JP2011144113A - Active oxygen scavenger, radical scavenger and oxidative cell damage inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an active oxygen scavenger containing a highly safe natural extract as an active ingredient; to provide a radical scavenger; and to provide an oxidative cell damage inhibitor. <P>SOLUTION: The active oxygen scavenger, the radical scavenger and the oxidative cell damage inhibitor contain the extract from picked fruits of kiwi. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an active oxygen scavenger, a radical scavenger, and an oxidative cytotoxicity inhibitor.

In recent years, active oxygen has attracted attention as a factor that particularly oxidizes biological components, and its adverse effect on living organisms has become a problem. Active oxygen is generated in the process of energy metabolism in living cells. Examples of active oxygen include superoxide (that is, superoxide anion generated by one-electron reduction of oxygen molecules: .O ₂ ⁻ ), hydrogen peroxide (H ₂ O ₂ ), hydroxy radical (.OH), singlet oxygen ( ¹ O ₂ ) and the like. These active oxygens are essential for the phagocytic sterilization mechanism and play an important role in removing viruses and cancer cells.

  However, excessive production of active oxygen attacks in vivo molecules constituting membranes and tissues in the living body and induces various diseases. Normally, superoxide produced in vivo and used as a starting material for other active oxygen is sequentially eliminated by the catalytic action of superoxide dismutase (SOD) contained in the cells. Is excessive or when the SOD action is reduced, superoxide elimination is insufficient, and the superoxide concentration increases, which may cause tissue damage such as rheumatoid arthritis and Behcet's disease, myocardial infarction, stroke Cause cataracts, wrinkles, diabetes, arteriosclerosis, stiff shoulders, coldness.

  In particular, since the skin is directly affected by environmental factors such as ultraviolet rays, it is an organ that easily generates superoxide, and therefore, by increasing the concentration of superoxide, for example, the biological tissue such as collagen is decomposed and denatured or It is thought to crosslink and oxidize fats and oils to produce lipid peroxides that damage cells, and the damage caused by active oxygen is the cause of aging such as skin wrinkle formation and skin elasticity reduction It is thought to become. Therefore, by inhibiting / suppressing the generation of active oxygen and in vivo radicals, skin aging such as wrinkle formation and reduced elasticity, tissue disorders such as rheumatoid arthritis and Behcet's disease, myocardial infarction, stroke, cataracts, diabetes, arteries It is considered that various disorders involving active oxygen such as hardening, stiff shoulders, and coldness can be prevented, treated or improved.

  In addition, active oxygen is known to cause inflammation, production of lipid peroxide, inactivation of various enzymes and DNA damage, and these oxidative cell damage is caused by rough skin, wrinkle formation, skin elasticity. It is considered to be one of the causes of skin aging such as decline.

  Therefore, attempts have been made to obtain active oxygen scavenging substances, radical scavenging substances, etc. from natural products advantageous in terms of safety. Extracts from Brassica family Brassica (Patent Documents) 1) and the like are known. In addition, as an extract capable of suppressing oxidative cell damage caused by active oxygen, an extract from Japanese black beetle, mulberry, water hyssop (see Patent Document 2) and the like are known.

JP 2003-81848 A JP 2009-227598 A

  The present invention finds a highly safe natural extract having an active oxygen scavenging action, radical scavenging action, and oxidative cell damage suppressing action, and an active oxygen scavenger, radical scavenger and oxidative active ingredient containing the same. It aims at providing a cytotoxicity inhibitor.

  In order to achieve the above object, the active oxygen scavenger, radical scavenger, or oxidative cytotoxicity inhibitor of the present invention is characterized by containing an extract from the fruit of kiwi fruit as an active ingredient.

  In the present invention, “kiwi fruit fruit” refers to a fruit that is thinned (pruned) for the purpose of increasing the sugar content and size of the cultivated kiwi fruit to a desired range in the cultivation process of kiwi fruit. Specifically, the sugar content is 3.0 to 7.0%, preferably 4.0 to 5.5%, and the size (major axis) is 1 to 4 cm, preferably 2 to 2%. It means kiwi fruit which is 3cm.

  In the present invention, “oxidative cell damage” means cell damage caused by active oxygen, particularly oxidative damage such as inflammation, lipid peroxide production, inactivation of various enzymes, DNA damage, etc. Organizational disorders such as organizational disorders are also included. The cells that are damaged by active oxygen are not particularly limited as long as they are living cells that are damaged by active oxygen.

