KR20220093477A - Cosmetic composition for anti-aging containing prunus yedoensis flower biorenovate extract - Google Patents

Abstract

The present invention relates to a cosmetic composition containing a biorenovation extract of Prunus × yedoensis flowers as an active ingredient and, more specifically, to a technique configured such that a biorenovation extract of Prunus × yedoensis flowers, which is highly effective in ameliorating skin wrinkles and improving elasticity, is prepared by fermenting an extract of Prunus × yedoensis flowers with Bacillus amyloliquefaciens, and that the biorenovation extract is used as an anti-aging cosmetic composition.

Description

Cosmetic composition for anti-aging containing Yoshino cherry tree flower biorenovate extract as an active ingredient {Cosmetic composition for anti-aging containing prunus yedoensis flower biorenovate extract}

The present invention relates to a cosmetic composition for anti-aging containing a Yoshino cherry tree flower bioconversion extract as an active ingredient, and specifically, an extract having excellent skin wrinkles and elasticity improvement activity by applying a bio-renovation technique to Yoshino cherry blossom flowers. It relates to a technology for manufacturing and using the same as a cosmetic composition for anti-aging.

In human skin aging, the secretion of various hormones that control metabolism decreases, the function and activity of immune cells and skin cells are lowered, and the biosynthesis of immune proteins and bio-constituting proteins necessary for the living body is reduced, resulting in endogenous aging and various It is divided into exogenous aging caused by external factors such as pollutants, gravity, and ultraviolet rays.

Both intrinsic aging and extrinsic aging are caused by structural changes in the extracellular matrix present in the dermal layer of the skin, and among its components, the degeneration of elastic fibers and the decrease in the amount of collagen are cause wrinkles A typical symptom of aging skin is the occurrence of fine lines and wrinkles caused by loss of elasticity, which may be caused by a significant decrease in collagen protein related to skin elasticity in the collagen of the dermal tissue of the skin. Collagen occupies more than 90% of the dermis and protects and maintains the skin from external stimuli because it provides tension and strength to the skin. Therefore, the decrease in collagen has a very deep relationship with skin aging and wrinkle formation.

The degradation of the extracellular matrix and type I procollagen occurs by matrix metalloproteinases (MMPs) induced by the transcription factor AP-1 (activating protein-1). Among the MMP family, MMP-1 is known as a representative degrading enzyme that degrades collagen. In addition, as another factor causing skin aging, the generation of active oxygen by ultraviolet rays or stress in relation to the decrease in collagen. The generation of active oxygen in the skin causes damage to the cells by attacking the cell membrane and causes the skin to lose its function, thereby causing wrinkles. The generation of free radicals in vivo induces the chain activation of the MMPs gene and collagen degrading enzyme, thereby causing structural destruction of collagen and the formation of skin wrinkles that reduce the elasticity of the skin. Therefore, inhibition of collagen degrading enzymes and production of reactive oxygen species, and promotion of collagen synthesis are important factors in inhibiting skin aging.

Recently, research on the development of anti-aging cosmetics derived from natural products has been actively conducted. As an example, Korean Patent Application Laid-Open No. 10-2020-0057677 discloses, 'A cosmetic composition for improving skin elasticity or preventing skin wrinkles, containing, as an active ingredient, a mixed extract of at least one selected from the group consisting of Centella asiatica and green tea and a mixed extract or a fraction thereof. ' is disclosed, and Korean Patent Application Laid-Open No. 10-2020-0031055 discloses 'a cosmetic composition for improving skin wrinkles or increasing elasticity, including a fermented extract of reed (Phragmites Communis)'. In addition, Korean Patent Publication No. 10-2020-0072842 discloses that the fermented water parsley extract obtained by fermenting 'Oenanthe 　javanica 　 D.C. with a mycelium of oyster mushroom is included as an active ingredient. Cosmetic composition for improvement, elasticity improvement or whitening' is disclosed.

The present inventors have searched for materials derived from natural products that have anti-aging activity for improving skin wrinkles and elasticity, and skin wrinkle and elasticity improvement activity from bioconversion extracts prepared by applying a specific bio-renovation technique to Yoshino cherry blossoms The present invention was completed by confirming that this significantly increased.

