KR20210114081A - Hydrolysate of whole plant of Hydrangea macrophylla and Novel use of the same - Google Patents

Abstract

The present invention provides a cosmetic composition for anti-oxidation, whitening, photo-aging resistance, moisturizing, and anti-inflammation, containing flowers and leaves of Hydrangea macrophylla, a whole extract thereof, and hydragenol separated and refined therefrom as active ingredients.

Description

Hydrolysate of hydrangea whole plant and its use {Hydrolysate of whole plant of Hydrangea macrophylla and Novel use of the same}

The present invention relates to a hydrolyzate of hydrangea outpost and its use, and more particularly, to a hydrolyzate of hydrangea outpost containing hydrangenol prepared by treating an ethanol extract of hydrangea with the complex enzyme biscozyme and its antioxidant, whitening, photoaging, and moisturizing And it relates to a cosmetic composition for anti-inflammatory against autoimmune factors.

Hydrangea belongs to the family Hydrangeaceae and its scientific name is Hydrangea macrophylla . Hydrangea means water in Greek, and macrophylla means very small, meaning that it is a flower that likes water.

The flowers of hydrangea are distributed in Korea, China and Japan, and the stem is 1-3 m high, and the leaves are opposite, ovate or broad ovate, thick and shiny. There are saw teeth on the edge of the leaf, and the flowers hang a lot in the corymbal inflorescence at the tip of the stem in June-July, and there are light purple, blue, and light red flowers. The inflorescence is round and 10-15 cm in diameter. The calyx has 4-5 pieces, looks like a petal, and is very small. Flowers bloom in round inflorescences at the tip of branches in early summer. The flowers start to bloom white at first, but gradually turn blue, add red again, and later turn purple, and the color changes depending on the soil composition. If the component is strong, the color is pink, if the acid is strong, it becomes indigo, and if it is neutral, it becomes white. Halofuginone, which is mainly used for ornamental purposes and contained in the root, is known to inhibit the progression of various autoimmune diseases.

The leaves of hydrangea (Hydrangea macrophylla) are deciduous shrubs of the dicotyledonous plant Rosaceae Rosaceae, and the leaves are opposite, egg-shaped, thick, and have serrated edges. The leaves of hydrangea have been used as tea since ancient times, and have been used as an antipyretic agent in folklore. In addition, antimalarial action, immunosuppressive action and antiobesity, skin whitening effect, etc. have been reported.

Recently, various attempts have been made to develop cosmetics that can impart anti-inflammatory functions to the skin using natural extracts, and as a typical example, Korean Patent Publication No. 2007-0011939 proposes a cosmetic composition containing camellia extract , Korean Patent Publication No. 2010-0086698 proposes a cosmetic composition for skin soothing and irritation relief containing mung bean fermentation-enzyme extract, and Korean Patent Publication No. 2013-0016930 discloses sage, lemon balm, agar agar and onion extract. A cosmetic composition for alleviating atopic skin and skin irritation containing as an active ingredient is mentioned. In addition, Korean Patent Application Laid-Open No. 2015-0078948 proposes a cosmetic composition for improving skin inflammation or alleviating skin irritation comprising a mixed extract of mistletoe, pine bark, and rock pine as an active ingredient, and Korean Patent Publication No. 2009-0091547 discloses that A cosmetic composition for anti-inflammatory and skin irritation relief containing a mixed extract of the casualty is proposed.

In addition, the prevention and treatment of neuropsychiatric diseases through LTP inhibition of the concentrated powder of the filtrate obtained by filtering an aqueous solution of 70% ethanol extraction of dried hydrangea leaves is described in Korean Patent Registration No. 171962. And a cosmetic composition for skin irritation and inflammation relief by the function of inhibiting the expression of cytokines IL-6, IL-8, IL-1β, which induces inflammation due to fine dust stimulation of yeast liquid fermented product of hydrangea leaf extract is disclosed in Korean Patent No. 1867308 , Hydrangea leaves, stems, and flowers above-ground parts of the outpost were dried and pulverized to prepare methanol extract, and the phenolic component and flavonoid substance of the hydrangea outpost extract concentrated under reduced pressure for whitening cosmetic composition according to the inhibitory function of tyrosinase activity 1492074. In addition, the antioxidant, wrinkle improvement, and whitening functional cosmetics of a mixed extract of zebra, sage and hydrangea are disclosed in Korean Patent Registration No. 1738075.