  According to the present invention, it is possible to provide an active oxygen scavenger, a radical scavenger and an oxidative cytotoxicity inhibitor that contain an extract from the fruit of kiwi, which is a natural product, as an active ingredient. .

Hereinafter, an embodiment of the present invention will be described.
The active oxygen scavenger, radical scavenger, or oxidative cytotoxicity inhibitor of the present embodiment contains an extract from the fruit of kiwi (Actinidia chinensis Planch. (Actinidiaceae)) as an active ingredient.

  Here, in the present embodiment, the “extract” includes an extract obtained by using a fruit of kiwi (Actinidia chinensis Planch. (Actinidiaceae)) as an extraction raw material, a diluted or concentrated solution of the extract, and the extract. A dried product obtained by drying, or a crude product or a purified product thereof is included.

  Kiwi (Actinidia chinensis Planch. (Actinidiaceae)) is a deciduous vine shrub belonging to the genus Matatabidae, and its fruits are cultivated in various parts of Japan and can be easily obtained from these areas. The component part of kiwi used as an extraction raw material is a fruit. The cultivar of kiwi (Actinidia chinensis Planch. (Actinidiaceae)) that can be used as an extraction raw material in the present invention is not particularly limited, but is rainbow red, apple kiwi, red heart, golden king, zestpri gold, Kobayashi 39, viewer country, first emperor, teardrop, red, grape kiwi, sanuki gold, honey, etc. can be suitably used.

  The fruit of kiwi used as an extraction raw material has a sugar content of 3.0 to 7.0% and a size (major axis) of 1 to 4 cm, preferably a sugar content of 4.0 to 5 0.5% and a size (major axis) of 2 to 3 cm.

  The extract from the fruit of kiwi fruit can be obtained by pulverizing the extraction raw material using a crusher and subjecting it to extraction with an extraction solvent.

  As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. It is preferable.

  Water that can be used as the extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, acidic / alkaline ionized water, deep ocean water, and various other treatments. Is included. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, the 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 hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic 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. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, 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 a mixed liquid of water and a lower aliphatic alcohol, it is preferable to mix 1 to 100 parts by volume of a lower aliphatic alcohol with respect to 1 part by volume of water. When using a mixed liquid, it is preferable to mix 1 to 100 parts by volume of a lower aliphatic ketone with respect to 1 part by volume of water, and when using a mixed liquid of water and a polyhydric alcohol, water 1 It is preferable to mix 1 to 100 parts by volume of a polyhydric alcohol with respect to the volume part.

  The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in an extraction solvent 5 to 15 times (mass ratio) of the extraction raw material, the soluble components are extracted at room temperature or under reflux, and then filtered to remove the extraction residue. A liquid can be obtained. The obtained extract is diluted, concentrated, dried, purified, etc. according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude purified product or a purified product thereof. Processing may be performed.

  Purification can be performed by, for example, activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, or the like. The obtained extract can be used as it is as an active ingredient of an active oxygen scavenger, a radical scavenger, or an oxidative cytotoxicity inhibitor, but a concentrate or a dried product is easier to use.

  Since the extract from the fruit of kiwi fruit has a unique odor, it can be purified for the purpose of decolorization, deodorization, etc. within a range that does not cause a decrease in its physiological activity. Since it is not used in a large amount when blended into a material, there is no practical problem even if it is not purified.

  Since the extract from the fruit of kiwi fruit obtained as described above has an active oxygen scavenging action, a radical scavenging action or an oxidative cell damage inhibiting action, the active oxygen scavenger is used by utilizing each action. It can be used as an active ingredient of a radical scavenger or an oxidative cytotoxicity inhibitor.

  In addition, as will be apparent from the examples described later, the extract from the fruit of kiwi fruit also has an action to promote epidermal keratinocyte proliferation, an action to promote expression of profilagrin mRNA, or an action to suppress cell damage caused by ultraviolet irradiation. Therefore, each action can be used as an active ingredient of an anti-aging agent, an epidermal keratinocyte proliferation promoter, a profilagrin mRNA expression promoter, or a cytotoxicity inhibitor by ultraviolet irradiation.

  Each of the above-mentioned drugs may consist only of an extract from the fruit of extracting kiwi, or may be a preparation of an extract from the fruit of extracting kiwi.

