JP2010254667A - Collagen gel shrinking agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a collagen gel shrinking agent to shrink collagen gel containing collagen and a fibroblast cell. <P>SOLUTION: There is provided the collagen gel shrinking agent containing an apple extract and effective for shrinking the collagen gel containing the collagen and the fibroblast cell. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a collagen gel shrinking agent.

In recent years, collagen gel has been used for drug evaluation as a dermal tissue model. The collagen gel is obtained by suspending fibroblasts in a collagen solution and gelling them under the fibroblast culture conditions. The fibroblasts are three-dimensionally cultured in the collagen gel, and the shape of the fibroblasts is different from that of the monolayer culture, and has a bipolar spindle shape similar to that in the living body. The collagen has a structure resembling a dermis tissue by re-arranging collagen fibers by gelation. The collagen gel is highly correlated with in vivo tests and has high reproducibility, so it is used for cytotoxicity tests and in vitro eye irritation tests. In recent years, the elasticity of skin, sagging, firmness, It is also used as a wrinkle improvement evaluation model (for example, Patent Document 1), wound healing promotion (for example, Patent Document 2) or wound contraction evaluation model (for example, Patent Document 3).

JP 2003-176208 A JP 2001-064196 A JP 2004-35526 A

  An object of the present invention is to provide a collagen gel contracting agent that contracts a collagen gel containing collagen and fibroblasts. It can be used for evaluation models of wound healing promotion and contraction.

Focusing on the apple extract, it was discovered that it has collagen contraction as a result of testing, and the present invention has been achieved. Furthermore, as a result of further research, it has been found that apple extract has a function of suppressing wrinkle formation, a function of suppressing transdermal moisture transpiration, a function of retaining stratum corneum moisture, and a function of suppressing epidermal thickening.

1. A collagen gel shrinking agent, which shrinks a collagen gel containing collagen and fibroblasts, comprising an apple extract as an active ingredient.
2. The apple extract contains an apple-derived polyphenol; The collagen gel shrinking agent described.
3. 1. The apple extract is an apple fruit extract Or 2. The collagen shrinkage agent described.
4.1. ~ 3. A wrinkle formation inhibitor comprising as an active ingredient any one of the collagen contractors described in 1.
5). 3. Prevents wrinkle formation due to UV exposure. The wrinkle formation inhibitor described.
6.1. ~ 3. A transdermal moisture transpiration inhibitor comprising any of the collagen contractors described in 1) as an active ingredient.
7). 5. Suppress transdermal moisture transpiration due to UV exposure. The transdermal moisture transpiration inhibitor as described.
8.1. ~ 3. A stratum corneum moisture-retaining agent containing any one of the collagen contractors described in 1) as an active ingredient.
9. 7. Suppresses the decrease in stratum corneum water content due to UV exposure. The stratum corneum moisture content retention agent as described.
10.1. ~ 3. An epidermis thickening inhibitor comprising any one of the collagen contractors described in 1) as an active ingredient.
11. 10. Suppresses thickening of epidermis caused by UV exposure. The epidermis thickening inhibitor as described.
12 The wrinkle formation inhibitor which uses an apple extract as an active ingredient.
13. A transdermal moisture transpiration inhibitor comprising apple extract as an active ingredient.
14 A stratum corneum moisture-retaining agent containing apple extract as an active ingredient.
15. An epidermis thickening inhibitor comprising apple extract as an active ingredient.

  The apple extract of the present invention can contract a collagen gel containing collagen and fibroblasts according to the collagen gel contracting agent. In particular, an effect was observed in apple fruit extract. Furthermore, the apple extract was found to be effective in suppressing wrinkle formation, suppressing transdermal moisture transpiration, maintaining stratum corneum moisture, and suppressing epidermal thickening. These functions are particularly effective against damage caused by exposure to ultraviolet rays contained in sunlight or the like.

It is a graph which shows the result of the collagen gel shrinkage rate of each evaluation sample. It is a photograph which shows the collagen gel contraction test result of control and the collagen gel contraction agent of this invention. The graph which shows a wrinkle volume ratio. Graph showing transdermal moisture transpiration TEWL. A graph showing the stratum corneum moisture content SC hydration. The graph which shows skin thickness. Graph showing DNA damage (8-OH dG). The graph which shows PCNA (cell proliferation marker). The graph which shows skin Hyp (hydroxyproline).