However, so far, there has been no disclosure in any literature on a cosmetic composition containing hydrangenol derived from an enzymatic hydrolyzate of hydrangea whole plant for moisturizing, photoaging, and anti-inflammatory functional cosmetic composition against various immune factors.

Korean Patent No. 10-1719162 Korean Patent Registration No. 10-1738075 Korean Patent Registration No. 10-1867308 Korean Patent Registration No. 10-1492074 Korean Patent Publication No. 10-2011-0063269 Korean Patent Publication No. 10-2014-0055049 Korean Patent Publication No. 10-2015-0078948 Korean Patent Registration No. 10-681638

Efficient syntheses of (6)-hydrangenol, (6)-phyllodulcin and (6)-macrophyllol, Tetrahedron, 2003, 59, 879-802 New type of anti-diabetic compounds from the processed leaves of macrophylla var. thunbergii (Hydrangea Dulcis Folium), Bioorganic & Medicinal Chemistry Letters, 2007, 17, 4972-4976 Medicinal Flowers. XXXX.1) Structures of Dihydroisocoumarin Glycosides and Inhibitory Effects on Aldose Reducatase from the Flowers of Hydrangea macrophylla var. thunbergii, Chemical and Pharmaceutical Bulletin, 2013, 61, 655-661 Water extract of processed Hydrangea macrophylla (Thunb.) Ser. Leaf attenuates the expression of pro-inflammatory mediators by suppressing Akt-mediated NF-κB activation, Environmental Toxicology and Pharmacology, 2013, 35, 311-319 Hydrangenol inhibits lipopolysaccharide-induced nitric oxide production in BV2 microglial cells by suppressing the NF-κB pathway and activating the Nrf2-mediated HO-1 pathway, International Immunopharmacology, 2016, 35, 61-9 Chemical Constituents from Leaves of Hydrangea serrata and Their Anti-photoaging Effects on UVB-Irradiated Human Fibroblasts, Biological and Pharmaceutical Bulletin, 2019, 42, 424-431

An object of the present invention is to separate and purify a hydrangenol component from a mixture of dried hydrangea flowers and leaves in an equal ratio (w/w), and to provide use as a cosmetic composition comprising the same.

The above object of the present application is to measure the anti-inflammatory and photoaging inhibitory effects of Butylene Glycol extract of dried hydrangea flowers and the anti-inflammatory and moisturizing effects of Butylene Glycol extract of dried hydrangea leaves; Analyzing anti-inflammatory and photoaging inhibitory effects after performing HPLC of an equal ratio (w/w) mixture of Butylene Glycol extracts of dried hydrangea flowers and hydrangea leaves; A 50% ethanol (EtOH) extract was obtained from an equal ratio (w/w) mixture of dried hydrangea flowers and hydrangea leaves, and the concentrated solution filtered under reduced pressure was suspended in distilled water and sequentially fractionated in the order of hexane, chloroform, ethyl acetate, butanol. Then, column chromatography was performed using chloroform and methanol as mobile phases in an open column filled with ethyl acetate fractional layer with normal phase silica gel, and the precipitate obtained from the chloroform fractional layer was washed with methanol to recover Hydrangenol (Compound I) and ethyl Separating and purifying Hydrangenol 8-o-glucoside (Compound II) from the fractionation layer subjected to column chromatography on the acetate fraction; Measuring the anti-inflammatory effect of compound I and compound II, which are indicator components obtained in the separation and purification step; Separately, the concentrated solution of the 50% ethanol (EtOH) extraction filtrate of the dried hydrangea flower and leaf in an equal ratio (w/w) mixture is most preferably added with 3.5% of the complex hydrolase viscozyme and 50 After hydrolysis at ℃ for 24 hours, the enzyme was inactivated at 100℃/1 hour, and then centrifuged to obtain a precipitate, and again 100% ethanol (EtOH) extract was obtained and centrifuged to obtain a supernatant. Concentrating and performing HPLC analysis on the products obtained in each step; It is achieved through the step of evaluating the antioxidant, whitening, photoaging, skin moisturizing and anti-inflammatory effects of the complex enzyme-treated extract.

