KR102351384B1 - Cosmetic composition containing fermented extract using paeonia suffruticosa, camphor tree leaf, vitamin tree leaf, centella asiatica and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a method for preparing a cosmetic composition which contains a fermented extract using Paeonia suffruticosa, leaves of Cinnamomum camphora, leaves of Hippophae rhamnoides, and Centella asiatica as an effective ingredient, thereby having skin regeneration, anti-inflammatory and anti-oxidant effects. The method comprises: a step A of preparing a mixture by mixing Paeomia suffruticosa, flowers, leaves, and stems of Paeonia suffruticosa, leaves of Hippophae rhamnoides, leaves of Cinnamomum camphora, and Centella asiatica; a step B of adding the mixture prepared through step A to a broth-type medium and sterilizing the medium in an environment where the temperature is 120-122 degrees Celsius and the atmospheric pressure is 1.5; a step C of inoculating the medium to which the mixture was added and sterilized through step B with pre-cultured lactic acid bacteria and fermenting the medium in an environment where the temperature is 37 degrees Celsius using a shaking incubator at a shaking agitation speed of 50-250 rpm for five days; a step D of sterilizing the medium fermented through step C in an environment where the temperature is 120 degrees Celsius and the atmospheric pressure is 1.5 for 15 minutes; and a step E of recovering supernatant which is the fermented extract by performing centrifuging on the medium that is completely sterilized through step D in an environment where the temperature is 4 degrees Celsius using a high-speed centrifugal separator at a rotation speed of 8000 rpm for 30 minutes.

Description

Cosmetic composition containing fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica as an active ingredient to have skin regeneration, anti-inflammatory and antioxidant effects, and manufacturing method thereof {COSMETIC COMPOSITION CONTAINING FERMENTED EXTRACT USING PAEONIA SUFFRUTICOSA, CAMPHOR TREE LEAF, VITAMIN TREE LEAF, CENTELLA ASIATICA AND METHOD FOR MANUFACTURING THE SAME}

The present invention relates to a cosmetic composition comprising a fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica as an active ingredient and having skin regeneration, anti-inflammatory and antioxidant effects, and a method for manufacturing the same.

In the manufacture of cosmetics, as more and more interest in environmental aspects and the degree of harm to the human body appears, various cosmetic products using natural plants that are environmentally friendly and harmless to the human body as active ingredients are being developed.

In manufacturing a cosmetic composition using these natural plants as an active ingredient, it provides a variety of effects from skin beauty-related effects such as whitening and wrinkle improvement to effects such as antioxidants and anti-inflammatory properties, and natural to further enhance the level of effects. Various types of research have been attempted in which plants are not simply added, but are subjected to additional treatment processes such as mixing, drying, pulverization, hot water extraction, fermentation or steaming.

In this regard, in the prior literature on the prior art of providing a cosmetic composition comprising a peony root extract (Paeonia Suffruticosa Root Extract) used as a preservative component as an active ingredient, the "fermented peony root extract of Korean Patent Publication No. 10-2293081" There is a moisturizing cosmetic composition for skin improvement used" (hereinafter referred to as 'prior art').

However, in the case of a cosmetic composition using an existing natural plant, including the prior art, because it concentrates on one natural plant and relies on a single active ingredient, the effects other than a specific cosmetic effect are only insignificant, and various effects are not provided at once. There was a problem that I couldn't.

In addition, in the case of a cosmetic composition using an existing natural plant, including the prior art, even when various natural plants are used in combination, the balanced effect cannot be improved through the mutual sum of the plants, and the function is inhibited due to the collision between specific plant-derived components. There was a problem in that the effects of whitening, wrinkle improvement, antioxidant, anti-inflammatory, etc. provided through this were only at a negligible level, as induced or simply mixed with natural plants after post-treatment according to the practice.

The present invention was created to solve the above problems, and an object of the present invention is to improve skin regeneration, anti-inflammatory and antioxidant effects by going through a fermentation process using natural plants and lactic acid bacteria such as peony, camphor leaf, vitamin leaf, and Centella asiatica. It is to provide a cosmetic composition that can increase the customer's satisfaction with the use of a single level.

In order to achieve the above object, the method for preparing a cosmetic composition having skin regeneration, anti-inflammatory and antioxidant effects by including a fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica as an active ingredient of the present invention is, Step A of preparing a mixture by mixing peony stems, vitamin leaves, camphor leaves and centella; B step of adding the mixture prepared through step A to the Broth-type medium and then sterilizing for 15 minutes in a temperature of 120 degrees to 122 degrees and 1.5 atmospheres; After inoculating the pre-cultured lactic acid bacteria into the medium to which the sterilized mixture was added through step B, using a shaking incubator in a 37 degree temperature environment at a shaking agitation speed of 50 rpm to 250 rpm For 5 days through the main culture Step C to proceed with the fermentation; D step of sterilizing the medium fermented through step C for 15 minutes in an environment of 120 degrees Celsius and 1.5 atmospheres; And step E of recovering the fermented extract supernatant by centrifuging the medium sterilized through step D for 30 minutes at a rotation speed of 8000 rpm using a high-speed centrifuge in a temperature environment of 4 degrees Celsius.

