whole composition, Preferably it is about 0.1-0.5 mM.

  The cell culture composition of the present invention can be blended with other components as long as the effects of the present invention are not impaired. Examples of other components include carbon sources, inorganic salts, various vitamins, amino acids, buffers, cell growth factors, and serum. In addition, a compound that can improve collagen production ability such as DTPA, TPEN, or ACA, or a compound that enhances collagen production such as vitamin C or a derivative thereof may be added.

  The cell culture composition of the present invention has the effect of remarkably accelerating collagen production of cultured cells, and can be used, for example, for culturing artificial skin and for promoting the formation of artificial tissue. In addition, it can be used for preparation of a scaffold material for regenerative medicine and used for promoting the formation of a collagen-containing tissue.

4). Pharmaceutical composition for wound healing The pharmaceutical composition for wound healing of the present invention comprises the above-mentioned present production promoter. The pharmaceutical composition of the present invention is used to promote wound healing on the skin surface or the like.

The pharmaceutical composition of the present invention can be prepared according to a conventional method. For example, it can be produced by preparing the collagen production promoter together with a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier, known additives, other pharmaceutically active ingredients, and the like as long as the effects of the present invention are achieved. In addition, a compound that can improve collagen production ability such as DTPA, TPEN, or ACA, or a compound that enhances collagen production such as vitamin C or a derivative thereof may be added.

The compounding ratio of the compound represented by the general formula (1) in the pharmaceutical composition of the present invention varies depending on the application site, the age of the application target, the form of the pharmaceutical composition, etc., and cannot be specified uniformly. In the case of HPA or 2,4-HPA, it is about 0.1 to 2.0 mM, preferably about 0.1 to 0.5 mM, with respect to the whole composition.

The pharmaceutical composition of the present invention can be set in an appropriate form as desired. For example, it can be in the form of a dry powder, gel, cream, ointment, suspension, solution or biocompatible synthetic or natural solid matrix.

The pharmaceutical composition for wound healing of the present invention has an effect of significantly promoting collagen production of tissue cells, and can be used for rapid healing of wounds, improvement of symptoms, and the like.

The collagen promoter of the present invention comprises the compound represented by the general formula (1) as an active ingredient, has an excellent promotion of collagen production, is excellent in stability in aqueous solution, sustained action, and has low toxicity. It is. Furthermore, the present invention provides a cosmetic composition, a cell culture composition, and a wound healing pharmaceutical composition containing the above collagen production promoter and having an excellent collagen production promoting action.

It is a figure which shows the type I collagen dyeing | staining image of the one-derived fibroblast of the sample group regarding HPA. It is a figure which shows the type I collagen dyeing | staining image of the 1-derived fibroblast of the sample group regarding 2,4-HPA. It is drawing which shows the chromatogram by the HPLC analysis after the non-process of the sample group regarding ACA, or heat processing. It is drawing which shows the cell death induction activity with respect to the HeLa cell of racemic ACA and chemically synthesized HPA. It is a figure which shows the inhibitory effect of various ACA and chemically synthesized HPA in NF-kB activation by LPS stimulation using RAW264.7 cells. 1 is a drawing showing the chemical structure of racemic 1'-acetoxychabicol acetate (ACA).

EXAMPLES Hereinafter, although an Example and a comparative example are given and this invention is demonstrated, this invention is not limited to these.

1. HPA preparation
HPA was prepared according to the method shown below. In addition, 2M, 4-HPA or 3M, 4-HPA can be synthesized by using 4-Iodo-3-methylphenol or 4-Iodo-2-methylphenol as a starting material in the same manner.

4-Iodophenol
(Wako,3 g, 13.6 mmol) を乾燥CH₂Cl₂に溶解し、Et₃N (Wako, 2.75 g, 2 equiv)、4-dimethylaminopyridine
(Wako, 83 mg, 0.05 equiv)、Ac₂O (Wako, 0.93 g, 2 equiv) を加え、室温で一晩撹拌した。溶媒を減圧留去し、クロロホルムに溶解した後、1 M HCl水溶液で洗浄した。有機層を無水硫酸ナトリウムで乾燥後、濃縮し、シリカゲルカラムクロマトグラフィー（展開溶媒：Hexane : EtOAc = 10 : 1）にて精製を行い、4-Iodophenyl acetateを得た (3.3 g, 92.5%)。