The present invention obtains an ethanol extract of dried hydrangea flower and leaf outpost, and treats the filtrated concentrate with a complex hydrolase to purify and isolate the natural compound hydrangenol, and a novel skin photoaging, moisturizing, whitening and anti-inflammatory of this compound It has the effect of confirming the efficacy and providing a novel functional cosmetic composition containing this compound.

1 is a graph showing the results of an anti-inflammatory test of a flower extract using Butylene Glycol, an organic solvent of hydrangea;
2 is a photoaging test result of a flower extract using Butylene Glycol, an organic solvent of hydrangea.
3 is an anti-inflammatory test result of an organic solvent Butylene Glycol extract of hydrangea leaves.
Figure 4a is an anti-inflammatory immune factor TNF-α / ELISA of the organic solvent Butylene Glycol extract of hydrangea leaves,
FIG. 4b shows the results of PGE2/ELISA, and FIG. 4c shows the results of COX-2/qPCR and AQP-3/qPCR experiments.
5 is an HPLC analysis chromatogram of Butylene Glycol extract of a 1:1 (w/w) mixture of flowers and leaves of hydrangea.
6 is a graph of the anti-inflammatory effect of Butylene Glycol extract of a 1:1 (w/w) mixture of flowers and leaves of hydrangea.
7 is a graph of the photoaging effect of Butylene Glycol extract of a 1:1 (w/w) mixture of flowers and leaves of hydrangea.
8a, 8b, 8c and 8d show the anti-inflammatory effects (TNF-α/ELISA), (PGE2/ELISA), (COX-2/ qPCR) and (COX-2/Western blot).
9 is a diagram showing the separation and purification steps of indicator components of a 1:1 (w/w) mixture of hydrangea flowers and leaves according to the present invention.
10 is a chromatogram showing HPLC analysis of compounds I and II isolated and identified according to the present invention.
11A and 11B are experimental results of anti-inflammatory effects of the index components I and II isolated and identified according to the present invention.
12 is a diagram showing the most preferred complex enzymatic hydrolysis process of the present invention.
13A and 13B are chromatograms showing HPLC analysis of an ethanol extract of hydrangea whole plant and a hydrolyzed extract of hydrangea whole plant.
14A to 14E are graphs showing the skin antioxidant, whitening, photoaging and moisturizing effects of the hydrolyzed ethanol extract of hydrangea whole plant.
15A to 15D are graphs showing the skin anti-inflammatory effect of an extract treated by hydrolysis of an ethanol extract of hydrangea whole plant.
16 is a western blot of the skin anti-inflammatory effect of the hydrolyzed ethanol extract of hydrangea whole plant.

Hereinafter, the specific content of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited thereto.

Preparation of flower extract using hydrangea Butylene Glycol (hereinafter referred to as B.G)

The dried hydrangea flowers were extracted with 40% butylene glycol 100 times the dry weight (kg) at 60°C for 24 hours, and then filtered with 0.45um. The following experiment was performed with the obtained flower B.G extract as a test material.

[Experimental Example 1-1] Anti-inflammatory test

Using the hydrangea flower BG extract obtained in the above Example as a test material, it was evaluated whether there was an anti-inflammatory effect after artificially treating RAW 264.7 cells with LPS, an inflammatory substance. ^{It was aliquoted to 2 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and BG extract of flowers were administered by concentration and cultured for 24 hours. After incubation, the medium supernatant was taken and centrifuged at 13,000 rpm for 3 minutes to collect only the supernatant, and then reacted with Griess reagent 1:1 and absorbance was measured at 570 nm. As a result of the experiment, it showed excellent NO production inhibitory efficacy even at a low concentration of 72% at a concentration of 0.5% (FIG. 1).