  The collagen gel shrinking agent of the present invention contains an apple extract as an active ingredient. In the collagen gel shrinking agent of the present invention, the apple extract is preferably an apple fruit extract. In particular, it is preferable that polyphenol contained in the apple fruit extract is the main component.

It is known that apple extracts contain polyphenols, organic acids, amino acids and the like.
An apple extract is a BNP regulator and a blood circulation promoter (JP 2007-008837 A, JP 2006-265220 A) containing an extract as an active ingredient as a BNP regulator capable of enhancing the vascular function improving action. No.), beverages (Japanese Unexamined Patent Publication No. 2004-305087), cosmetic ingredients (Japanese Unexamined Patent Publication No. 2001-187724), deodorant components (Japanese Unexamined Patent Publication No. 11-319051), perfume ingredients (Japanese Unexamined Patent Publication No. 08-2008). No. 023939) and the like.
However, the collagen gel contraction effect has not yet been reported for apple extracts and apple-derived polyphenols. Moreover, it has not been reported that the apple extract has an effect of suppressing wrinkle formation, suppressing transdermal moisture transpiration, maintaining stratum corneum moisture, and suppressing epidermal thickening.

Below, the collagen gel shrinkage | contraction agent and wrinkle formation inhibitor of this invention, a transdermal moisture transpiration amount inhibitor, a stratum corneum moisture content retention agent, and an epidermis thickening inhibitor are demonstrated in detail.
The collagen gel contraction agent of the present invention is a collagen gel contraction agent that contracts a collagen gel containing collagen and fibroblasts, and is characterized by containing an apple extract as an active ingredient. The apple extract is an active ingredient for suppressing wrinkle formation, suppressing transdermal moisture transpiration, maintaining stratum corneum moisture, and suppressing epidermal thickening. In particular, it functions effectively as a suppressive action against disorders such as wrinkle formation, transdermal moisture transpiration, stratum corneum moisture reduction, and epidermal thickening caused by exposure to ultraviolet rays contained in sunlight.

  In the apple extract, examples of apple (Malus pumilla) varieties include, but are not particularly limited to, Fuji, Kunimitsu, Wang Lin, Benitama, Jonagold, Delicious, Sansa, and Chiaki. The apple extraction site is not particularly limited, and examples thereof include fruits, leaves, trunks, flowers, and the like, preferably fruits. The fruit may be, for example, immature fruit (young fruit) or fully ripe fruit, and is not particularly limited. The part of the fruit used for extraction is not particularly limited, and examples thereof include whole fruit, pulp, pericarp, and seed. The apple extract may be extracted from these parts alone or in combination of two or more.

  The method for extracting the apple extract is not particularly limited, and a conventionally known method can be adopted. A specific example of the extraction method may be as follows, for example. First, the whole fruit of an apple is washed with water and then pulverized with a grinder or the like. The pulverized product may be subjected to pectinase treatment, centrifuged, and then partitioned and filtered with an extraction solvent to prepare an apple extract. Although it does not restrict | limit especially as said pectinase process, For example, you may carry out by adding 10-50 ppm of pectinases under the temperature conditions of 20-60 degreeC. Although it does not restrict | limit especially as an extraction solvent, For example, organic solvents, such as hexane and chloroform, are mentioned.

In the present invention, the apple extract may be, for example, a commercially available apple extract or may be prepared by extracting from an apple fruit, and is not particularly limited.
The apple extract content contained in the collagen gel shrinking agent of the present invention is not particularly limited, but is, for example, 0.01 to 99% by weight, preferably 0.1 to 50% by weight.
The collagen gel shrinking agent of the present invention is preferably an apple extract containing an apple-derived polyphenol.

  The polyphenol fractionation method is not particularly limited, and a conventionally known method can be employed. For example, the polyphenol may be fractionated by passing the apple extract through a column and then eluting the adsorbate on the column and concentrating the eluted fraction under reduced pressure. Furthermore, a powder auxiliary agent may be added to this concentrated liquid, and freeze-dried or spray-dried to prepare polyphenol powder.

