JP2012097028A - Anti-aging agent, anti-oxidant, arginase activity accelerator, slimming agent and whitening agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anti-aging agent, an anti-oxidant, an arginase activity accelerator, a slimming agent and a whitening agent which exhibit high anti-aging effect, anti-oxidation effect, arginase activity acceleration effect, slimming effect and whitening effect.SOLUTION: The anti-aging agent, anti-oxidant, arginase activity accelerator, slimming agent and whitening agent comprise the extract of Hosta sieboldii var. rectifolia as an active ingredient.

Description

  The present invention relates to an anti-aging agent, an antioxidant, an arginase activity promoter, a slimming agent, and a whitening agent that exhibit a high anti-aging effect, antioxidant effect, arginase activity promoting effect, slimming effect, and whitening effect.

  Conventionally, women's interest in maintaining the aesthetics of the skin is very high, and wrinkles, spots, tarmi, etc. are always at the top of women's skin problems. Among these wrinkles, wrinkles and tarmi have decreased dermal fibroblast function due to aging, etc., accompanied by decrease or degeneration of dermal matrix such as collagen and elastin, and oxidative damage due to external stress such as ultraviolet rays. It is an important factor. The other big problem is the darkening of the skin, although there are some unclear points, it is caused by hormonal abnormalities and the production of melanin by stimulation of ultraviolet rays of sunlight. Among them, spots and buckwheat are melanin. The cause is abnormal pigmentation.

  So far, various cell activators, antioxidants, and melanin production inhibitors have been searched and formulated for the purpose of preventing or ameliorating various symptoms that impair the above-described skin aesthetics.

  For example, as a cell activator, essence of Ponkan (see Patent Document 1), genus Genus, Wedge and extracts thereof (see Patent Document 2), chlorella extract with an organic solvent (see Patent Document 3), etc., antioxidants As an agent, an extract of a plant belonging to the genus Heteroteca genus (see Patent Document 4) or an extract of Kayunangin (see Patent Document 5) and the like, and as a melanin production inhibitor, an extract of Honda walla (see Patent Document 6), ginger An extract of a genus plant (see Patent Document 7) and the like are known.

JP 2001-131045 A JP 2000-178198 A JP 11-335293 A JP-A-11-180886 Japanese Patent Laid-Open No. 10-182413 JP 10-330220 A

  Naturally-derived components are known to have various pharmacological and cosmetic effects, and so far many plants and fungi have been widely applied to fields such as external preparations for skin and foods and drinks. However, there are many naturally-derived ingredients whose effects are not yet known, and the development of effective ingredients with excellent arginase activity promoting action, cell activation action, antioxidant action, whitening action, etc. is expected. It was. The present invention was made in order to find such an active ingredient, and can be widely applied in the field of external preparations for skin and foods and drinks, an antioxidant, an arginase activity promoter, a slimming agent, And to provide a whitening agent.

  In order to find an anti-aging agent, an antioxidant, an arginase activity promoter, a slimming agent, and a whitening agent that can be widely applied to fields such as external preparations for skin and foods and beverages, the present inventors The substance was examined. As a result, an excellent anti-aging effect, antioxidant effect, arginase activity promoting effect, slimming effect, and whitening effect were found in the extract of the prickly cattle, and further studies were made to complete the present invention. That is, the present invention provides an anti-aging agent, an antioxidant, an arginase activity promoter, a slimming agent, and a whitening agent, which contains an extract of a spotted bovine as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the anti-aging agent, antioxidant, arginase activity promoter, slimming agent, and whitening agent which have the outstanding effect can be provided.

A plant used as a raw material of the present invention, Hostectobori Nakai ( Hosta lectifolia Nakai ), is a herb belonging to the genus Liboaceae and is widely distributed from Hokkaido to Honshu and Kyushu.

  In the present invention, the active body or the dried product may be used for the prickly cattle, but it is preferable to use extracts extracted using various solvents. For extraction, any part of the stem, leaves, flowers, seeds, roots, rhizomes, fruits, buds, etc. of the prickly cattle may be used, but whole plants may be used from the viewpoint of effectiveness. In the extraction, it may be used as it is, but considering the extraction efficiency, it is preferable to perform the extraction after performing processing such as shredding, drying, and pulverization. The extraction can be performed by immersing in an extraction solvent or by an extraction method using a supercritical fluid or a subcritical fluid. In order to increase the extraction efficiency, the mixture may be homogenized in stirring or an extraction solvent. The extraction temperature is suitably about 5 ° C. to the boiling point of the extraction solvent. The extraction time varies depending on the type of extraction solvent and the extraction temperature, but it is appropriate to set it to about 1 hour to 14 days.