  The extract from the fruit of kiwi fruit is any powder, granule, tablet, liquid, etc., using a pharmaceutically acceptable carrier such as dextrin, cyclodextrin, etc. It can be formulated into a dosage form. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent and the like can be used. In addition, the extract from the fruit of kiwi fruit can be used by blending with other compositions (for example, skin cosmetics).

  In addition, the active oxygen scavenger, radical scavenger, or oxidative cytotoxicity inhibitor of the present embodiment is another natural extract having an active oxygen scavenging action, radical scavenging action, or oxidative cytotoxicity inhibitory action as necessary. Etc. can be blended and used as an active ingredient.

  The administration method of the active oxygen scavenger, radical scavenger, or oxidative cytotoxicity inhibitor of the present embodiment generally includes transdermal administration, etc., but it is suitable for its prevention and treatment depending on the type of disease. A method (for example, oral administration, transmucosal administration, etc.) may be appropriately selected.

  Further, the dose of the active oxygen scavenger, radical scavenger or oxidative cytotoxicity inhibitor of the present embodiment may be appropriately increased or decreased depending on the type of disease, severity, individual differences among patients, administration method, administration period, etc. .

  The active oxygen scavenger of this embodiment is a skin aging such as wrinkle formation and elasticity reduction, tissue damage such as rheumatoid arthritis and Behcet's disease, myocardial infarction through the active oxygen scavenging action of the extract from the fruit of kiwi fruit It is possible to prevent and improve various disorders involving active oxygen such as stroke, cataract, diabetes, arteriosclerosis, stiff shoulders, and coldness. However, the active oxygen scavenger of the present embodiment can be used for all purposes other than these uses that are meaningful for exhibiting the active oxygen scavenging action.

  The radical scavenger of this embodiment is a skin scavenging process such as wrinkle formation and reduced elasticity, tissue damage such as rheumatoid arthritis and Behcet's disease, myocardial infarction, stroke through the radical scavenging action of the extract from the fruit of kiwi It is possible to prevent and improve various disorders involving active oxygen (radicals) such as cataract, diabetes, arteriosclerosis, stiff shoulders, and coldness. However, the radical scavenger of the present embodiment can be used for all uses other than these uses that are meaningful for exhibiting radical scavenging action.

  The oxidative cytotoxicity inhibitor of the present embodiment can suppress the cellular damage caused by active oxygen through the oxidative cytotoxicity-inhibiting action of the extract from the fruit of kiwi fruit, resulting in rough skin. It can prevent and improve skin aging symptoms such as wrinkle formation and skin elasticity reduction. However, the oxidative cytotoxicity-suppressing agent of this embodiment can be used for all uses that are meaningful for exerting an oxidative cytotoxicity-inhibiting action in addition to these uses.

  The extract from the fruit of kiwi fruit has an active oxygen scavenging action, radical scavenging action or oxidative cell damage inhibiting action, and is excellent in use feeling or safety when applied to the skin. It is suitable for blending into the material.

  In this case, the skin cosmetic may contain an extract from the fruit of kiwi fruit as it is, or an active oxygen scavenger, a radical scavenger or an oxidative agent formulated from the extract from the fruit of kiwi fruit. You may mix | blend a cytopathic inhibitor.

  Reactive oxygen is added to skin cosmetics by adding an active oxygen scavenger, radical scavenger, or oxidative cytotoxicity inhibitor formulated from an extract from the fruit of kiwi fruit extract or from the fruit of the kiwi fruit. An erasing action, a radical erasing action or an oxidative cell damage inhibiting action can be imparted.

  There are no particular limitations on the type of skin cosmetic that can be blended with extracts from kiwi fruit, etc. Examples include ointments, creams, emulsions, lotions, packs, foundations and the like.

  When the extract from the fruit of kiwi fruit is blended in the skin cosmetic, the blending amount can be appropriately adjusted according to the type of the skin cosmetic, but the preferred blending ratio is 0.0005-0. .45 mass% (in terms of solid content), and a particularly suitable blending ratio is 0.005 to 0.05 mass% (in terms of solid content). In addition, as will be apparent in the examples described later, the extract from the fruit of kiwi fruit is less active oxygen scavenging, radical scavenging, oxidative at lower concentrations than the extract from the ripe fruit of kiwi. It has the effect of inhibiting cell damage, inhibiting cell damage caused by UV irradiation, promoting keratinocyte proliferation, and promoting profilaggrin mRNA expression. Even if the concentration is as low as .0005 to 0.001% by mass (in terms of solid content), the desired effect can be imparted to the skin cosmetic.