  In the present invention, for example, a commercially available apple-derived polyphenol-containing product may be used as the polyphenol, and it may be prepared by extraction and fractionation from apple fruit, and is not particularly limited. Although the said polyphenol content contained in the collagen gel contraction agent of this invention is not restrict | limited in particular, For example, it is 0.01 to 99 weight%, Preferably, it is 0.1 to 50 weight%.

  The collagen gel contracting agent of the present invention contracts a collagen gel containing collagen and fibroblasts. Collagen gel contraction refers to, for example, a reduction in the size of the gel. The index of the size of the collagen gel is not particularly limited, and examples thereof include the diameter, surface area, and volume of the collagen gel.

  In this invention, the collagen gel should just be a shape which can be shrunk | reduced, For example, solid form may be sufficient besides gel form.

  The type of collagen contained in the collagen gel is not particularly limited and includes, for example, type I, type II, type III, type IV, type V collagen, and the like, preferably type I collagen. The collagen may be, for example, processed collagen. The processing is not particularly limited, and examples thereof include heat treatment and enzyme treatment. Examples of the heat-treated collagen include gelatin and the like, and examples of the enzyme-treated collagen include atelocollagen and collagen peptide. The collagen concentration in the collagen gel is not particularly limited, and can be appropriately set according to, for example, the shape.

In collagen gel, the tissue derived from fibroblasts is not particularly limited, for example, skin,
Examples include lung, heart and the like, preferably skin. Fibroblast-derived species is not particularly limited, and examples thereof include humans, pigs, cows, rabbits, rats, mice, and the like, and preferably humans. The cell density of fibroblasts in the collagen gel is not particularly limited and can be set as appropriate.

  The collagen gel may contain other components other than collagen and fibroblasts, for example. Other components are not particularly limited, and examples thereof include a culture solution and serum.

[Collagen shrinkage test]
In this example, apple phenon SH (manufactured by Asahi Food and Healthcare) was used as a collagen gel contraction agent, and the collagen gel contraction effect was measured as follows. In this example, as comparative examples, Agni dry extract (E10063, manufactured by Ask Chemical Co., Ltd.), hop dry extract (E10004, manufactured by Ask Chemical Co., Ltd.), Naringin (manufactured by Sigma Co.), chlorogenic acid (ICN Biomaterials) As a positive target, EXTRASOME (registered trademark) CP7-L (manufactured by NOF Corporation) was used, and the collagen contraction effect was measured in the same manner as Applephenon SH.

  First, each evaluation agent described in Table 1 below was added to 10% fetal bovine serum-containing Dulbecco's modified Eagle medium (DMEM) so as to have a predetermined concentration to prepare an evaluation sample.

Next, the neonatal skin-extracellular cells were suspended in DMEM containing 10% fetal bovine serum so as to be 6 × 10 ⁶ cells / mL to prepare a cell suspension. Under ice cooling, 20 mL of a collagen solution having the composition shown in the following table was mixed with 4 mL of the cell suspension to prepare a cell collagen solution. 1 mL of the cell collagen solution was dispensed into each well of a flat-bottom 12-well plate and gelled by culturing at 37 ° C. under 5% CO ₂ for 6 hours to prepare a collagen gel. After completion of the culture, the collagen gel was peeled from the well plate using a sterile spatula. To this collagen gel, 1 mL of each evaluation sample was added, and cultured for 7 days under conditions of 37 ° C. and 5% CO ₂ . During the culture period, the medium was changed every other day. After completion of the culture, the culture solution in each well was removed, and the collagen gel was washed with PBS. This collagen gel was infiltrated into a 10% neutral formalin solution (Wako Pure Chemical Industries, Ltd.) for 24 hours and fixed, and then immersed in a 1 w / v% Triton-X (registered trademark) solution to obtain the formalin solution. Was replaced. As a control, collagen gel untreated with the evaluation sample was cultured, fixed and replaced in the same manner except that DMEM containing 10% fetal bovine serum was added and cultured instead of the evaluation sample.