  Extraction solvents include water, lower alcohols such as methanol, ethanol, propanol and isopropanol, polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, dipropylene glycol and glycerin, ethers such as ethyl ether and propyl ether. And solvents such as esters such as butyl acetate and ethyl acetate, and ketones such as acetone and ethyl methyl ketone can be used, and one or more of these can be selected and used. Further, physiological saline, phosphate buffer, phosphate buffered saline, or the like may be used. Furthermore, you may use 1 type, or 2 or more types of supercritical fluids and subcritical fluids, such as water, a carbon dioxide, ethylene, propylene, ethanol, methanol, ammonia.

  The extract of the above-mentioned solvent of the prickly cattle can be used as it is, but the concentrated and dried product can be used again by dissolving it in water or a polar solvent, and it is decolorized as long as these physiological functions are not impaired. It may be used after a purification treatment such as deodorization and desalting or a fractionation treatment by column chromatography or the like. The above-mentioned extract of Tachigiboushi, its processed products and fractions can be freeze-dried after each treatment and fractionation and dissolved in a solvent before use.

  The extract of Tachigiboushi has excellent anti-aging action, antioxidant action, arginase activity promoting action, slimming action, and whitening action, and contains anti-aging agent, antioxidant, arginase containing Tachigiboushi extract as an active ingredient It can be used as an activity promoter, slimming agent, and whitening agent. Anti-aging agents, antioxidants, arginase activity promoters, slimming agents, and whitening agents, which are made from Tachigiboushi extract as an active ingredient, can be used not only for the skin but also for hair and can be taken orally. It can also be applied to foods, beverages, pharmaceuticals, and the like.

  Tachigibobo extract has cell activation (anti-aging), collagen production promotion (anti-aging), superoxide anion scavenging (antioxidant), free radical scavenging (antioxidant), lipid peroxide resistance Demonstrates promoting action (antioxidant action), arginase activity promoting action, neutral fat accumulation inhibiting action (slimming action), tyrosinase activity inhibiting action (whitening action), melanin production inhibiting action (whitening action) Useful as an oxidizing agent, arginase activity promoter, slimming agent, and whitening agent.

  Hereinafter, production examples of the guinea pig extract and tests for evaluating each action will be described in more detail. However, the technical scope of the present invention is not limited thereto.

[Extract 1]
100 g of the dry ground pulverized material of the ground plant including stems, buds, flowers, and sprouts of the prickly cattle was heated and extracted with 2.0 kg of hot water for 20 minutes. The extract supernatant was filtered and lyophilized to obtain extract 1.

[Extract 2]
100 g of a dry pulverized product of the above-ground whole plant including stems, buds, flowers and shoots of Tachigiboushi was dispersed in 2.0 kg of 50 vol% ethanol aqueous solution and extracted at room temperature for 2 hours with stirring. The extract supernatant was filtered off, concentrated under reduced pressure, and lyophilized to obtain extract 2.

[Extract 3]
A dry pulverized product of whole ground plant including stems, buds, flowers, and shoots of prickly cattle is put into a pressure cell, and liquefied carbon dioxide is continuously poured by a pump, and the conditions are kept under conditions of 25 MPa, 5 mL / min, and 40 ° C. Carbon dioxide was allowed to flow, and the extract components were collected in a gas-liquid separator to obtain an extract.

  Using the above extract 1, extract 2 and extract 3, dermal fibroblast activation action, dermal fibroblast type I collagen production promotion action, human epidermal keratinocyte activation action, superoxide anion scavenging action, DPPH radical The evaluation was made on the erasing ability, lipid peroxide resistance improving action using epidermal cells, arginase activity promoting action, neutral fat accumulation inhibiting action, tyrosinase activity inhibiting action using human epidermal melanocytes, and melanin production inhibiting action. Note that * and ** described in each evaluation result indicate that the significance probability P value in the t-test is less than 5% (P <0.05), and less than 1% significance (P <0.01). ) Is represented by **.