4-Iodophenol
(Wako, 3 g, 13.6 mmol) was dissolved in dry CH ₂ Cl ₂ and Et ₃ N (Wako, 2.75 g, 2 equiv), 4-dimethylaminopyridine
(Wako, 83 mg, 0.05 equiv) and Ac ₂ O (Wako, 0.93 g, 2 equiv) were added, and the mixture was stirred overnight at room temperature. The solvent was distilled off under reduced pressure, dissolved in chloroform, and washed with 1 M HCl aqueous solution. The organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography (developing solvent: Hexane: EtOAc = 10: 1) to give 4-Iodophenyl acetate (3.3 g, 92.5%).

4-Iodophenyl
acetate (500 mg, 1.91 mmol) をDMF（脱水，Wako）4 mLに溶解し、allyl alcohol (Wako, 260 mL, 3 equiv), AgOAc (Wako, 318 mg, 1 equiv), Ph₃P
(Wako, 50 mg, 0.1 equiv), Pd(OAc)₂ (Wako, 21.4 mg, 0.05 equiv) を加え、N₂下、70℃で一晩撹拌した。セライトで沈殿物を除去し、EtOAcを加え、1 M HCl、続いて飽和NaHCO₃水溶液で洗浄した。有機層を 無水硫酸ナトリウムで乾燥後、濃縮し、シリカゲルカラムクロマトグラフィー（展開溶媒：Hexane : EtOAc = 2 : 1）にて精製を行い、得られた粗結晶をHexane-EtOAc (5 : 1) より再結晶して4-((E)-3-hydroxyprop-1-enyl)phenyl acetate（ＨＰＡ）を得た (150 mg,
41%)。

4-Iodophenyl
Acetate (500 mg, 1.91 mmol) is dissolved in 4 mL of DMF (dehydrated, Wako), allyl alcohol (Wako, 260 mL, 3 equiv), AgOAc (Wako, 318 mg, 1 equiv), Ph ₃ P
(Wako, 50 mg, 0.1 equiv) and Pd (OAc) ₂ (Wako, 21.4 mg, 0.05 equiv) were added, and the mixture was stirred overnight at 70 ° C. under N ₂ . The precipitate was removed with celite, EtOAc was added and washed with 1 M HCl followed by saturated aqueous NaHCO ₃ . The organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography (developing solvent: Hexane: EtOAc = 2: 1). The resulting crude crystals were purified from Hexane-EtOAc (5: 1). Recrystallization gave 4-((E) -3-hydroxyprop-1-enyl) phenyl acetate (HPA) (150 mg,
41%).

In addition, it does not specifically limit about the manufacturing method of HPA, You may use another method. For example, it can also be prepared by the following method.

ACA 200 mg (0.85
mmol) was dissolved in 80 mL of a 20% ethanol-containing aqueous solution and stirred at 80 ° C. for 2 days. The solvent was concentrated and then lyophilized, and the residue was separated and purified by silica gel column chromatography (developing solvent: Hexane: EtOAc = 2: 1). The slightly reddish crystals were recrystallized from Hexane-EtOAc (5: 1) to give colorless crystals (yield 87 mg).

The mass spectrum of this compound and the results of ¹ H NMR spectrum in deuterated methanol are shown below. MS shows that the molecular formula is C ₁₁ H ₁₂ O ₃ and that the compound obtained by analysis of the ¹ H NMR spectrum is HPA. That is, it can be confirmed that the compound obtained by thermal decomposition of ACA is HPA.

MS: JMS-700T
(JEOL)
HRMS (CI, direct) calcd for C ₁₁ H ₁₃ O ₃ ,
[M + H] ⁺ 193.0865; found, 193.0873.
NMR: AVANCE 300N
(BRUKER)
¹ H NMR (300 MHz, MeOD); d 2.26 (s, 3 H, a ), 4.22 (dd, 2 H, J
= 1.5, 5.4 Hz, b ), 6.34 (dt, 1 H, J = 15.9, 5.4 Hz, c ), 6.61
(dd, 1 H, J = 1.5, 15.9 Hz, d ), 7.04 (d, 2 H, J = 8.7 Hz, e ),
7.43 (d, 2 H, J = 8.7 Hz, f) .