An object of the present invention is to provide a cosmetic composition containing a Yoshino cherry tree flower bioconversion extract as an active ingredient to exhibit excellent skin wrinkle and elasticity improvement effects.

According to the present invention in order to achieve the above object, there is provided a cosmetic composition containing, as an active ingredient, a Yoshino cherry blossom bioconversion extract prepared by fermenting an extract of Yoshino cherry blossoms with Bacillus amyloliquefaciens.

The Yoshino cherry tree flower bioconversion extract is prepared in the following way.

(A) adding an extraction solvent to the dried Yoshino cherry tree flowers to prepare a Yoshino cherry tree flower extract;

(B) mixing the Yoshino cherry blossom extract and Bacillus amyloliquefaciens and culturing at 30-40° C. for 60-80 hours; and

(C) After the end of the culture, centrifugation is performed at 5000 ~ 6000 rpm for 10 to 15 minutes, and the supernatant is collected to prepare a Yoshino cherry blossom bioconversion extract.

The extraction in step (A) is characterized in that it is extracted 2-3 times at 100 ~ 125 ℃ by adding 5 to 10 times the weight of purified water to the dried Yoshino cherry blossoms.

The Yoshino cherry tree flower bioconversion extract is contained in an amount of 0.1 to 30% by weight based on the total weight of the cosmetic composition.

The cosmetic composition is characterized in that it is for improving skin wrinkles and elasticity.

The Yoshino cherry blossom bioconversion extract prepared according to the biorenovation technique of the present invention can be used as a safe and excellent anti-aging cosmetic because it exhibits improved skin wrinkles and elasticity improvement activity.

1 is a graph showing the collagen production promoting effect of Yoshino cherry tree flower bioconversion extract according to an embodiment of the present invention.
2 is a graph showing the effect of increasing collagen 1 gene expression of the Yoshino cherry tree flower bioconversion extract according to an embodiment of the present invention.
3 is a graph showing the effect of increasing collagen 3 gene expression of the Yoshino cherry tree flower bioconversion extract according to an embodiment of the present invention.
4 is a graph showing the effect of increasing collagen 4 gene expression of the Yoshino cherry tree flower bioconversion extract according to an embodiment of the present invention.
5 is a graph showing the effect of increasing the elastin gene expression of the Yoshino cherry tree flower bioconversion extract according to an embodiment of the present invention.
6 is a graph showing the effect of increasing collagen 1 protein expression of a Yoshino cherry tree flower bioconversion extract according to an embodiment of the present invention.
7 is a graph showing the effect of increasing collagen 3 protein expression of the Yoshino cherry tree flower bioconversion extract according to an embodiment of the present invention.
8 is a graph showing the effect of increasing elastin protein expression of Yoshino cherry tree flower bioconversion extract according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

The Yoshino cherry tree (Prunus yedoensis), which is native to Jeju Island in Korea, is a deciduous tree in the Rosaceae family of the Rosaceae family of the dicotyledonous plant. It is a deciduous broad-leaved tree widely planted as an ornamental tree and roadside tree nationwide. It reaches 10 m in height. The leaves are alternate phyllotaxis, oval egg-shaped or inverted egg-shaped, 6-12 cm long, with hairs on the back, sharp saw teeth on the edge, and 2 nectar glands at the tip of petiole. The flowers bloom before the leaves in April, and 3 to 6 flowers are hung in umbel, and the small peduncle has hairs. The flower buds are pink and white when in full bloom. The flower stalk has hairs along with the calyx, the petal has a concave tip, and the style has spread hairs. The fruit is a drupe, round and ripens from reddish red to purple black in June to July.

In oriental medicine, the bark of the Yoshino cherry tree has long been used to treat respiratory and skin diseases. In a recent study, it was reported that the ethanol extract of Yoshino cherry tree bark or Yoshino cherry leaf had anti-inflammatory and vascular relaxing effects (Yang G, Ham I, Choi HY. Anti-inflammatory effect of prunetin via the suppression of NF-κB). pathway.Food Chem Toxicol.2013;58:124-132).