[Anti-aging effect: human dermal fibroblast activation effect]
Normal human dermal fibroblasts were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells per well. Dulbecco's modified Eagle medium (DMEM) supplemented with 1% by weight fetal bovine serum (FBS) was used as the seeding medium. After culturing for 24 hours, the medium was replaced with 1% by mass FBS-added DMEM medium to which the extract 3 was added so as to have each concentration shown in Table 1, and further cultured for 48 hours. Next, the MTT reagent adjusted with the culture medium so that it might become 400 micrograms / mL was added to the cell except the supernatant, and it culture | cultivated for about 2 hours. Finally, formazan produced in 2-propanol was extracted and the absorbance at 550 nm was measured. At the same time, the absorbance at 650 nm was measured as turbidity, and the cell activation effect was evaluated using the difference between the two measured values. The evaluation results are shown in Table 1 as relative values with the cell activation effect in the control with no sample added being taken as 100.

  As is clear from Table 1, it was revealed that the medium supplemented with the guinea pig extract exhibits a significant dermal fibroblast activation effect.

[Anti-aging effect: human dermal fibroblast type I collagen production promoting effect]
Normal human dermal fibroblasts were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells per well. Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS) was used as the seeding medium. After culturing for 24 hours, the culture medium was replaced with 0.5% by mass FBS-added DMEM medium to which the extract 1 was added so as to have each concentration shown in Table 2, and further cultured for 24 hours. The ELISA method was used for the quantification of type I collagen secreted into the culture supernatant. Finally, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt ( ABTS) and hydrogen peroxide were added and reacted, and then the absorbance at 405 nm was measured. The amount of protein in each well was measured with BCA Protein Assay Kit manufactured by PIERCE, and the amount of type I collagen produced per unit protein was determined. The evaluation results are shown in Table 2 as relative values when the type I collagen production amount per unit protein amount in the control with no sample added is defined as 100.

  As is clear from Table 2, it was revealed that the medium supplemented with the guinea pig extract exhibits a significant dermal fibroblast type I collagen production promoting action.

[Anti-aging effect: human epidermal keratinocyte activation effect]
Human epidermal keratinocytes HaCaT were seeded in a 96-well microplate so as to be 2.0 × 10 ⁴ cells per well. Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS) was used as the seeding medium. After culturing for 24 hours, the medium was replaced with 5% by mass FBS-added DMEM medium to which the extract 2 was added so as to have each concentration shown in Table 3, and further cultured for 24 hours. Next, the MTT reagent adjusted with the culture medium so that it might be set to 100 microgram / mL was added to the cell except the supernatant, and it culture | cultivated for about 2 hours. Finally, formazan produced in 2-propanol was extracted and the absorbance at 550 nm was measured. At the same time, the absorbance at 650 nm was measured as turbidity, and the cell activation effect was evaluated using the difference between the two measured values. The evaluation results are shown in Table 3 as relative values when the cell activation effect in the control with no sample added is taken as 100.

  As is clear from Table 3, it was revealed that the medium supplemented with the extract of the spotted bovine shows a significant human epidermal keratinocyte activation effect.

  As shown in Tables 1 to 3, the Tachibo bovine extract exhibits excellent anti-aging because it exhibits a human dermal fibroblast activation effect, a human dermal fibroblast type I collagen production promoting effect and a human epidermal keratinocyte activation effect. Demonstrate the effect.

[Antioxidant action: SOD-like activity]
Extract 25 was added to a HANK'S (+) solution at a concentration of 25 μL shown in Table 4 in a sample solution of 25 μL, and a HANK ′S (+) solution containing 75 μL of 0.25 mM WST-1 and 1 mM hypoxanthine. Was added. Furthermore, 25 μL (0.0075 Units) of xanthine oxidase was added, and after reacting at 37 ° C. for 15 minutes, the absorbance at 450 nm was measured. When the absorbance when no sample is added is (A) and the absorbance when the sample is added is (B), the superoxide anion elimination rate is defined by the following equation.
Erase rate (%) = {1- (B) / (A)} × 100
The evaluation results are shown in Table 4.

  As is clear from Table 4, it was revealed that the guinea pig extract exhibits a significant superoxide anion scavenging action.