2. Preparation of 2,4-HPA, 2H, 4-HPA, 2,4H-HPA
2,4-ACA (240 mg, 0.82 mmol) was dissolved in 80 mL of a 20% ethanol-containing aqueous solution and stirred at 80 ° C. for 2 days. After concentrating the solvent, lyophilization was performed, and the residue was separated and purified by silica gel column chromatography (developing solvent: CHCl ₃ : MeOH = 10: 1), and 2,4-HPA (yield 50 mg) and 2H, 4-HPA,
A 2,4H-HPA mixture (abundance ratio 6: 4, yield 25 mg) was obtained. This summary is shown in Equation 3.

The results of ¹ H NMR spectrum analysis of 2,4-HPA are shown below.
¹ H NMR (300 MHz, CDCl ₃ ); d 2.29 (s, 3 H), 2.33 (s, 3
H), 2.45 (brs, 1 H), 4.29 (dd, 2 H, J = 1.5, 5.4 Hz), 6.32 (dt, 1 H, J
= 15.9, 5.4 Hz), 6.59 (dt, 1 H, J = 15.9, 1.5 Hz), 6.88 (d, 1 H, J
= 2.1 Hz), 6.98 (dd, 1 H, J = 8.7, 2.1 Hz), 7.54 (d, 1 H, J = 8.7 Hz).

3. Evaluation of collagen production promoting action The collagen production promoting action of the compounds was evaluated by the following method.

(1) Preparation of sample Human normal skin-derived fibroblasts (CCD-1059SK, Dainippon Pharmaceutical Co., Ltd.) are subcultured 3-6 times in EMEM medium containing 10% FBS (fetal bovine serum). did. Next, it is prepared on a culture slide (Culture slide: manufactured by Falcon) so that the number of cells becomes 1 × 10 ⁶ , cultured in EMEM medium containing 10% FBS for 24 hours, and the cells are fixed to the slide. In order to match the cell cycle, the cells were cultured in EMEM medium alone for 24 hours. Thereafter, the medium was replaced with an EMEM medium containing 10% FBS, and at the same time, the above test compound was added and cultured for 24 hours to prepare each sample group shown below. As test compounds, HPA and 2,4-HPA prepared by the method described above were used. For comparison, racemic ACA (see FIG. 6), which is a similar compound, was used. Moreover, the group which does not add a test compound was prepared as control.

<Sample group>
Sample 1) Control (no compound added)
Sample 2) HPA 0.1 μM added sample 3) HPA 1.0 μM added sample 4) 2,4-HPA 0.1 μM added sample 5) 2,4-HPA 1.0 μM added Sample 6) Racemic ACA 0.1 μM added (Comparative Example 1)
Sample 7) Racemic ACA 1.0 μM added (Comparative Example 2)

(2) Measurement of production amount of collagen With respect to the sample group prepared in (1), the production amount of collagen was analyzed immunohistochemically by the following procedure.

After the culture slide was washed 3 times with PBS solution for 5 minutes, a 4% paraformaldehyde solution was added and allowed to stand at 4 ° C. overnight to fix the sample. After washing 3 times with PBS solution containing 0.1% Triton-X for 5 minutes, blocking of endogenous peroxidase was performed with 3% H ₂ O ₂ solution for 5 minutes. Subsequently, non-specific reaction was blocked for 5 minutes using 10% normal goat serum. Thereafter, the reaction of the primary antibody was performed for 60 minutes using an anti-rat type I collagen antibody (Anti-rat type I collagen antibody (LSL) 200-fold diluted solution). After washing with PBS solution for 5 minutes 3 times, secondary antibody reaction was performed for 30 minutes using biotinylated goat anti-rabbit immunoglobulin antibody (Biotinylated Goat anti-rabbit immunogloblins antibody (DAKO) 400-fold diluted solution) It was. After washing 3 times with PBS solution for 5 minutes, the enzyme solution (Horseradish
peroxidase-labelled streptavidine-biotine complex (DAKO) 400-fold dilution) was performed for 30 minutes. After washing with PBS solution for 5 minutes 3 times, DAB (3,3-diaminobenzidin
Tetra-hydrocheloride) solution was reacted for 5 minutes to perform peroxidase color reaction. After washing with PBS solution for 3 minutes for 5 minutes, it was sealed with a water-soluble mounting medium to prepare a specimen. Image analysis was performed on the staining intensity at a positive reaction (expression of type I collagen) in the obtained specimen.