[Experimental Example 1-2] Photoaging experiment

After inducing ROS generation in Human Dermal Fibroblasts with UVB, it was evaluated whether the ROS generation inhibitory effect was achieved through fluorescent substances. ^{It was aliquoted to 3 x 10 4} cells per well in a 48-well plate, and then incubated at 37°C, 5% CO _{2 in an incubator for 24 hours.} After overnight, the medium was replaced with a new medium, and BG extracts of flowers were administered by concentration and cultured for 1 hour, followed by DCFH-DA treatment and cultured for 1 hour. After incubation, the medium was removed and 100 μL PBS was dispensed and UVB ^{was irradiated with an intensity of 100 mJ/cm 2} , and the excitation wavelength at 485 nm and emission wavelength at 530 nm were measured in a microplate reader at 37°C. As a result of the experiment, it showed the efficacy of inhibiting ROS induced by UVB by 26% at a concentration of 2% (FIG. 2).

Preparation of Leaf Extract Using Butylene Glycol of Hydrangea

The dried hydrangea leaves were extracted with 40% butylene glycol 100 times the dry weight (kg) at 60°C for 24 hours, and then filtered with 0.45um. The following experiment was performed using the B.G extract of the obtained leaf as a test material.

[Experimental Example 2-1] Anti-inflammatory test

After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether there was an anti-inflammatory effect. ^{It was aliquoted to 2 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and BG extract were administered by concentration and cultured for 24 hours. After incubation, the medium supernatant was taken and centrifuged at 13,000 rpm for 3 minutes to collect only the supernatant, and then reacted with Griess reagent 1:1 and absorbance was measured at 570 nm. As a result of the experiment, 87% at a concentration of 1% and 56% at a concentration of 0.5% showed excellent NO production inhibitory efficacy (FIG. 3).

[Experimental Example 2-2] Anti-inflammatory immune factor test

After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether they had an inhibitory effect on inflammation through enzyme-linked immunoprecipitation assay. ^{It was aliquoted to 1 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and BG extract of leaves were administered by concentration and cultured for 24 hours. After incubation, the medium supernatant was taken and the amount of inflammatory cytokine production was measured by performing TNF-α, PGE2 ELISA kit (R&D systems). As a result of the TNF-α inhibition test, the 2% concentration showed 86% inhibition, and the PGE2 inhibition experiment showed 26% inhibition at the 0.5% concentration. In addition, after artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether there was an effect of suppressing the expression of immune factors at the gene level. ^{It was aliquoted to 1 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and extract were administered and incubated for 24 hours. After incubation, cDNA was synthesized by washing thoroughly with PBS, and quantitative reverse transcription PCR (RT-qPCR) was performed by quantifying 100 ng/μL each. The relative expression rate was normalized with a housekeeping gene (β-actin). As a result of the experiment, it showed an inhibitory effect of 67% at a concentration of 2% of the extract ( FIGS. 4a to 4c ).

Preparation of organic solvent sequential fractions of ethanol extract of 1:1 (w/w) mixture of flowers and leaves of hydrangea

Dried hydrangea flowers and leaves were soaked in 50% ethanol 30 times the dry weight and extracted at room temperature. After the extract obtained above was concentrated using a vacuum suction filter, only the filtrate was suspended in 100% distilled water, and fractionation was performed in the order of hexane, chloroform, ethyl acetate, butanol, and distilled water. HPLC analysis was performed using the extract obtained above as a test material, and then the following experiment was performed (FIG. 5).

[Experimental Example 3-1] Anti-inflammatory (NO assay, ELISA, qPCR, Western blot), photoaging test