[Antioxidant action: DPPH radical scavenging action]
Extract 1 was adjusted to each concentration shown in Table 5 with 50% by mass ethanol, and 100 μL was added to each 96-well microplate. Further, 100 μL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution was added each time, and after mixing well, left at room temperature in the dark for 24 hours, derived from DPPH radicals Absorbance at 516 nm was measured. When the absorbance when no sample is added is (A) and the absorbance when the sample is added is (B), the DPPH radical elimination rate is defined by the following equation.
Erase rate = {1- (B) / (A)} × 100
The evaluation results are shown in Table 5.

  As is clear from Table 5, it was revealed that the Tachigiboushi extract exhibits a high DPPH radical scavenging action.

[Antioxidant action: lipid peroxide resistance test of epidermal cells]
Human epidermal keratinocytes HaCaT were seeded in a 96-well microplate so as to be 2.0 × 10 ⁴ cells per well. Dulbecco's modified Eagle medium (DMEM) supplemented with 10% by weight fetal bovine serum (FBS) was used as the seeding medium. After culturing for 24 hours, the medium was replaced with 10% by mass FBS-added DMEM medium to which the extract 2 was added so as to have each concentration shown in Table 6, and further cultured for 24 hours. The solution was replaced with a HANK'S (+) solution to which an arbitrary concentration of t-butyl hydroperoxide was added, and cultured for 2 hours. Furthermore, it replaced | exchanged for PBS (-) containing 150 microgram / mL neutral red, and culture | cultivated at 37 degreeC for 2 hours. Next, it exchanged for 50 volume% ethanol aqueous solution containing 1 volume% acetic acid, neutral red taken in the cell was extracted, and the light absorbency of 540 nm of the extract was measured. The evaluation results are shown in Table 6 as relative values when the cell viability in the control with no t-butyl hydroperoxide added is taken as 100.

  As is clear from Table 6, it was revealed that the cell viability when lipid peroxide was added was significantly improved in the medium supplemented with the spotted bovine extract.

  As shown in Tables 4 to 6, the guinea pig extract exhibits an excellent antioxidant effect since it exhibits a superoxide anion scavenging action, a DPPH radical scavenging action, and a lipid peroxide resistance improving action on epidermal cells.

[Arginase activity promoting effect]
Human skin keratinocytes were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells per well. As the seeding medium, Dulbecco's modified Eagle medium (DMEM) with 5% fetal bovine serum (FBS) added was used. After 24 hours, the medium was replaced with 5% FBS-added DMEM medium containing 1.2 mM CaCl ₂ to which the extract 2 was added so as to have each concentration shown in Table 7, and further cultured for 9 days. The medium was changed once every 3 days. After completion of the culture, the culture supernatant was collected and evaluated for its ability to promote arginase activity. Arginase hydrolyzes arginine to produce ornithine and urea. Urea is decomposed into ammonia by urease, and ammonia reacts with salicylic acid and hypochlorous acid in the presence of sodium pentacyanonitrosyl iron (III) dihydrate (sodium nitroprusside) to produce indophenol. The absorption (570 nm) of indophenol was measured under alkaline conditions, the urea concentration was determined, and the arginase activity was quantified. For determination of urea, the same measurement was performed using urea nitrogen B-Test Wako manufactured by Wako Pure Chemical Industries, and a calibration curve was prepared. Moreover, the protein amount of each well was measured with BCA Protein Assay Kit, and the ability to promote arginase activity per unit protein amount was determined. The evaluation results are shown in Table 7 as relative values when the arginase activity per unit protein amount in the control with no sample added is defined as 100.

  As is apparent from Table 7, it was revealed that the guinea pig extract exhibits a significant arginase activity promoting effect.

[Slimming action: Inhibition of neutral fat accumulation]
Subcutaneous fat-derived normal human preadipocytes Cryo HPRAD-SQ (Sanko Junyaku Co., Ltd.) were seeded on a 96-well microplate so that the number was 1.0 × 10 ⁴ per well. PGM medium (containing 10% by mass FBS, 2 mM L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin) was used as the seeding medium. Immediately before the cells became confluent, the medium was replaced with PGM-differentiation medium (containing 10 μg / mL insulin, 1 μM dexamethasone, 200 μM indomethacin, 500 μM isobutylmethylxanthine) supplemented with extract 2 to induce differentiation into adipocytes. After initiation of differentiation induction, the cells were cultured for 10 to 14 days until the control group matured and many lipid droplets accumulated in the cells. After the cells were collected, the cells were fixed using a 10% neutral buffered formaldehyde solution. After washing with PBS (−), 0.5 w / v% Oil Red O solution was added and cultured at 37 ° C. for 2 hours. After washing with PBS (−), methanol was added to extract the dye. After extraction, the absorbance at 550 nm was measured with a microplate reader. At the same time, the absorbance at 650 nm was measured as turbidity, and the neutral fat accumulation inhibitory action was evaluated using the difference between the two measured values. The evaluation results are shown in Table 8 as relative values when the neutral fat accumulation amount in the control with no sample added is defined as 100.