In the present invention, a bioconversion extract is prepared by applying a bio-renovation technique to the flower of the Yoshino cherry tree.

In the present invention, the term "bio-renovation" refers to a process of converting an added substance into a chemically modified form using the physiological function of an organism. In the present invention, a Bacillus amyloliquefaciens strain is used for the bioconversion. The technical feature is to produce a cosmetic with excellent skin wrinkle and elasticity improvement activity through bioconversion by mixing and culturing Yoshino cherry blossom extract with Bacillus amyloliquefaciens.

According to an embodiment of the present invention, the Yoshino cherry tree flower bioconversion extract is prepared by a method comprising the following steps.

(A) adding an extraction solvent to the dried Yoshino cherry tree flowers to prepare a Yoshino cherry tree flower extract;

(B) mixing the Yoshino cherry blossom extract and Bacillus amyloliquefaciens and culturing at 30-40° C. for 60-80 hours; and

(C) After culturing, centrifugation is performed at 5000 ~ 6000 rpm for 10 ~ 15 minutes, and the supernatant is collected to prepare a Yoshino cherry blossom bioconversion extract.

At this time, as the extraction solvent in step (A), from the group consisting of water, anhydrous or hydrous alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol, dipropylene glycol and butylene glycol At least one selected may be used.

It is more preferable that the extraction in step (A) is to add 5 to 10 times the weight of purified water to the dried Yoshino cherry blossoms and extract 2-3 times at 100 to 125°C.

The Yoshino cherry tree flower extract prepared by the above method has a superior collagen production promoting effect (Test Example 1), collagen 1, 3, and 4 gene expression increase effect (Test Example 2, 3, 4), elastin gene expression increase effect (Test Example 5), collagen 1, 3 protein expression increase effect (Test Examples 6 and 7) and elastin protein expression increase effect (Test Example 8), thereby improving skin wrinkles and elasticity was confirmed to be excellent.

The Yoshino cherry tree flower bioconversion extract prepared by the above preparation method is contained as an active ingredient in an amount of 0.1 to 30% by weight based on the total weight of the cosmetic composition.

The cosmetic composition of the present invention may be prepared in any conventionally prepared formulation, for example, lotion, cream, essence, cleansing foam, cleansing water, pack, body lotion, body oil, body gel, shampoo, conditioner, hair conditioners, hair gels, foundations, lipsticks, mascaras, makeup bases, microneedles, and the like.

[Example]

Hereinafter, the present invention will be described in detail through the following examples and test examples, but the present invention is not limited by these examples.

Preparation Example 1: Preparation of Yoshino cherry tree flower extract

The washed and dried Someiyoshino cherry blossoms were put into an extraction tank, and purified water 10 times by weight was added. The primary extract was recovered by extraction at 120° C. for 6 to 8 hours, and 10-fold purified water was added to the extraction tank again.

The secondary extract was recovered by extraction at 120° C. for 6 to 8 hours. The recovered primary and secondary extracts were mixed, filtered through a filter having a pore size of 1.0 μm (Rainbow, Korea), and concentrated to prepare a Yoshino cherry tree flower extract.

Example 1: Preparation of Yoshino cherry tree flower bioconversion extract

Microbial culture

Bacillus amyloliquefaciens (KCTC 13588) strain was distributed from the Microbial Resource Center and cultured for 20 hours according to optimal growth conditions in Nutrient broth (beef extract 3.0g/L and peptone 5.0g/L).

After culturing for 20 hours, centrifugation was performed at 5,000 rpm for 10 minutes to obtain a microbial pellet.

Preparation of bioconversion extracts

The obtained microbial pellet was washed with PG buffer (50 mM phosphate (pH 7.2) and 2% glycerol), and suspended in 5 ml of PG buffer. Thereafter, the suspension and the Yoshino cherry tree flower extract prepared in Preparation Example 1 were mixed and incubated at 30° C. for 72 hours.

After incubation, centrifugation was performed at 5000 to 6000 rpm for 10 minutes. After that, the supernatant was collected to prepare a Yoshino cherry blossom bioconversion extract.

Test Example 1: Collagen production ability evaluation

In order to evaluate the collagen production ability before and after application of the biorenovation technique of the present invention, the following tests were performed.

