JP5322599B2 - Antioxidant - Google Patents

Description

  The present invention relates to an anti-aging agent, an anti-inflammatory agent, a whitening agent, and an antioxidant that contain giraffe extract as an active ingredient.

  Causes of skin symptoms such as aging, disease, stress, UV wrinkles, stains, reduced skin elasticity, dryness, decreased cellular function, melanin production and pigmentation due to UV rays, decrease and degeneration of dermal matrix components, UV rays, etc. Oxidative damage of cells due to. In order to prevent and ameliorate such skin symptoms, various active ingredients have been searched and formulated. In particular, naturally-derived components are known to have various pharmacological and cosmetic effects, and the application of extracts such as plants and fungi to skin external preparations and oral compositions has been studied so far.

  For example, in order to obtain an extract of a plant belonging to the genus Halibut (see Patent Document 1) as a moisturizing agent excellent in skin moisturizing effect and safety, and to obtain a skin external preparation having an anti-aging and improving action on the skin, The essence of Ponkan as a component having an activation or growth promoting action (see Patent Document 2), an extract of plants belonging to the genus Genus ginseng (see Patent Document 3), a chlorella extract (see Patent Document 4), and a loquat extract (see Patent Document 5) ) Etc. are disclosed. As a whitening agent, an extract of white crane reishi (see Patent Document 6) and the like, and as an antioxidant, an extract of a plant belonging to the genus Sarogase (see Patent Document 7) and the like suppress fat accumulation in the living body. As components, mammalian milk-derived phospholipids (see Patent Literature 8), brown algal enzyme degradation products (see Patent Literature 9) and the like are already known.

JP 2007-77072 A JP 2001-131045 A JP 2000-178198 A JP 11-335293 A Japanese Patent Publication No. 5-17206 JP 2003-89630 A Japanese Patent Laid-Open No. 10-182413

  Thus, various naturally-derived components have been applied so far. However, there are many naturally-derived components whose effects are not yet known, and the development of active ingredients having excellent anti-aging, antioxidant, and whitening effects has been expected.

  As a result of examining various components derived from nature, the present inventors have found that an excellent anti-aging action, anti-inflammatory action, whitening action, and antioxidant action are present in giraffe extract, and further investigation is made. Over time, the present invention has been completed.

  That is, the present invention relates to an anti-aging agent, an anti-inflammatory agent, a whitening agent, and an antioxidant that contain giraffe extract as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the anti-aging agent, anti-inflammatory agent, whitening agent, and antioxidant which have the outstanding effect can be provided by using giraffe extract as an active ingredient.

  By blending these anti-aging agents, anti-inflammatory agents, whitening agents, and antioxidants into skin external preparations and oral compositions, various skin symptoms such as wrinkles, tarmi, reduced skin elasticity, spots and dullness can be obtained. Various compositions that exhibit an excellent effect in preventing and improving expression can be provided.

The giraffe used in the present invention is a dicotyledonous plant belonging to the genus Amanengusa, distributed from Kyushu to Hokkaido, and has many mutant species. In the present invention, the giraffe is not particularly limited. Manabu name of Kirinsou is, Sedum kamtschaticum Fisch., Sedum aizoon L., Sedum aizoon L. var. Ru floribundum Naka i der.

  When these giraffes are used, there are no particular restrictions on the site of use, and any part such as leaves, stems, flowers, roots, etc. can be used. A plurality of parts may be used in combination.

  In the extraction, giraffe may be used as it is, but considering the extraction efficiency, it is preferable to perform the extraction after processing such as shredding, drying, and pulverization.

  The extraction can be performed by immersing in an arbitrary extraction solvent for a predetermined time. The extraction solvent may be heated as necessary. Alternatively, extraction can be performed using a supercritical fluid or a subcritical fluid. In order to increase the extraction efficiency, stirring or homogenization in an extraction solvent may be performed. 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 is suitably about 1 hour to 14 days.

  As an extraction solvent, in addition to 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 Solvents such as esters; esters such as butyl acetate and ethyl acetate; ketones such as acetone and ethyl methyl ketone can be used. These may be used alone or in combination of any two or more. Saline, phosphate buffer, phosphate buffered saline, and the like may be used. Furthermore, you may use 1 type, or 2 or more types of supercritical liquids and subcritical liquids, such as water, a carbon dioxide, ethylene, propylene, ethanol, methanol, ammonia.