(Composition of collagen solution)

The immobilized collagen gel was perfectly round like the well shape. Therefore, the diameter of each evaluation sample treated or untreated collagen gel was measured and the area was calculated as follows. That is, the diameter of the fixed collagen gel was measured in two different directions, and the average value was calculated. The collagen gel radius (r, cm) is calculated by halving the average value, the obtained gel radius (r, cm) is substituted into the following formula (1), and the gel area (S, cm ² ) is calculated. Calculated. Furthermore, the collagen gel shrinkage rate (%) of each evaluation sample was calculated using the following formula (2).

Collagen gel area (cm ² ) = r × r × 3.14 (1)
r = collagen gel radius (cm)
Collagen gel shrinkage (%) = (ab) / a × 100 (2)
a = Analysis sample untreated gel area (cm ² )
b = collagen gel area (cm ² ) of the evaluation sample treatment

[Test results]
FIG. 1 and Table 3 below show the results of the collagen gel contraction rate (%) of each evaluation sample. In the graph of the figure, the vertical axis represents the collagen gel contraction rate (%), and the horizontal axis represents the name of each evaluation sample and its concentration. As shown in the figure and Table 3 below, the 0.1 w / v% hop dry extract treatment group showed 17.9% shrinkage, and the 0.1 w / v% Agni dry extract treatment group showed −33. A shrinkage of 2% was exhibited. Moreover, the maximum contraction rate of the naringin treatment group was 22.0% at 30 μmol / L, and the maximum contraction rate of the chlorogenic acid treatment group was 24.5% at 30 μmol / L. On the other hand, the apple phenone SH, which is the collagen shrinkage agent of this example, showed a contraction rate of 24.1% in the 0.006 w / v% treatment group and 47.1% in the 0.018 w / v% treatment group. The shrinkage rate was shown, and the 0.06 w / v% treatment group showed a shrinkage rate of 82.1%. That is, the apple phenone SH exhibits collagen contraction activity higher than that of polyphenols naringin and chlorogenic acid, and particularly at a treatment concentration of 0.1 w / v%, it is more prominent than EXTRASOME (registered trademark) CP7 which is a positive target. It showed contractile activity.

  FIG. 2 shows collagen gel photographs of Applephenon SH 0.06 w / v% treatment and control. In the photograph in the figure, each well in the left row contains a control collagen gel, and each well in the right row contains a collagen gel treated with applephenon SH 0.1 w / v%. The control collagen gel had the same area as the well, and no contraction was observed. The collagen gel treated with Applephenon SH 0.06 w / v% contracted into a gel significantly smaller than the well area indicated by an arrow. That is, the remarkable shrinkage | contraction of the collagen gel was confirmed by the apple phenone SH0.1% treatment.

[Photoaging test with apple extract]
It has been pointed out that there is a relationship between photoaging due to sun exposure (ultraviolet rays) and pigmentation of wrinkles, rough skin, stains and freckles (Gilchrest; Photodamage: Blackwell Science, Inc., 1995). It is known that long-term exposure to the sun (ultraviolet rays) increases deep wrinkles on the face and neck, and also causes dry skin and pigmentation such as rough skin, spots and freckles. As a countermeasure, a utilization test of the collagen contractility of the newly discovered apple extract was conducted. That is, the test was conducted by paying attention to an apple extract containing polyphenol as a main component as a component for alleviating, preventing, suppressing and improving skin aging and photoaging of the skin.
Skin aging generally refers to a physiological phenomenon that occurs when physiological aging associated with aging and photoaging due to sun exposure (ultraviolet rays) influence each other. With regard to this skin aging phenomenon, in the dermis tissue, the collagen component of the dermis is reduced, oxidized, saccharified, or denatured, so that the skin structure cannot withstand gravity and loses its elasticity, which can be the main cause of skin wrinkles and sagging. We also focused on exposure to ultraviolet rays as a major factor in the denaturation of collagen.

<Test method>
In a test system in which hairless mice were irradiated with UVA waves and UVB waves to form wrinkles, test substances were orally ingested to examine the effects of suppressing wrinkles and suppressing skin aging.
The test was conducted under the following conditions 1) to 11) using a 6-week-old hairless mouse (Hos: HR-1 female) at the time of introduction.