After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether there was an anti-inflammatory effect. ^{It was aliquoted to 2 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and extract were administered by concentration and incubated for 24 hours. After incubation, the medium supernatant was taken and centrifuged at 13,000 rpm for 3 minutes to collect only the supernatant, and then reacted with Griess reagent 1:1 and absorbance was measured at 570 nm. As a result of the experiment, 94% at the 2% concentration and 54% at the 1% concentration showed excellent NO production inhibitory efficacy (FIG. 6). After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether they had an inhibitory effect on inflammation through enzyme-linked immunoprecipitation assay. ^{It was aliquoted to 1 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and extract were administered by concentration and incubated for 24 hours. After incubation, the medium supernatant was taken and the amount of inflammatory cytokine production was measured by performing TNF-α, PGE2 ELISA kit (R&D systems). As a result of the TNF-α inhibition test, the 0.5% concentration showed 50% inhibition (8a), and in the PGE2 inhibition experiment, the 2% concentration showed 78% inhibition (8b). After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether there was an effect of suppressing the expression of immune factors at the gene level. ^{It was aliquoted to 1 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and extract were administered and incubated for 24 hours. After incubation, cDNA was synthesized by washing thoroughly with PBS, and quantitative reverse transcription PCR (RT-qPCR) was performed by quantifying 100 ng/μL each. The relative expression rate was normalized with a housekeeping gene (β-actin). As a result of the experiment, hydrangea extract showed an inhibitory effect to 56% at a concentration of 2% (8c). After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether there was an effect of suppressing the expression of immune factors at the protein level. ^{It was aliquoted to 5 x 10 5} cells per well in a 6-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and extract were administered by concentration and incubated for 24 hours. After incubation, the cells were sufficiently washed with PBS and treated with a lysis buffer to obtain a cell lysate. Only the supernatant was collected by centrifugation at 13500 rpm for 15 minutes, and protein was quantified using Bradford reagent. Proteins were separated by electrophoresis, blotted on Polyvinylidene Difluoride (PVDF) membrane, and antibodies were detected by chemiluminescence through ECL. As a result of the COX-2 (Cyclooxygenase-2) experiment, the 1% concentration of the extract showed 71% inhibitory efficacy (8d). After inducing ROS generation in Human Dermal Fibroblasts with UVB, it was evaluated whether the ROS generation inhibitory effect was achieved through fluorescent substances. ^{It was aliquoted to 3 x 10 4} cells per well in a 48-well plate, and then incubated at 37°C, 5% CO _{2 in an incubator for 24 hours.} After overnight, it was replaced with a new medium, and the extracts were administered by concentration and cultured for 1 hour, treated with DCFH-DA and incubated for an additional hour. After incubation, the medium was removed and 100 μL PBS was dispensed and UVB ^{was irradiated with an intensity of 100 mJ/cm 2} , and the excitation wavelength at 485 nm and emission wavelength at 530 nm were measured in a microplate reader at 37°C. At a concentration of 2% hydrangea extract, an inhibition rate of 28% was confirmed (FIG. 7).

HPLC analysis of compounds I and II according to the present invention

HPLC analysis was performed to identify the compounds separated and purified from the hydrangea flower and leaf 1:1 (w/w) extract, and the following conditions were used (FIG. 9).

Detector: PDA UV 311nm

Flow rate: 1.0ml/min

Column Temperature : 30.0 ℃

Sample Temperature: 30.0 ℃

Running Time : 60 min

Column: Kromasil C18, 5㎛, 4.6×250㎜

Injection volume: 10 μl

Solvent condition : acetonitrile : water (0.1% Acetic acid)=10:90 - 100% acetonitrile

[Experimental Example 4-1] Separation and purification of indicator components (I) and (II)

As a result of HPLC analysis, as shown in FIG. 10, the structure was determined to be Compound I (Hydragenol 8-glucoside) and Compound II (Hydragenol).

[Experimental Example 4-2] Anti-inflammatory test

After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether there was an anti-inflammatory effect. ^{It was aliquoted to 2 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and extract were administered by concentration and incubated for 24 hours. After incubation, the medium supernatant was taken and centrifuged at 13,000 rpm for 3 minutes to collect only the supernatant, and then reacted with Griess reagent 1:1 and absorbance was measured at 570 nm. As a result of the experiment, only hydrangenol showed excellent NO production inhibitory efficacy as 75% at a concentration of 100 μg/mL and 44% at 50 μg/mL ( FIGS. 11a and 11b ).