  The above extract of giraffe with the above solvent can be used as it is, but it may be left standing for a certain period of time and aged, or the concentrated and dried product may be dissolved again in water or a polar solvent. It can also be used. Alternatively, it may be used after performing purification treatment such as decolorization, deodorization, desalting, etc., or fractionation treatment by column chromatography or the like, as long as these physiological functions are not impaired. The above-mentioned extract of giraffe and its treated and fractionated products can be freeze-dried after each treatment and fractionation and dissolved in a solvent before use. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

  Giraffe extract has excellent anti-aging action, anti-inflammatory action, whitening action, and antioxidant action, and can be used as an anti-aging agent, anti-inflammatory agent, whitening agent, and antioxidant.

  Anti-aging agent containing giraffe extract as an active ingredient has excellent cell activation effect of human dermal fibroblasts, type I collagen production promotion effect in dermal fibroblasts, and excellent effect in preventing and improving aging symptoms Demonstrate.

  An anti-inflammatory agent containing giraffe extract as an active ingredient has an excellent hyaluronidase inhibitory effect, and exhibits an excellent effect in preventing and improving inflammation.

  A whitening agent containing giraffe extract as an active ingredient has an excellent inhibitory effect on melanin production, and prevents and improves pigmentation, blemishes, freckles, etc., and exhibits an excellent whitening effect.

  An antioxidant containing giraffe extract as an active ingredient has an excellent radical scavenging effect and a superoxide anion scavenging effect, and exhibits an excellent antioxidant action.

[Extract 1]
The ground part of giraffe was dried and pulverized, purified water of 20 times the mass of the sample was added, and the mixture was extracted by heating to 120 ° C. for 20 minutes with an autoclave. From the obtained extract, insoluble matters were removed by suction filtration while maintaining a high temperature state, followed by freeze-drying to obtain a hot water extract of Giraffe (Extract 1).

[Extract 2]
The above-ground part of giraffe was dried and pulverized, 50% by mass of 20 times the mass of the sample was added, and extracted for 2 hours while stirring at room temperature. The obtained extract was filtered to remove insoluble matters, concentrated under reduced pressure, and freeze-dried to obtain an ethanol extract of giraffe (extract 2).

  Each effect was evaluated using the said extract. 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) with a significance probability of *, and less than 1% significance probability (P <0.01). ) Is represented by **.

[Dermal fibroblast activation]
Normal human dermal fibroblasts were seeded in a 96-well microplate at 2.0 × 10 ⁴ cells per well. The seeding medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with 1% by weight fetal bovine serum (FBS). After 24 hours, the culture medium was replaced with a sample culture solution containing the extract 1 adjusted to each concentration in 1% by mass FBS-added DMEM medium, and further cultured for 48 hours. Next, the MTT reagent was adjusted in the medium to 400 μg / mL, added to the cells from which the supernatant was removed, and cultured for about 2 hours. Finally, formazan produced in 2-propanol was extracted, and 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 cell activation effect was evaluated using the difference between the two measured values. Evaluation was carried out by obtaining a relative value when the cell activation effect in the control when no extract 1 was added was taken as 100, and the results are shown in Table 1.

  The results are as shown in Table 1, and the giraffe extract showed a significant dermal fibroblast activation effect.

[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. The seeding medium used was Dulbecco's modified Eagle (DMEM) medium supplemented with 5% by weight fetal bovine serum (FBS). After 24 hours, the culture medium was replaced with a sample culture solution containing the extract 2 adjusted to each concentration in a DMEM medium supplemented with 0.5 mass% FBS, and further cultured for 24 hours.
The amount of type 1 collagen secreted into the culture supernatant was determined by ELISA, and finally 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) against labeled peroxidase. And after adding hydrogen peroxide and making it react, the light absorbency of 405 nm was measured with the microplate reader. In the evaluation, in addition to the sample culture solution, a DMEM medium supplemented with 0.5% FBS was used as a negative control.
The evaluation was performed by obtaining a relative value when the cell activation effect when no extract was added was taken as 100. Specifically, the amount of protein was determined by measuring the amount of protein with BCA Protein Assay Kit manufactured by PIERCE, and the amount of collagen production per unit cell or unit protein was calculated. The value was determined.

  The results are as shown in Table 2. The giraffe extract showed a significant dermal fibroblast type I collagen production promoting action.

  As shown in Tables 1 and 2, the giraffe extract has a high dermal fibroblast activation action and dermal fibroblast type I collagen production promoting action, and exhibits an anti-aging effect.