1) Preparation and administration of test object In the grouping of hairless mice, animals having a good general condition were divided into 6 groups per group so that there was no difference between the groups according to body weight on the day of administration start. Each individual was raised at 1 gauge / group.
Mice feed MF (manufactured by Oriental Bioservice) was subjected to a mixing treatment so that each test substance shown in Table 4 was uniformly mixed, and allowed to freely ingest by mixing. A list of administration groups is shown. An apple extract was used, and Pomativive HFV (manufactured by Val De Ville Bioactives (VVB) of France (Japan Agency Unitech Foods)) was used. Pomactive HFV is an apple polyphenol product made from French cider apples. The standard component is 90% or more of all polyphenols (calculated by the UV method). The main component is procyanidin, quercetin (glycoside), phlorizin, and catechins.
Feeding was carried out from immediately after animal delivery to 18 hours before dissection.

2) UV irradiation (Table 4; Test summary)
During UV irradiation, the animals were transferred to special cages, were irradiated with UVB20mJ / cm ² and UVA10J / cm ² by one group. Irradiation was carried out three times a week for 10 weeks on Monday, Wednesday and Friday.

3) Measurement of transcutaneous water transpiration (TEWL) Measurement of transcutaneous water transpiration was made using a vapometer (manufactured by Keystone Scientific), 2cm from the tail of the back to the neck and 0.5cm from the lumbar spine to the right. The measurement was performed by applying a terminal to the terminal. The measurement day was carried out on the test start day, and it was confirmed that there was no variation in the TEWL value of each group.

4) Measurement of stratum corneum moisture content Measurement of stratum corneum moisture content is performed using MoistureCheckerMY-808S (manufactured by SCALAR) with the terminal placed 2cm from the tail of the back to the neck and 0.5cm on the right side from the lumbar spine. did.

5) Dissection After fasting for 18 hours from the day after the end of the main breeding period in each group, anesthesia was introduced by intraperitoneal administration of 0.5 mg / kg body weight of ice-cooled Avertin (2,2,2-tribromoethanol). Thereafter, a reflective replica (manufactured by Asahi Biomed) was collected in a range of 2 cm from the tail of the back of the skin toward the neck and 0.5 cm on the right side from the lumbar spine. Thereafter, the abdomen was dissected and dissected. The skin tissue was infiltrated with 10% neutral formalin, embedded in paraffin, and stained with hematoxylin-eosin.

6) Wrinkle volume ratio measurement of skin morphology observation Photographing of the skin surface part and determination of the collected replica were performed. It was calculated ASA-03RXD wrinkle formation degree as an index wrinkle volume percent with ((Yes) Asahi Bio Ltd. ^{Med) (μm 3 / mm 2/} 100).

7) Thickening of epidermis The thickness of the epidermis was measured by hematoxylin-eosin staining of the skin tissue performed in the section “5) Dissection”. Six locations were arbitrarily selected from the same stained image of 500 μm, the thickness of the epidermis at each location was measured, and the average value of the 6 points was taken as the thickness of the individual epidermis.

8) 8-Hydroxy-2′-deoxyguanosine (8-OH dG) measurement 8-OH dG, which is a biomarker of DNA damage, was performed by immunohistochemical staining, focusing on epidermal cells.
Paraffin-embedded skin tissue prepared in the section “5) Dissection” was appropriately prepared with a 5 μm-thick section, and deparaffinization and hydrophilization were performed based on known methods. Antigen activation was carried out by microwave treatment for 5 minutes in boiling 0.01 M citrate buffer (pH 6.0). After cooling to room temperature, the reaction was carried out with methanol containing 0.3% hydrogen peroxide at room temperature for 30 minutes to inhibit endogenous peroxidase. After washing with water and 10 mM PBS (−), blocking reaction was performed by leaving at room temperature for 30 minutes with rabbit serum 75 times (diluted solution was PBS (−)). The serum was dropped from the slide glass, and the primary antibody (N45.1: manufactured by Nikken Zeil Co., Ltd.) was left at 5 μg / ml at 4 ° C. overnight to react with the primary antibody. After washing twice with PBS (−), a biotinylated secondary antibody (biotinylated rabbit immunoglobulin M; manufactured by DAKO) diluted 300-fold was reacted with the secondary antibody at room temperature for 30 minutes. The plate was washed twice with PBS (−) and reacted with ABC reagent (ABC-HRP; manufactured by Vectastein) for 30 minutes at room temperature. The reaction was performed at room temperature for 3 minutes and 30 seconds using DAB (3,3-diaminobenzidine tetrahydrochloride: manufactured by DAKO) as a coloring reagent. After washing with water, dehydration and encapsulation were performed based on a known method. The state of staining of the skin tissue was observed under a microscope, and scored based on the following criteria. Those having difficulty in scoring were added with an intermediate value of 0.5, and the score of each group was calculated.