Preparation of viscozyme hydrolyzate of ethanol extract of hydrangea whole plant according to the present invention

The dried hydrangea flower and leaf outpost were soaked in 50% ethanol 30 times the dry weight of the dried hydrangea and extracted at room temperature. After the extract obtained by filtration was concentrated enough to completely remove ethanol, viscozyme, a complex hydrolytic enzyme, was added with 3.5% of the weight of hydrangea. After hydrolysis reaction at 50° C. for 24 hours, it was treated at 100° C. for 1 hour for inactivation. The inactivated hydrangea enzyme extract was centrifuged and the precipitate was concentrated.

Ethanol was added to the concentrated precipitate to extract the active ingredient, then centrifuged again, and only the supernatant was collected and concentrated to obtain a hydrolyzate having an increased active ingredient, hydrangenol (FIG. 12).

[Experimental Example 6-1] HPLC analysis

HPLC analysis was performed to confirm the change in hydrangenol content of hydrolysates of hydrangea flowers and leaves, and the following conditions were used.

Detector: PDA UV 311nm

Flow rate: 1.0ml/min

Column Temperature : 30.0 ℃

Sample Temperature: 30.0 ℃

Running Time : 60 min

Column: Kromasil C18, 5㎛, 4.6×250㎜

Injection volume: 10 μl

Solvent condition : acetonitrile : water (0.1% Acetic acid)=10:90 - 100% acetonitrile

As a result of the analysis experiment, it was confirmed that Hydragenol 8-glucoside (Compound I) and Hydragenol Compound II were separated and purified, respectively, as shown in the chromatograms shown in FIGS. 13A and 13B.

Functional test of viscozyme hydrolyzate of the present invention

The following experiments were performed using compounds I and II separated and purified according to Example 5 as test materials.

[Experimental Example 6-1] Antioxidant experiment

After artificially treating Human Dermal Fibroblast with H ₂ O ₂ , it was evaluated whether the ROS inhibitory effect was achieved through a fluorescent material. ^{It was aliquoted to 1.5 x 10 4} cells per well in a black 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was replaced with a new medium, and the extracts were administered by concentration and incubated for 1 hour. After incubation, the cells were washed with PBS, treated with H ₂ O ₂ and DCFH-DA, and excitation wavelengths at 485 nm and emission wavelengths at 530 nm were measured in a microplate reader at 37°C, once every 5 minutes for 30 minutes. As a result of the experiment, it showed excellent antioxidant efficacy with an inhibition rate of 67% at a concentration of 100 μg/mL (14a).

[Experimental Example 6-2] Whitening experiment

After artificially treating B16F1 cells with α-MSH, it was evaluated whether there was an effect of inhibiting melanogenesis. ^{It was aliquoted to 2 x 10 4} cells per well in a 24-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was replaced with a new medium, and the extracts were administered by concentration and cultured. After 72 hours, the culture medium was removed and treated with 1 N NaOH containing 10% DMSO to dissolve melanin in the cells at 60°C. This solution was placed in a 96-well plate and absorbance was measured at 405 nm. As a result of the experiment, it showed a whitening effect with an inhibition rate of 29% at a concentration of 50 μg/mL (14b).

[Experimental Example 6-3] Photoaging experiment

After inducing ROS generation in Human Dermal Fibroblasts with UVB, it was evaluated whether the ROS generation inhibitory effect was achieved through fluorescent substances. ^{It was aliquoted to 3 x 10 4} cells per well in a 48-well plate, and then incubated at 37°C, 5% CO _{2 in an incubator for 24 hours.} After overnight, it was replaced with a new medium, and the extracts were administered by concentration and cultured for 1 hour, treated with DCFH-DA and incubated for an additional hour. After incubation, the medium was removed and 100 μL PBS was dispensed and UVB ^{was irradiated with an intensity of 100 mJ/cm 2} , and the excitation wavelength at 485 nm and emission wavelength at 530 nm were measured in a microplate reader at 37°C. Inhibition rate of 41% was confirmed in 100 μg/mL of hydrangea leaf enzyme-treated extract (14c).