  The above skin cosmetics are active oxygen scavenging action, radical scavenging action, oxidative cell damage inhibiting action, cell damage inhibiting action by ultraviolet irradiation, epidermal keratinocyte proliferation promoting action or profilagrin Main agents, auxiliaries or other components used in the production of normal skin cosmetics, such as astringents, bactericides / antibacterial agents, UV absorbers, moisturizers, cell activators, anti-inflammatory agents, as long as they do not interfere with the mRNA expression promoting action -Antiallergic agents, antioxidant / active oxygen scavengers, fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances and the like can be used in combination. By using together, it becomes a more general product, and the synergistic action between the above-mentioned components used in combination may bring about a superior use effect than would normally be expected.

  The skin cosmetics have high safety, and also have an active oxygen scavenging action, a radical scavenging action, an oxidative cell damage inhibiting action, a cell damage inhibiting action by ultraviolet irradiation, an epidermal keratinocyte proliferation promoting action and a profilagrin. Skin aging such as wrinkle formation and decreased elasticity, tissue damage such as rheumatoid arthritis and Behcet's disease, myocardial infarction, stroke, cataract, diabetes through one or more actions selected from the group consisting of mRNA expression promoting action It is possible to prevent, treat or improve various disorders involving active oxygen such as arteriosclerosis, stiff shoulders, coldness, dry skin, rough skin, and the like.

  The active oxygen scavenger, radical scavenger, or oxidative cytotoxicity inhibitor of the present embodiment is preferably applied to humans. It can also be applied to animals.

  EXAMPLES Hereinafter, although an Example and a comparative example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all.

[Example 1] Manufacture of fruit extract of kiwi fruit extract (Actinidia chinensis Planch. (Actinidiaceae), rainbow red species) Fruit extract (sugar content: 4.3%, major axis: 2 cm) of crushed material 200g Water) 400 mL was added, and the mixture was heated and extracted at 30 ° C. for 1 hour, and filtered while hot. The residue was further extracted in the same manner. The obtained extracts were combined, concentrated under reduced pressure, and further dried to obtain 5.5 g of kiwi fruit extract.

[Comparative Example 1] Manufacture of kiwi fruit extract Implemented except that 200 g of ground ripened fruit (sugar content: 13.4%, major axis: 5 cm) of kiwi (Actinidia chinensis Planch. In the same manner as in Example 1, 18.5 g of kiwi fruit extract was obtained.

[Test Example 1] Superoxide scavenging action test (NBT method)
The kiwi fruit extract of Example 1 and the kiwi fruit extract of Comparative Example 1 were tested for superoxide scavenging action as follows.

In a test tube, 2.4 mL of 0.05 M NaHCO ₃ buffer (pH 10.2), 3 mM xanthine, 3 mM EDTA, 50 μg / mL bovine serum albumin solution and 0.75 mM NBT (nitroblue tetrazolium) 0.1 mL each was added, and 0.1 mL of the sample solution (Example 1 and Comparative Example 1, see Table 1 below for the sample concentration) was added thereto, and the mixture was allowed to stand at 25 ° C. for 10 minutes. After standing, 0.1 mL of xanthine oxidase as an enzyme solution was added and stirred rapidly and reacted at 25 ° C. for 20 minutes. Thereafter, 0.1 mL of 6 mM copper chloride was added to stop the reaction, and the absorbance at a wavelength of 560 nm was measured.

  Even when the enzyme solution was not added, the same operation and absorbance were measured, and the same measurement was performed when distilled water was added without adding the sample solution. From the obtained results, the superoxide elimination rate (%) was calculated by the following formula.

Superoxide erasure rate (%) = {1- (AB) / (CD)} × 100
In the formula, A represents “absorbance when enzyme solution is added / sample solution added”, B represents “absorbance when enzyme solution is not added / sample solution is added”, and C is “when enzyme solution is added / sample solution is not added” "D" represents "absorbance when no enzyme solution is added / sample solution is not added".
The results are shown in Table 1.