Score ×; Score 3; Cell nuclei above the epidermis cells stain in many layers Δ; Score 2; Cell nuclei above the epidermis cells stain ○: Score 1; Cell nuclei of epidermal cells stain ◎; Score 0; Epidermis Staining to the extent that cell nuclei are scattered

9) Measurement of Proliferation Cell Nuclear Antigen (PCNA; Proliferating Cell Nuclear Antigen) PCNA is a nuclear protein whose expression is recognized from the late cell growth phase to the early DNA synthesis phase of the cell cycle. DNA polymerase and RFC (replication factor C; Clamp attachment factor) and a new DNA are synthesized and repaired. However, in an environment where PCNA is constantly expressed, such as continuous irradiation of weak ultraviolet rays, the cell cycle becomes abnormal, and then cell proliferation increases and epidermal cell turnover increases, resulting in keratinization failure and epidermal thickening. Invited, ultimately, it is thought to cause a photoaging phenomenon.
Measurement of PCNA, which is a biomarker of cell proliferation, by immunohistochemical staining was performed focusing on epidermal cells.
Paraffin-embedded skin tissue prepared in the section “5) Dissection” was appropriately prepared with a 5 μm-thick section, and deparaffinization and hydrophilization were performed based on known methods. Antigen activation was performed by microwave treatment for 5 minutes in boiling 0.01 M citrate buffer (pH 6.0). After cooling to room temperature, the reaction was carried out with methanol containing 0.3% hydrogen peroxide at room temperature for 20 minutes to inhibit endogenous peroxidase. After washing with water and PBS (−), blocking reaction was performed by leaving rabbit serum at 75 times (diluted solution PBS (−)) for 30 minutes at room temperature. The serum was dropped from the slide glass, and the primary antibody (PC-10: manufactured by Santa Cruz) was left at 1 μg / ml at 4 ° C. overnight to react with the primary antibody. After washing twice with PBS (−), a biotinylated secondary antibody (biotinylated rabbit immunoglobulin M; manufactured by DAKO) diluted 300-fold was reacted with the secondary antibody at room temperature for 30 minutes. The plate was washed twice with PBS (−) and reacted with ABC reagent (ABC-HRP; manufactured by Vectastein) for 30 minutes at room temperature. The reaction was performed at room temperature for 3 minutes and 30 seconds using DAB (3,3-diaminobenzidine tetrahydrochloride: manufactured by DAKO) as a coloring reagent. After washing with water, dehydration and encapsulation were performed based on a known method. The state of staining of the skin tissue was observed under a microscope, and scored based on the following criteria. Those having difficulty in scoring were added with an intermediate value of 0.5, and the score of each group was calculated.

Score ×; Score 3; Cell nuclei above the epidermis cells stain in many layers Δ; Score 2; Cell nuclei above the epidermis cells stain ○: Score 1; Cell nuclei of epidermal cells stain ◎; Score 0; Epidermis Staining to the extent that cell nuclei are scattered

10) Skin hydroxyproline (Hyp)
Skin protein was hydrolyzed with hydrochloric acid and the amount of hydroxyproline was measured by LC / MS / MS.
10 mg was cut out from the mouse skin frozen in the section “5) Dissection” for collagen measurement. The skin piece was finely cut with a scissors and placed in a test tube with a screw cap, and 1 ml of 6N hydrochloric acid was added, followed by acid hydrolysis at 110 ° C. for 24 hours. The reaction solution was diluted 50 times with MilliQ water, then filtered (φ = 0.45 mm), and further diluted 100 times with MilliQ water was used as a sample for measuring hydroxyproline.
Hydroxyproline was determined using ACQUITY LC / MS / MS system ^(R) TQD ^(Waters).
LC condition-Column HSS T3 2.1X100mm (product made from Waters)
・ Flow rate 0.4mL / min
・ Column temperature 50 ℃
-Mobile phase A: 0.1% formic acid
B: 0.1% formic acid, 80% acetonitrile