FIG. 1 shows type I collagen-stained images of human-derived fibroblasts obtained for HPA (Sample 2 and Sample 3). Table 1 shows the analysis results of the staining intensity. The intensity values in the table are expressed as a ratio of the staining intensity of each sample (measured with 20 cells per slide and averaged) when the control is 100.

Expression level of type I collagen (NIH
Analysis by image) Average value

FIG. 1 and Table 1 confirm that HPA (samples 2 and 3) has significantly improved collagen production as compared to the control (sample 1). In addition, a comparable staining image was obtained even when compared with ACA (samples 6 and 7), suggesting that it has an activity to enhance collagen production ability.

FIG. 2 shows the results of a type I collagen-stained image of human-derived fibroblasts obtained for 2,4-HPA (sample 4 and sample 5).

From FIG. 2, it was suggested that 2,4-HPA (sample 4 and sample 5) also has a collagen-producing ability enhancing activity like HPA.

4). Evaluation of stability in aqueous solution
The stability of HPA in aqueous solution was evaluated using ACA, a precursor before decomposition. First, in order to investigate the stability of ACA in an aqueous solution, a stock solution of (S)-or (R) -ACA before heating was stored at room temperature for 4 days, and reverse phase HPLC analysis was performed under the following conditions.
column:
SHODEX Silica 5C8 4E (particle size 5 mm, 4.6 mmφ´ 250 mm)
Column temperature: 40 ° C
Mobile phase: 10 mM KH ₂ PO ₄ / acetonitrile = 30/70
Flow rate: 1 mL / min
Measurement wavelength: 210 nm
The result of reverse phase HPLC analysis is shown in FIG. As a result, it was confirmed that ACA was completely decomposed in an aqueous solution in 4 days even at room temperature.

In contrast, HPA and 2,4-HPA are much more stable in aqueous solutions than ACA and 2,4-ACA, and are not expected to decompose easily even when heated. As shown in the above-mentioned method for preparing HPA and 2,4-HPA, the compound obtained by thermal decomposition of ACA and separated and purified is HPA, and the compound obtained by thermal decomposition of 2,4-ACA and separated and purified Is confirmed to be 2,4-HPA.

  Hereinafter, the thermal decomposition mechanism of ACA will be described. ACA is structurally unstable, and a rearrangement reaction of an acetoxyl group between 1 'and 3' tends to occur as shown in Formula 4.

However, since the acetyl group on the phenol side remains without being hydrolyzed by the heat treatment of ACA, a thermal decomposition mechanism as shown in Formula 5 is expected.

Since ACA is decomposed even in an aqueous solution, HPA may be generated in the cell by the mechanism of Formula 5. In addition, 3'-ACA has no action to enhance collagen production, and there is no significant difference in activity even when chiral (S) -ACA and (R) -ACA are compared. Expected to be a truly active molecule involved.

5. Evaluation of other physiological activities
ACA is known to have various physiological activities such as the ability to induce apoptosis and the suppression of NF-kB activation. Similar to the collagen production enhancement effect, it was evaluated whether HPA also has these physiological activities.

(1) Preparation of stock solutions Various ACA were prepared to be 5, 10, 20 mM, and HPA was 5, 10, 20, 40 mM DMSO or ethanol solution to obtain a stock solution. At the time of use, it added so that it might become 1,000 times dilution with respect to a culture solution.

(2) Evaluation of cell death-inducing effect by WST assay Human cervical cancer-derived HeLa cells (0.5 x 10 ⁵ cells / mL, RPMI1640 containing 10% FBS) were seeded in 100-mL plates in 96-well plates and cultured for 24 hours The medium was replaced with a medium containing a predetermined concentration of ACA or HPA and incubated at 37 ° C. for 22 hours. After adding 10 mL each of WST reagent (Cell Counting Kit-8, DOJINDO), measure the difference in absorbance between 620 nm and 450 nm using a 96-well microplate reader, and treat untreated cells as 100% viability. Rate was evaluated.

The analysis result of the obtained WST assay is shown in FIG.