  As is clear from Table 8, the Tachigiboushi extract exhibits a significant neutral fat accumulation inhibitory action, and thus it has been clarified that it exhibits an excellent slimming effect.

[Whitening action: Tyrosinase activity inhibitory action]
Normal human epidermal melanocytes were seeded in a 96-well microplate so as to be 3.0 × 10 ⁴ cells per well. Medium 154S was used as the seeding medium. After culturing for 24 hours, the medium was replaced with Medium 154S to which the extract 1 was added so that the concentrations shown in Table 9 were obtained, followed by further culturing for 48 hours. Next, it was replaced with 75 μL of a phosphate buffer containing 1% by weight Triton-X to completely lyse the cells, and 50 μL was used as a crude enzyme solution. To the crude enzyme solution, 50 μL of a 0.05% by mass L-dopa-containing phosphate buffer as a substrate was added and allowed to stand at 37 ° C. for 2 hours. The absorbance at 405 nm was measured immediately after the addition of the substrate and at the end of the reaction, and the amount of dopamelanin produced was determined by introducing the difference between the two measured values into the following equation.
Amount of dopamelanin produced = {(405 nm value after reaction−405 nm value before reaction) −2.166} /5.238
In addition, the amount of protein in each well was measured with BCA Protein Assay Kit manufactured by PIERCE, and the amount of dopamelanin produced per unit protein amount was determined. The evaluation results are compared with the amount of dopamelanin produced per unit protein amount in the control with no sample added, and are shown in Table 9.

  As is clear from Table 9, it was revealed that the medium supplemented with the extract of Tachibobo exhibits a significant tyrosinase activity inhibitory action.

[Whitening effect: Suppressing melanin production]
B16 mouse melanoma cells (B16F0 cells) were seeded in a 90 mm dish so that the number of B16 mouse melanoma cells was 1.8 × 10 ⁴ per dish. Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by weight fetal bovine serum (FBS) was used as the seeding medium. After culturing for 24 hours, the medium was replaced with a 5% by mass FBS-added DMEM medium to which the extract 2 was added so as to have each concentration shown in Table 11, and further cultured for 5 days. After completion of the culture, cells were peeled off by trypsin treatment, transferred to a 1.5 mL microtube, and centrifuged to obtain a cell precipitate. The obtained precipitate was visually determined based on the following determination table. In the evaluation, a 5% FBS-added DMEM medium containing 5% by mass FBS was used as a negative control, and a 5% FBS-added DMEM medium containing 50 mM sodium lactate was used as a positive control. These visual determination results were determined as determination 5 and determination 1, and used as an index for sample determination. As shown in Table 10, the visual judgment was evaluated in five stages. At the same time, the tissue solubilizer (trade name Solvable) was added to the precipitate, boiled, returned to room temperature, and the absorbance at 500 nm was measured with a spectrophotometer (HITACHI spectrophotometer U-3010) to determine the total amount of melanin. Asked. The evaluation results are shown in Table 11.

  As is clear from Table 11, it was revealed that the addition of the guinea pig extract exhibits a significant melanin production inhibitory action.

  As shown in Tables 9 and 11, the Tachigibo bovine extract exhibits an excellent antioxidant effect because it exhibits a tyrosinase activity inhibitory action and a melanin production inhibitory action.

Claims (5)

  An anti-aging agent comprising, as an active ingredient, an extract of the Lily family Hosta genus Tativiboshi.   An antioxidant containing, as an active ingredient, an extract of the Lily family Hosta sp.   An arginase activity promoter comprising, as an active ingredient, an extract of a lily family Hosta genus Tachigiboushi.   A slimming agent containing, as an active ingredient, an extract of the Lilyaceae genus Tachigiboushi.   A whitening agent containing, as an active ingredient, an extract of the Lilyaceae genus Tativogoshi.