MS / MS conditions ・ Ionization ESI + (Positive)
・ Capillary voltage 0.5kV
・ Desolvent gas 1,000 L / Hr
・ Corn gas 50L / Hr
・ Ion source heater 150 ℃
・ Monitor MRM
Precursor ion (m / z) 132.16
Product ion (m / z) 86.1
-Dwell time (seconds) 0.05
・ Cone voltage (V) 28
Collision energy (eV) 12
The amount of hydroxyproline in the skin collagen was calculated by comparing the peak area with the standard hydroxyproline (manufactured by Wako).

11) Statistical processing The test results were expressed as mean ± standard deviation (mean ± SD), and multiple comparisons (Steel's test) were tested for significance with the analysis software of Excel Statistics 2008. ** ⇒p <0.01, * ⇒p <0.05, and p <0.1 was noted as a significant trend.

The results of Test Example 2 are sequentially shown below.
1. General condition observation and measurement of wrinkle volume ratio In the UV irradiated animals from the 4th week of UV irradiation, light browning of the skin of the head, wrinkle depth of the neck, and wrinkles of the back of the hind limbs were compared with those of the UV non-irradiated animals. It came to stand out.
In the appearance observation at the time of dissection, wrinkles on the face, neck and back of the hind limb were clearly confirmed in the UV-irradiated animals, but also in the UV irradiation, high concentration (0.06%), medium concentration (0.012%) and The symptom was mild in the mice ingested at a low concentration (0.006%). Particularly, the wrinkle depth of the animals ingested at a high concentration was shallow, and there was no degree of wrinkle macroscopically. In addition, the apple extract intake animals had a moist feeling on the skin as compared to the control group, and the symptoms were particularly noticeable in the high concentration intake group.
As shown in the graph of wrinkle volume ratio in Table 6 and FIG. 3, clear wrinkle formation was observed by ultraviolet irradiation, and the wrinkle suppression effect was significantly recognized in a mixed diet group under the same ultraviolet irradiation conditions. . On the other hand, the wrinkle-suppressing effect was negligible in the mixed diet group, and the wrinkle volume ratio was significantly suppressed by the 0.06% and 0.012% mixed diets by a significant difference test.

2. Body weight transition As shown in Table 6, there was no significant difference in body weight transition between the groups.

3. Food intake As shown in Table 6, there was no significant difference in food intake as in the case of body weight change. The amount of food intake was calculated from the average value from 6 weeks to 10 weeks of ultraviolet irradiation when the weight gain became slow.
The equivalent amount when a human ingests can be calculated from “2. Weight transition” and “3. Food intake”. In terms of body surface area, the daily intake per person (60 kg body weight) can be estimated from the conversion formula (body surface area) = (body weight) ^2/3 (cm ² ). A list is shown in Table 8. From this result, the mixed diet administration to 0.06% and 0.012% mice showing significant suppression of the wrinkle volume ratio was 412.8 mg / day / kg human 60 kg body weight and 82.75 mg / It became 60 kg body weight per day / kg, and it was considered that wrinkles associated with the photoaging phenomenon could be suppressed with an amount that could be easily consumed by humans.

4). Liver weight There was no significant difference in liver weight, and no abnormal symptoms were observed. No abnormalities were observed in other organs.

5). Transdermal moisture transpiration (TEWL)
The results are shown in Table 6.
It is considered that the higher the value of transdermal moisture transpiration, the more water evaporates from the stratum corneum and causes the skin to dry. The increase in the amount of transdermal water transpiration due to UV irradiation is generally known. In this test, the amount of transdermal water transpiration on the day of dissection was higher in the UV + group than in the UV-group, confirming the dry state. At this time, the UV + irradiation and pomactive HFV intake groups showed low TEWL values. Also in the measurement of the dissection date, the UV-group and the pomictive HFV showed significantly lower TEWL values in the 0.06%, 0.012% and 0.006% diet groups compared to the UV + irradiation group.