[Hyaluronidase inhibitory action]
Commercially available hyaluronic acid potassium salt (derived from human umbilical cord) was dissolved in 0.1 M phosphate buffer (pH 7.0) to a concentration of 0.9 mg / mL to obtain a substrate solution. A commercially available hyaluronidase (derived from bovine testis) was dissolved in 0.1 M phosphate buffer (pH 7.0) so as to be 5,3000 units / mL to obtain an enzyme solution. The enzyme solution was prepared at the time of use. In a test tube, 0.1 mL of the extract 2-containing solution prepared at each concentration with a buffer solution and 0.03 mL of the enzyme solution were taken and reacted at 37 ° C. for 20 minutes. Next, 0.06 mL of an activator was added and reacted at 37 ° C. for 20 minutes. Further, 0.15 mL of the substrate solution was added and reacted at 37 ° C. for 1 hour. 0.06 mL of 0.4N NaOH was added to stop the reaction, and the mixture was immediately cooled with ice. Then, 0.06 mL of borate buffer (pH 9.1) was added, and the mixture was boiled for 3 minutes and further cooled with ice. After 2.0 mL of p-dimethylbenzaldehyde (p-DABA) solution was added and reacted at 37 ° C. for 20 minutes, the solution was transferred from each test tube to a 96-well microplate, and the absorbance at 585 nm was measured using a microplate reader. It was measured. As a control, a solution to which only an extract-free buffer solution was added was used. When the activity of hyaluronidase is inhibited, the degradation product N-acetylglucosamine decreases. Absorbance by p-DABA is lowered. Utilizing this fact, the inhibitory activity was obtained from the following equation. The results are summarized in Table 3.
Inhibition rate (%) = (control absorbance−sample absorbance) / control absorbance × 100

  As shown in Table 3, the giraffe extract has a hyaluronidase inhibitory action and exhibits an anti-inflammatory effect.

[Inhibition of melanin production]
B16 mouse melanoma cells were seeded at 1.8 × 10 ⁴ per 90 mm dish, and cultured using Dulbecco's modified Eagle medium (DMEM) supplemented with 5% by mass of fetal bovine serum (FBS). After 24 hours, the sample was added to 5% by mass FBS-added DMEM medium, and the medium was replaced with a culture solution containing the extract 2 adjusted to each concentration. After further culturing for 5 days, the cells were detached and collected with trypsin after completion of the culture. The collected cells were centrifuged to obtain a cell precipitate. The resulting precipitate was visually judged for its blackening condition according to the criteria shown below. In the evaluation, the extract 2 was not added and cultured in only 5% by mass FBS-added DMEM medium as a negative control, and 50 mM sodium lactate was added instead of the extract 2 and cultured as a positive control.
Evaluation criteria judgment 1: Same as positive control (almost white)
Judgment 2: Slightly blacker than the positive control (light brown)
Judgment 3: Between positive control and negative control (brown)
Judgment 4: Slightly whiter than the negative control (blackish brown)
Judgment 5: Same as negative control (almost black)

  The results are as shown in Table 4. The giraffe extract exhibits a clear melanin production inhibitory effect and exhibits a whitening effect.

[DPPH radical scavenging action]
Extract 2 was adjusted to each concentration using a 50% by mass aqueous ethanol solution, and 100 μL was added to each 96-well microplate. Further, 100 μL each of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) ethanol solution was added, mixed well, allowed to stand at room temperature in the dark for 24 hours, and the absorbance at 516 nm was measured. . When the absorbance of the blank with no extract 2 added is (A) and the absorbance when the extract 2 is added is (B), the value of the following formula (2) is defined as the radical elimination rate.
DPPH radical scavenging rate = {1- (B) / (A)} × 100 (%) (2)

  The results are as shown in Table 5, and the giraffe extract showed a clear DPPH radical scavenging action.

[SOD-like activity]
To 75 μL of the HANK ′S (+) solution containing 0.25 mM WST-1 and 1 mM hypoxanthine, 25 μL of the sample solution prepared by adjusting the extract 2 to each concentration using the HANK ′S (+) solution is added. Further, 25 μL (0.0075 units) of xanthine oxidase was added and reacted at 37 ° C. for 15 minutes, and then the absorbance at 450 nm was measured. When the absorbance when only the HANK'S (+) solution is added instead of the sample solution is (A) and the absorbance when the sample solution is added is (B), the superoxide anion elimination rate is expressed by the following formula (3) ). Superoxide anion elimination rate (%) = [1- (B) / (A)] × 100 (3)

  The results are as shown in Table 12, and the giraffe extract showed a clear superoxide anion scavenging action.

  As shown in Tables 11 and 12, the giraffe extract has a DPPH radical scavenging effect and a superoxide anion scavenging effect, and exhibits an antioxidant action.

Claims (1)

An antioxidant containing an extract of giraffe (Sedum kamtschaticum Fisch., Sedum aizoon L., Sedum aizoon L. var. Floribundum Nakai) as an active ingredient.