In order to measure the amount of procollagen type I synthesis when HDF cells were treated with Yoshino cherry tree flower and Yoshino cherry tree flower bioconversion extract by concentration, they were seeded in a 48 well plate at a concentration of 2 × 10 ⁴ cells/well and then stabilized for 24 hours. Thereafter, after washing the cells with DPBS, the test substance was diluted in DMEM medium and cultured for 24 hours in a medium treated with concentrations of 1, 10 and 100 μg/mL, respectively. After recovering the supernatant from each well and adding the procollagen type I C-peptide assay kit to each well, the total amount of procollagen type I was measured. The results are shown in FIG. 1 .

As can be seen in FIG. 1 , it was confirmed that the collagen production ability was increased in the sample of Example 1, which was bioconverted by applying the bio-renovation technique compared to the sample of Preparation Example 1.

Test Example 2: Collagen 1 gene expression evaluation

In order to examine COL1A1 activity, HDF cells were seeded in a 60 mm tissue culture dish at a concentration of 2×10 ⁵ cells/well, and then cultured for 24 hours to stabilize the cells. After removing the medium, it was washed with DPBS, and the test substance was incubated for 24 hours in a medium treated with each concentration of 1, 10, and 100 μg/mL, and then the medium was removed and washed with DPBS. RNA was extracted using the RNeasy Mini Kit. cDNA was synthesized from the extracted RNA using AccuPower RT PreMix. 1μL cDNA, 1μL COL1A1 primer, and 10μL SYBR Green PCR supermix were mixed and put in a Real-Time PCR machine, and 35-40 cycles were checked in real time. The results are shown in FIG. 2 .

As can be seen in FIG. 2 , it was confirmed that Collagen 1 gene expression was significantly increased in the sample of Example 1, which was bioconverted by applying the bio-renovation technique compared to the sample of Preparation Example 1.

Test Example 3: Collagen 3 gene expression evaluation

In order to check COL3A1 activity, HDF cells were seeded in a 60 mm tissue culture dish at a concentration of 2×10 ⁵ cells/well, and then cultured for 24 hours to stabilize the cells. After removing the medium and washing with DPBS, the test substances were cultured for 24 hours in a medium treated with 1, 10 and 100 μg/mL, respectively, and then the medium was removed and washed with DPBS. RNA was extracted using the RNeasy Mini Kit. cDNA was synthesized from the extracted RNA using AccuPower RT PreMix. 1μL cDNA, 1μL COL3A1 primer, and 10μL SYBR Green PCR supermix were mixed and put in a Real-Time PCR machine, and 35 to 40 cycles were checked in real time. The results are shown in FIG. 3 .

As can be seen in FIG. 3 , it was confirmed that Collagen 3 gene expression was significantly increased in the sample of Example 1, which was bioconverted by applying the bio-renovation technique compared to the sample of Preparation Example 1.

Test Example 4: Collagen 4 expression evaluation

To check COL4A1 activity, HDF cells were seeded in a 60 mm tissue culture dish at a concentration of 2×10 ⁵ cells/well, and then cultured for 24 hours to stabilize the cells. After removing the medium and washing with DPBS, the test substances were cultured for 24 hours in a medium treated with 1, 10, and 100 μg/mL, respectively, and then the medium was removed and washed with DPBS. RNA was extracted using the RNeasy Mini Kit. cDNA was synthesized from the extracted RNA using AccuPower RT PreMix. 1μL cDNA, 1μL COL4A1 primer, and 10μL SYBR Green PCR supermix were mixed and put in a Real-Time PCR machine, and 35-40 cycles were checked in real time. The results are shown in FIG. 4 .

As can be seen in FIG. 4 , it was confirmed that Collagen 4 gene expression was significantly increased in the sample of Example 1, which was bioconverted by applying the bio-renovation technique compared to the sample of Preparation Example 1.