6). Table 7 shows the stratum corneum moisture content.
It is considered that the lower the numerical value of the stratum corneum water content, the lower the moisture content of the stratum corneum or the natural moisturizing factor (NMF) related to water retention, which causes the dry state of the skin. It is generally known that the stratum corneum moisture content is reduced by UV irradiation. In this test, the stratum corneum moisture content on the day of dissection was lower in the UV + group than in the UV- group, confirming the dry state. At this time, the UV irradiation + pomatic HFV intake group showed recovery of the stratum corneum water content. Compared with the UV + irradiated group, the UV irradiated + pomatic HFV showed significantly higher keratin water content in the 0.06%, 0.012% and 0.006% fed groups.

7). The skin thickening results are shown in Table 7.
In contrast to the [UV + / control diet] group, significant suppression of epidermal thickening (acanthosis) was demonstrated in the [UV− / control diet] group, the pomactive HFV 0.06%, and 0.012% diet groups.

8.8-OHdG
The results are shown in Table 7.
In contrast to the [UV + / control diet] group, 8-OHdG was significantly suppressed in the [UV− / control diet] group and the pomactive HFV 0.06% mixed diet group. Moreover, 8-OHdG was suppressed as a significant tendency (p = 0.0926) in the pomactive HFV 0.012% mixed group.
8-OHdG is an indicator of cellular DNA damage, and is formed by oxidation of guanine in DNA by exposure to oxidative stress such as ultraviolet rays, radiation, chemical pollutants, and tobacco smoke. It was suggested that 8-OHdG was increased in the basal cell layer of the mouse skin due to ultraviolet irradiation in this test, and that DNA damage was caused, and that 8-OHdG was significantly suppressed by the feeding of pomactive HFV 0.06%. is doing. Moreover, 8-OHdG was suppressed as a significant tendency (p = 0.0926) with 0.012% diet.

9. PCNA
The results are shown in Table 7.
In contrast to the [UV + / control diet] group, PCNA was significantly suppressed in the [UV− / control diet] group, the pomactive HFV 0.06%, 0.012% and 0.006% mixed diet groups.

10. Skin hydroxyproline (Hyp)
The results are shown in Table 7.
Compared with the [UV + / control diet] group, Hyp was significantly increased in the pomactive HFV 0.06% and 0.012% mixed groups. An increase in Hyp suggests an increase in the amount of collagen in the skin tissue.
To summarize the results of this study, the administration of dietary feed to mice with 0.06% and 0.012% of poctive HFV that showed a significant suppression of the wrinkle volume ratio was 412.8 mg / day / kg 60 kg for humans, respectively. The body weight was 82.75 mg / day / kg and the human body weight was 60 kg, and it was considered that wrinkles associated with photoaging could be suppressed with an amount that can be easily consumed by humans.

Claims (15)

  A collagen gel shrinking agent, which shrinks a collagen gel containing collagen and fibroblasts, comprising an apple extract as an active ingredient.   The collagen gel contractor according to claim 1, wherein the apple extract contains polyphenol derived from apple.   The collagen contractor according to claim 1 or 2, wherein the apple extract is an extract of apple fruit.   The wrinkle formation inhibitor which uses any collagen contraction agent described in Claims 1-3 as an active ingredient.   The wrinkle formation inhibitor according to claim 4, which suppresses wrinkle formation caused by ultraviolet exposure.   The transdermal moisture transpiration inhibitor which uses any collagen contraction agent described in Claims 1-3 as an active ingredient.   The transdermal moisture transpiration inhibitor according to claim 6, which suppresses transdermal moisture transpiration due to ultraviolet exposure.   A stratum corneum moisture-retaining agent comprising as an active ingredient any one of the collagen contractors according to claim 1.   The stratum corneum moisture-retaining agent according to claim 8, which suppresses a decrease in stratum corneum moisture due to ultraviolet exposure.   An epidermis thickening inhibitor comprising any one of the collagen contractors according to claim 1 as an active ingredient.   The epidermis thickening inhibitor of Claim 10 which suppresses the thickening of the epidermis by ultraviolet exposure.   The wrinkle formation inhibitor which uses an apple extract as an active ingredient.   A transdermal moisture transpiration inhibitor comprising apple extract as an active ingredient.   A stratum corneum moisture-retaining agent containing apple extract as an active ingredient.   An epidermis thickening inhibitor comprising apple extract as an active ingredient.