[Experimental Example 6-4] Moisturizing effect test

It was evaluated whether the expression of hyaluronic acid synthase (HAS-2) and aquaporin (AQP-3) was increased when the human dermal fibroblast was treated with the whole plant extract. ^{It was aliquoted to 1.5 x 10 4} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and the extract was administered and incubated for 24 hours. After incubation, cDNA was synthesized by washing thoroughly with PBS, and quantitative reverse transcription PCR (RT-qPCR) was performed by quantifying 100 ng/μL each. The relative expression rate was normalized with a housekeeping gene (β-actin). As a result of the moisturizing factor test of the hydrangea whole plant enzyme-treated extract, it was confirmed that it was increased by 39% at 10 μg/mL in HAS-2, and increased by 29% at 10 μg/mL in AQP-3 (14d, 14e).

[Experimental Example 6-5] Skin anti-inflammatory test (NO assay + ELSIA + qPCR + Western blot)

After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether there was an anti-inflammatory effect. ^{It was aliquoted to 2 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and extract were administered by concentration and incubated for 24 hours. After incubation, the medium supernatant was taken and centrifuged at 13,000 rpm for 3 minutes to collect only the supernatant, and then reacted with Griess reagent 1:1 and absorbance was measured at 570 nm. As a result of the experiment, it showed excellent NO production inhibitory efficacy even at a low concentration of 30% at a concentration of 10 μg/mL (15a). After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether there was an anti-inflammatory effect. ^{It was aliquoted to 1 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and extract were administered by concentration and incubated for 24 hours. After incubation, the medium supernatant was taken and the amount of inflammatory cytokine production was measured by performing TNF-α, PGE2 ELISA kit (R&D systems). As a result of the TNF-α inhibition test, the inhibition rate was 42% at the concentration of 10 μg/mL (15b), and in the PGE2 inhibition experiment, the inhibition rate was 93% at the concentration of 10 μg/mL (15c). It can be confirmed that this shows better efficacy than the existing butylene glycol extract. After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether there was an anti-inflammatory effect. ^{It was aliquoted to 1 x 10 5} cells per well in a 96-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and extract were administered and incubated for 24 hours. After incubation, cDNA was synthesized by washing thoroughly with PBS, and quantitative reverse transcription PCR (RT-qPCR) was performed by quantifying 100 ng/μL each. The relative expression rate was normalized with a housekeeping gene (β-actin). As a result of the experiment, 58% at a concentration of 10 μg/mL showed excellent TNF-α expression inhibitory efficacy even at a low concentration (15d). After artificially treating RAW 264.7 cells with LPS, an inflammatory substance, it was evaluated whether there was an anti-inflammatory effect. ^{It was aliquoted to 5 x 10 5} cells per well in a 6-well plate, and then incubated at 37° C., 5% CO _{2 in an incubator for 24 hours.} After overnight, it was exchanged with a new medium, and LPS (1 ug/mL) and extract were administered by concentration and incubated for 24 hours. After incubation, the cells were sufficiently washed with PBS and treated with a lysis buffer to obtain a cell lysate. Only the supernatant was collected by centrifugation at 13500 rpm for 15 minutes, and protein was quantified using Bradford reagent. Proteins were separated by electrophoresis, blotted on Polyvinylidene Difluoride (PVDF) membrane, and antibodies were detected by chemiluminescence through ECL. As a result, IL-6 (Interleukin-6) was 71% at a concentration of 10 μg/mL, TNF-α (Tumor necrosis factor-α) was 50% at a concentration of 10 μg/mL, and COX-2 (Cyclooxygenase- 2) showed an inhibitory efficacy of 54% at a concentration of 10 μg/mL ( FIG. 16 ).

The efficacy of each extract or enzymatic hydrolyzate according to the present invention is summarized in [Table 1] below.

Claims (2)

A hydrolyzate obtained by obtaining an ethanol extract of dried hydrangea flowers and leaf stalks and adding the complex hydrolase viscozyme to the filtered concentrate. A cosmetic composition for skin antioxidant, whitening, photoaging, anti-inflammatory and moisturizing, comprising a compound represented by the following formula II derived from the hydrolyzate of claim 1.

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