  As shown in Table 1, it was confirmed that the kiwi fruit extract (Example 1) has an extremely excellent superoxide scavenging action (active oxygen scavenging action), particularly at a high concentration (100 μg / mL). On the other hand, the kiwi fruit extract (Comparative Example 1) showed almost no superoxide scavenging action. In addition, the strength of the superoxide scavenging action of the kiwi fruit extract increases depending on the extract (sample) concentration, so the degree of superoxide scavenging action can be adjusted by the concentration of the kiwi fruit extract. Was confirmed.

[Test Example 2] Radical scavenging action test The kiwi fruit extract of Example 1 and the kiwi fruit extract of Comparative Example 1 were tested for radical scavenging action as follows.

Add 3 mL of sample solution (Example 1 and Comparative Example 1, see Table 2 below for sample concentration) to 3 mL of 1.5 × 10 ⁻⁴ M DPPH (diphenyl-p-picrylhydrazyl) ethanol solution, and then shake and mix. Left for 30 minutes. After standing, the absorbance at a wavelength of 520 nm was measured. As a control, the same operation was performed using only the solvent in which the sample was dissolved instead of the sample solution, and the absorbance at a wavelength of 520 nm was measured. Further, as a blank, after adding 3 mL of the sample solution to ethanol, the absorbance at a wavelength of 520 nm was measured immediately. From the obtained results, the radical scavenging rate (%) was calculated by the following formula.

Radical scavenging rate (%) = {1- (BC) / A} × 100
In the formula, A represents “absorbance of control”, B represents “absorbance upon addition of sample solution”, and C represents “absorbance of blank”.
The results are shown in Table 2.

  As shown in Table 2, it was confirmed that the kiwi fruit extract (Example 1) has an excellent radical scavenging action particularly at a high concentration (200 μg / mL). On the other hand, the kiwi fruit extract (Comparative Example 1) did not show radical scavenging action. In addition, since the strength of radical scavenging action of kiwi fruit extract increases depending on the extract (sample) concentration, the degree of radical scavenging action can be adjusted by the concentration of kiwi fruit extract. It is done.

[Test Example 3] Oxidative Cytotoxicity Inhibitory Action Test The kiwi fruit extract of Example 1 and the kiwi fruit extract of Comparative Example 1 were tested for oxidative cytotoxicity inhibitory action as follows.

DMEM (Dulbecco's) containing 10% FBS (Fetal Bovine Serum, manufactured by JRH Biosciences), 40-year-old healthy male skin-derived fibroblasts (NBKN, established with research consent, passage number: 13 or less) The modified minimum essential medium (manufactured by Gibco) was used under the conditions of 37 ° C. and 5% CO ₂ -95% air.

  The plasmid pGL2 was diluted to 31 μg / mL with sterilized water, transferred to a 60 mm dish, rose Bengal (manufactured by Kanto Chemical Co., Ltd., 100 μg / mL) was added, and visible light (xenon lamp, manufactured by USHIO INC., 370 to 760 nm) was added. , 30 cm) for 10 minutes.

Cells (2 × 10 ⁴ cells / well) were seeded in a 24-well plate and cultured for 18 hours. A gene introduction agent (manufactured by Qiagen) was added to a plasmid (0.2 μg DNA / well) with or without rose bengal, and introduced at 37 ° C. for 24 hours. After exchanging the medium, a sample solution (Example 1 and Comparative Example 1, refer to Table 3 below for the sample concentration) was added, and further cultured for 24 hours.

  The cells were washed with PBS (−) (phosphate buffered saline) and then diluted 5-fold with sterilized water (Toyo Benet Inc., Picker Gene, 25 mM Tris (7.5), 2 mM DTT, 10% Glycerol, 1% Triton X-100) was added at 50 μL / well. Thereafter, the cells were stored at −80 ° C. for 30 minutes and allowed to stand at room temperature for 15 minutes to thaw the cells.

After stirring the plate (room temperature, 15 minutes), the cell lysate was collected in a 1.5 mL centrifuge tube with a scraper and centrifuged (15,000 rpm, 2 minutes). 20 μL of supernatant and luminescent substrate (Toyo Benet Corporation, Picker Gene, 20 mM Tricine / 1.07 mM (MgCO ₃ ) 4Mg (OH) ₂ .5H ₂ O / 2.67 mM MgSO ₄ /0.1 mM EDTA / 33.3 mM DTT / 270 μM Coenzyme A / 470 μM luciferin / 530 μM ATP) 100 μL was added, mixed at room temperature, set in a luminometer (GENELIGHT55, manufactured by Microtech Nichion), and measured at 560 nm.