As a result, as shown in FIG. 4, the ACA 20 mM treatment decreased the survival rate to about 50%, whereas HPA showed no cell death even at 40 mM. It was suggested that there was not.

(3) Evaluation of inhibition of NF-kB activation during LPS stimulation
It is known that when NF-kB is activated, its inhibitor protein IkBa is rapidly degraded. Therefore, inhibition of NF-kB activation was evaluated using IkBa degradation as an index. Mouse macrophage-like RAW262.7 cells (0.5 × 10 ⁶ cells / mL, RPMI1640 containing 10% FBS) were seeded in 1 mL each in a 30 mm dish and cultured for 24 hours.

Various ACA and HPA were added to 5 or 10 mM and incubated at 37 ° C. for 30 minutes. LPS (Sigma) solution was added to each dish at 10 mg / mL and incubated at 37 ° C. for 15 minutes. The cells were detached with a cell scraper, collected in a 1.5 mL Eppendorf tube using 1 × PBS, and centrifuged at 400 g for 5 minutes at 4 ° C. Discard the supernatant, add sample buffer 50 mM (2% SDS, 6 M urea, 1.1 M glycerol, 62.5 mM Tris-HCl (pH 6.8)) to suspend, sonicate at 4 ° C, and mix with the sample solution. did. SDS-PAGE (14%) was performed using 20 mL of the sample solution, transferred to a PVDF membrane, and then immunostained.

Block the PVDF membrane after transfer in 0.1% Tween 20 TBS solution (pH 7.5) containing 5% skim milk at 0 ° C overnight, and anti-IkBa antibody (Anti-IkBa antibody
The primary antibody reaction was performed for 60 minutes using produced in rabbit (manufactured by Sigma, diluted 1,000 times). After washing twice with 0.1% Tween 20 TBS solution (pH 7.5) for 10 minutes, secondary using an alkaline phosphatase labeled anti-rabbit IgG antibody (Anti-rabbit IgG antibody, alkaline phosphatase conjugated, Sigma, diluted 5,000 times) The antibody reaction was performed for 60 minutes. 0.1% Tween 20
TBS solution (pH
7.5) After washing twice with the solution for 10 minutes, an NBT-BCIP solution (manufactured by Sigma) was added to perform a color reaction to confirm the decomposition of IkBa.

The obtained immunostaining results are shown in FIG. As a result, as shown in FIG. 5, both (S) -ACA and (R) -ACA were confirmed to have an inhibitory effect on the degradation of IkBa by LPS stimulation, but not in HPA. Therefore, it was suggested that HPA has no inhibitory effect on NF-kB activation.

From the above, it was found that HPA has the same level of collagen production enhancement effect as ACA. In addition, HPA is a degradation product of ACA in aqueous solution, so it is expected to be more stable in aqueous solution than ACA. It was also confirmed that HPA does not exhibit the apoptosis induction and suppression of NF-kB activation that ACA originally has. Suppression of NF-kB activation works to inhibit the production of apoptosis-suppressing factors, and is closely linked to cytotoxicity as well as apoptosis-inducing ability. Therefore, HPA with lower toxicity is considered to be superior to ACA as a cosmetic composition. .

Claims (4)

General formula (1)

(In the formula, R ¹ , R ² , R ⁴ , R ⁵ Are the same or different and each represents hydrogen, a hydroxyl group, an alkyl group having 1 to 4 carbon atoms, or —O—C (═O) R ⁶ . R ⁶ represents an alkyl group having 1 to 4 carbon atoms. R ³ represents a -C (= O) ^{R 7.} R ⁷ represents an alkyl group having 1 to 8 carbon atoms. The collagen production promoter which uses the compound represented by this as an active ingredient. In general formula (1), R ¹ , R ² , R ⁴ , R ⁵ But,
(I) R ¹ , R ² , R ⁴ , R ⁵ All represent hydrogen, or
(Ii) R ¹ Is —O—C (═O) R ⁶ R ² , R ⁴ , R ⁵ Are the same or different and are hydrogen or —O—C (═O) R ⁷ Showing,
The collagen production promoter according to claim 1. The collagen production promoter according to claim 1, wherein the compound represented by the general formula (1) is HPA or 2,4-HPA. A cosmetic composition, a composition for cell culture, or a pharmaceutical composition for wound healing , comprising the collagen production promoter according to any one of claims 1 to 3 .

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