Test Example 5: Elastin gene expression evaluation

In order to check the elastin activity, HDF cells were seeded in a 60 mm tissue culture dish at a concentration of 2 × 10 ⁵ cells/well, and then cultured for 24 hours to stabilize the cells. After removing the medium and washing with DPBS, the test substances were cultured for 24 hours in a medium treated with 1, 10, and 100 μg/mL, respectively, and then the medium was removed and washed with DPBS. RNA was extracted using the RNeasy Mini Kit. cDNA was synthesized from the extracted RNA using AccuPower RT PreMix. 1μL cDNA, 1μL elastin primer, and 10μL SYBR Green PCR supermix were mixed and put in a Real-Time PCR machine, and 35-40 cycles were checked in real time. The results are shown in FIG. 5 .

As can be seen in FIG. 5 , it was confirmed that the elastin gene expression was significantly increased in the sample of Example 1, which was bioconverted by applying the bio-renovation technique compared to the sample of Preparation Example 1.

Test Example 6: Collagen 1 protein expression evaluation

In order to check COL1A1 activity, HDF cells were seeded in a 100 mm tissue culture dish at a concentration of 4 × 10 ⁵ cells/well, and then cultured for 24 hours to stabilize the cells. After removing the medium and washing with DPBS, the test substances were cultured for 24 hours in a medium treated with 1, 10 and 100 μg/mL, respectively, and then the medium was removed and washed with DPBS. Protein prep buffer is added, cells are lysed, centrifuged at 4°C and 13,000 rpm for 15 minutes, and the supernatant is collected. Then, the protein concentration is measured using a kit using the BCA method, and then sample buffer is added to contain the same concentration of protein. sample was prepared. 20 μL of protein was separated using SDS-PAGE, and the separated protein was transferred to a PVDF membrane and blocked in blocking buffer at room temperature for 1 hour. COL1A1 and β-actin were incubated at 4°C overnight, and then the secondary antibody was reacted at room temperature for 2 hours. After the reaction, ECL solution was used to confirm and quantify the protein band using ChemiDoc MP Imaging System. The results are shown in FIG. 6 .

As can be seen in FIG. 6 , it was confirmed that Collagen 1 protein expression was significantly increased in the sample of Example 1, which was bioconverted by applying the bio-renovation technique compared to the sample of Preparation Example 1.

Test Example 7: Collagen 3 protein expression evaluation

To check COL3A1 activity, HDF cells were seeded in a 100 mm tissue culture dish at a concentration of 4 × 10 ⁵ cells/well, and then cultured for 24 hours to stabilize the cells. After removing the medium and washing with DPBS, the test substances were cultured for 24 hours in a medium treated with 1, 10, and 100 μg/mL, respectively, and then the medium was removed and washed with DPBS. Protein prep buffer is added, cells are lysed, centrifuged at 4°C, 13,000 rpm for 15 minutes, and the supernatant is collected. Then, the protein concentration is measured using a kit using the BCA method. A sample containing 20 μL of protein was separated using SDS-PAGE, and the separated protein was transferred to a PVDF membrane and blocked in blocking buffer at room temperature for 1 hour. COL3A1 and β-actin were incubated at 4°C overnight, and then the secondary antibody was reacted at room temperature for 2 hours. After the reaction, ECL solution was used to confirm and quantify the protein band using ChemiDoc MP Imaging System. The results are shown in FIG. 7 .

As shown in FIG. 7 , it was confirmed that collagen 3 protein expression was significantly increased in the sample of Example 1, which was bioconverted by applying the bio-renovation technique compared to the sample of Preparation Example 1.

Test Example 8: Elastin protein expression evaluation

In order to check the elastin activity, HDF cells were seeded in a 100 mm tissue culture dish at a concentration of 4 × 10 ⁵ cells/well, and then cultured for 24 hours to stabilize the cells. After removing the medium and washing with DPBS, the test substances were cultured for 24 hours in a medium treated with 1, 10 and 100 μg/mL, respectively, and then the medium was removed and washed with DPBS. Protein prep buffer is added, cells are lysed, centrifuged for 15 minutes at 4°C and 13,000 rpm, the supernatant is collected, and the protein concentration is measured using a kit using the BCA method. A sample containing 20 μL of protein was separated using SDS-PAGE, and the separated protein was transferred to a PVDF membrane and blocked in blocking buffer at room temperature for 1 hour. Elastin and β-actin were incubated at 4°C overnight, and then the secondary antibody was reacted at room temperature for 2 hours. After the reaction, ECL solution was used to confirm and quantify the protein band using ChemiDoc MP Imaging System. The results are shown in FIG. 8 .