The inhibition rate (%) of oxidative cell damage was determined by calculating the amount of luciferase activity due to DNA repair after the addition of rose bengal to the absence of rose bengal.
The results are shown in Table 3.

  As shown in Table 3, it was confirmed that the kiwi fruit extract (Example 1) has an excellent inhibitory effect on oxidative cell damage compared to the kiwi fruit extract (Comparative Example 1).

[Test Example 4] Cytotoxicity inhibitory effect test by ultraviolet irradiation The kiwifruit extract of Example 1 and the kiwifruit extract of Comparative Example 1 were tested for the cytotoxicity inhibitory effect by ultraviolet irradiation as follows.

DMEM (Dulbecco's) containing 10% FBS (Fetal Bovine Serum, manufactured by JRH Biosciences), 40-year-old healthy male skin-derived fibroblasts (NBKN, established with research consent, passage number: 13 or less) The modified minimum essential medium (manufactured by Gibco) was used under the conditions of 37 ° C. and 5% CO ₂ -95% air.

The plasmid pGL2 was diluted to 31 μg / mL with sterilized water, transferred to a 60 mm dish, and then irradiated with ultraviolet rays of 1500 J / m ² on ice.

Cells (2 × 10 ⁴ cells / well) were seeded in a 24-well plate and cultured for 18 hours. A gene introduction agent (manufactured by Qiagen) was added to an ultraviolet-irradiated or unirradiated plasmid (0.2 μg DNA / well) and introduced at 37 ° C. for 24 hours. After exchanging the medium, a sample solution (Example 1 and Comparative Example 1, see Table 4 below for the sample concentration) was added, and further cultured for 24 hours.

  The cells were washed with PBS (−) (phosphate buffered saline) and then diluted 5-fold with sterilized water (Toyo Benet Inc., Picker Gene, 25 mM Tris (7.5), 2 mM DTT, 10% Glycerol, 1% Triton X-100) was added at 50 μL / well. Thereafter, the cells were stored at −80 ° C. for 30 minutes and allowed to stand at room temperature for 15 minutes to thaw the cells.

After stirring the plate (room temperature, 15 minutes), the cell lysate was collected in a 1.5 mL centrifuge tube with a scraper and centrifuged (15,000 rpm, 2 minutes). 20 μL of supernatant and luminescent substrate (Toyo Benet Corporation, Picker Gene, 20 mM Tricine / 1.07 mM (MgCO ₃ ) 4Mg (OH) ₂ .5H ₂ O / 2.67 mM MgSO ₄ /0.1 mM EDTA / 33.3 mM DTT / 270 μM Coenzyme A / 470 μM luciferin / 530 μM ATP) 100 μL was added, mixed at room temperature, set in a luminometer (GENELIGHT55, manufactured by Microtech Nichion), and measured at 560 nm.

The cell damage inhibition rate (%) due to ultraviolet irradiation was determined by calculating the amount of luciferase activity due to DNA repair after ultraviolet irradiation with respect to non-ultraviolet irradiation.
The results are shown in Table 4.

  As shown in Table 4, it was confirmed that the kiwi fruit extract (Example 1) has an excellent cell damage-suppressing effect by ultraviolet irradiation as compared with the kiwi fruit extract (Comparative Example 1).

[Test Example 5] Skin keratinocyte proliferation promoting action test The kiwi fruit extract of Example 1 and the kiwi fruit extract of Comparative Example 1 were tested for skin keratinocyte proliferation promoting action as follows.

Normal human newborn keratinocytes (NHEK) were cultured using a normal human epidermal keratinocyte long-term culture growth medium (EpiLife-KG2), and then cells were collected by trypsin treatment. The collected cells were diluted with EpiLife-KG2 to a cell density of 1.5 × 10 ⁴ cells / mL, then seeded at 100 μL per well in a collagen-coated 96-well plate, and cultured overnight. After completion of the culture, 100 μL of a sample solution dissolved in EpiLife-KG2 (Example 1 and Comparative Example 1, see Table 5 below for the sample concentration) was added to each well and cultured for 3 days.

  The epidermal keratinocyte proliferation promoting action was measured using the MTT assay. After completion of the culture, the medium was removed, and 100 μL of MTT dissolved in PBS (−) at a final concentration of 0.4 mg / mL was added to each well. After culturing for 2 hours, blue formazan produced in the cells was extracted with 100 μL of 2-propanol. After extraction, the absorbance at a wavelength of 570 nm was measured. At the same time, the absorbance at a wavelength of 650 nm was measured as turbidity, and the difference between the two was used as the amount of blue formazan produced. From each measured absorbance, the epidermal keratinocyte proliferation promotion rate (%) was calculated by the following formula.

Epidermal keratinocyte proliferation promotion rate (%) = St / Ct × 100
In the formula, St represents “absorbance when a sample solution is added”, and Ct represents “absorbance when no sample solution is added”.
The results are shown in Table 5.

  As shown in Table 5, it was confirmed that the kiwi fruit extract (Example 1) has an excellent epidermal keratinocyte proliferation promoting action as compared with the kiwi fruit extract (Comparative Example 1).

[Test Example 6] Profilaggrin mRNA expression promoting effect test The kiwi fruit extract of Example 1 and the kiwi fruit extract of Comparative Example 1 were tested for profilagrin mRNA expression promoting action as follows.

Normal human epidermal keratinocyte (NHEK) in a growth medium (EpiLife-KG2) for long-term culture of normal human epidermal keratinocytes in an 80 cm ² flask at 37 ° C., 5% CO ₂ -95% air The cells were precultured under the above conditions and cells were collected by trypsin treatment.

EpiLife-KG2 was used to seed 40 × 10 ⁴ cells / 2 mL / dish in 35 mm dishes (FALCON) and cultured overnight at 37 ° C. and 5% CO ₂ -95% air. After 24 hours, the culture solution was discarded, and 2 mL of the sample solution dissolved in EpiLife-KG2 to the required concentration (Example 1 and Comparative Example 1, see Table 6 below for sample concentration) was added to each dish at 37 ° C., 5 ° C. They were cultured for 24 hours under the conditions of% _CO 2 -95% air. After culturing, the culture solution is discarded, and total RNA is extracted with ISOGEN (NIPPON GENE, Cat. No. 311-02501), and the amount of each RNA is measured with a spectrophotometer so that it becomes 200 ng / μL. Total RNA was prepared.

  Using this total RNA as a template, the expression level of profilagrin and mRNA of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) which is an internal standard was measured. Detection was performed by real-time 2-step RT-PCR reaction using TaKaRa SYBR PrimeScript RT-PCR Kit (Perfect Real Time, code No. RR063A) using a real-time PCR device Smart Cycler (Cepheid). The expression level of mRNA for profilagrin is calculated based on the total RNA preparation prepared from the cells cultured with and without sample addition. The correction value of sample addition when 100 was calculated. From the obtained results, the profilaggrin mRNA expression increase promotion rate (%) was calculated by the following formula.

Profilaggrin mRNA expression increase promotion rate (%) = A / B × 100
In the formula, A represents “correction value when sample is added”, and B represents “correction value when sample is not added”.
The results are shown in Table 6.

  As shown in Table 6, the kiwi fruit extract (Example 1) is significantly superior to the kiwi fruit extract (Comparative Example 1) at a low concentration (12.5 μg / mL). It was confirmed to have a promoting action. That is, it was found that the kiwi fruit extract extracts promote the gene expression of profilagrin, which is a precursor of filaggrin involved in improving the skin moisturizing function, at a low concentration (12.5 μg / mL).

  As apparent from Test Examples 1 to 6 described above, the extract from the fruit of kiwi fruit does not have the extract from the ripe fruit of kiwi (superoxide scavenging action (reactive oxygen scavenging action), radical scavenging action. Remarkably superior even if it has an action) (or action that suppresses oxidative cell damage, suppresses cell damage by UV irradiation, promotes epidermal keratinocyte proliferation, promotes profilagrin mRNA expression) It was found to have an effect.

  The active oxygen scavenger, radical scavenger, and oxidative cytotoxicity inhibitor of the present invention can greatly contribute to the prevention and improvement of various disorders including cell damage caused by active oxygen species.

Claims (3)

  An active oxygen scavenger comprising an extract from a fruit of kiwi fruit as an active ingredient.   A radical scavenger comprising an extract from a fruit of kiwi fruit as an active ingredient.   An oxidative cytotoxicity inhibitor comprising an extract from a fruit of kiwi fruit as an active ingredient.