As can be seen in FIG. 8 , it was confirmed that the elastin protein expression was significantly increased in the sample of Example 1, which was bioconverted by applying the bio-renovation technique compared to the sample of Preparation Example 1.

Example 2: Preparation of serum containing Yoshino cherry tree flower bioconversion extract

A serum containing the Yoshino cherry tree flower bioconversion extract (Example 1) was prepared according to a conventional method with the composition and content of Table 1 below.

Raw material content (wt%) Yoshino cherry tree flower bioconversion extract (Example 1) 2.0 beeswax 1.0 Polysorbate 60 1.5 Sorbitan Sesquiolate 0.5 mineral oil 10.0 Sorbitan Stearate 0.5 lipophilic monostearate glycerin 1.0 stearic acid 1.5 Glyceryl Stearate/PEG-400 Stearate 1.0 propylene glycol 3.0 carboxy polymer 0.1 triethanolamine 0.1 Phenoxyethanol appropriate amount Purified water To 100

Example 3: Preparation of cream containing Yoshino cherry tree flower bioconversion extract

A cream containing the Yoshino cherry tree flower bioconversion extract (Example 1) was prepared according to a conventional method with the composition and content of Table 2 below.

Raw material content (wt%) Yoshino cherry blossom bioconversion extract (Example 1) 2.0 shea butter 2.0 Polysorbate 60 1.5 Sorbitan Sesquiolate 0.8 mineral oil 10.0 dimethicone 3.0 Sorbitan Stearate 0.5 lipophilic monostearate glycerin 1.0 cetearyl alcohol 2.0 Glyceryl Stearate/PEG-400 Stearate 1.0 propylene glycol 3.0 carboxy polymer 0.25 triethanolamine 0.25 Phenoxyethanol appropriate amount Purified water To 100

Test Example 9: Formulation stability confirmation

For the formulations prepared in Examples 2 and 3, stored in an opaque glass container at room temperature (25°C), refrigeration (4°C), and constant temperature (50°C) constantly in an opaque glass container and stored for 12 weeks Observation (discoloration, discoloration, and separation) was performed, and stability was confirmed. The results are shown in Table 3 below.

temperature condition Stability check (discoloration, discoloration and separation) Example 2 Example 3 Room temperature (25°C) 0 0 Refrigeration (4℃) 0 0 constant temperature (50℃) 0 0

< Formulation stability grade >

0: No change 1: Minor change 2: Change 3: Extreme change

As shown in the table above, it was confirmed that all formulations of Examples 2 and 3 were stable without discoloration, odor change and separation under the temperature conditions of 25°C, 4°C and 50°C.

Claims (5)

A cosmetic composition comprising a Yoshino cherry flower bioconversion extract as an active ingredient, which is produced by fermenting an extract of a Yoshino cherry tree flower with Bacillus amyloliquefaciens. According to claim 1, wherein the Yoshino cherry tree flower bioconversion extract
(A) preparing a Someiyoshino cherry tree flower extract by adding an extraction solvent to the dried Yoshino cherry blossoms;
(B) mixing the Yoshino cherry blossom extract and Bacillus amyloliquefaciens and culturing at 30-40° C. for 60-80 hours; and
(C) After culturing, centrifugation is performed at 5000 to 6000 rpm for 10 to 15 minutes, and the supernatant is collected to prepare a Yoshino cherry blossom bioconversion extract. Cosmetic composition, characterized in that it is prepared. According to claim 2, wherein the extraction in step (A) is a cosmetic composition, characterized in that the extraction is performed 2-3 times at 100 ~ 125 ℃ by adding 5 to 10 times the weight of purified water to the dried Yoshino cherry blossoms. The cosmetic composition according to claim 1, wherein the Yoshino cherry tree flower bioconversion extract is contained in an amount of 0.1 to 30% by weight based on the total weight of the cosmetic composition. The cosmetic composition according to claim 1, wherein the cosmetic composition is for improving skin wrinkles and elasticity.

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