Here, the A step, A-1 step of drying the mulberry bark, peony flower, peony leaf, peony stem, vitamin leaf, camphor leaf and centella; A-2 step of pulverizing the dried mulberry bark, peony flower, peony leaf and peony stem through the A-1 step; and A-3 step of preparing a mixture by mixing the dried and pulverized mulberry bark, peony flower, peony leaf and peony stem through the A-2 step and the vitamin leaf, camphor leaf and centella asiatica dried through the A-1 step; includes

In addition, in step A-3, 3 parts by weight of the dried and pulverized wood dandelion skin through step A-2, 1 part by weight of peony flower, 1 part by weight of peony leaf and 1 part by weight of peony stem, and 1 part by weight of peony stem dried through step A-1 This is a step of preparing a mixture by mixing 1 part by weight of vitamin leaves, 1 part by weight of camphor leaf, and 1 part by weight of Centella asiatica.

In addition, the step B is a step of adding 10 parts by weight of the mixture prepared through the step A to 1000 parts by volume of Lactobacilli MRS Broth medium, and then sterilizing it for 15 minutes in an environment of 121 degrees Celsius and 1.5 atmospheres, and the C step is Lactobacillus In this step, the lactic acid bacteria obtained by inoculating the lactic acid bacteria of casei KCTC 3109 into 400 parts by volume of Lactobacilli MRS Broth medium to which 0.5% glucose is added and pre-culturing for 12 hours in a temperature environment of 37 degrees are used for the main culture.

In addition, in step C, 50 parts by volume of the lactic acid bacteria obtained through pre-culture in the medium to which the sterilized mixture was added through step B was ^{inoculated based on the bacterial inoculation amount of 1×10 7} CFU/ml, and then the main culture was performed. It is an on-going step.

On the other hand, in order to achieve the above object, as another example, the present invention includes a fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica prepared by the method described above as an active ingredient, and has skin regeneration, anti-inflammatory and antioxidant effects. composition.

According to the present invention, there are the following effects.

First, it is possible to provide skin regeneration and whitening effects by including fermented extracts using peonies, camphor leaves, vitamin leaves and Centella asiatica as active ingredients.

Second, it is possible to provide anti-inflammatory and antioxidant effects by including a fermented extract using peony, camphor leaf, vitamin leaf and centella asiatica as an active ingredient.

Third, as an active ingredient, a fermented extract prepared through post-treatment and additional fermentation process using lactic acid bacteria, rather than simply mixing peony, camphor leaf, vitamin leaf and centella asiatica, is used as an active ingredient, providing effects such as skin regeneration, whitening, anti-inflammatory and antioxidant The level can be further advanced.

1 is a flowchart of a method for manufacturing a cosmetic composition having skin regenerating, anti-inflammatory and antioxidant effects by including a fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica as an active ingredient of the present invention.

A preferred embodiment of the present invention will be described in more detail with reference to the accompanying drawings, but already known technical parts will be omitted or compressed for the sake of brevity of description.

1. Description of the manufacturing method of the cosmetic composition

A method for preparing a cosmetic composition having skin regeneration, anti-inflammatory and antioxidant effects including fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica as an active ingredient of the present invention will be described in detail below with reference to FIG. 1 .

(1) Natural plant mixing step (Step A, S110)

In this step, the process of preparing a mixture by mixing mulberry bark, peony flower, peony leaf, peony stem, vitamin leaf, camphor leaf and centella asiatica is carried out.

Specifically, the natural plant mixing step (S110) proceeds in detailed steps of a drying step (A-1 step), a grinding step (A-2 step) and a mixing step (A-3).

First, in the drying step (step A-1), the process of drying the mulberry bark, peony flower, peony leaf, peony stem, vitamin leaf, camphor leaf and centella asiatica in a cool shade is carried out.

Here, the peony (Paeonia suffruticosa)-derived ingredient is mulberry skin, peony flower, and peony leaf peony stem, respectively, and is used separately.

In addition, as a component derived from peony (Paeonia suffruticosa), the peony skin, peony flower, and peony leaf peony stem are dried (step A-1) and then finely pulverized using a grinder (step A-2).

Next, the vitamin leaf means the leaf part of the vitamin tree (Hippophae rhamnoides L.), also called the buckthorn tree, and the camphor leaf means the leaf part of the camphor tree (Cinnamomum camphora (L.) J.Presl).

Next, Centella asiatica is a perennial plant of the Asteraceae family, which grows in a somewhat damp place, grows laterally, takes root at the node, and has scaly leaves.

Finally, through the drying step (Step A-1) and the pulverization step (Step A-2), the dried and pulverized bark, peony flower, peony leaf and peony stem and the vitamin leaf, camphor tree through the drying step (Step A-1) A mixture is prepared by mixing the leaves and Centella asiatica.

Specifically, the mixing ratio of mulberry skin, peony flower, peony leaf, peony stem, vitamin leaf, camphor leaf and centella asiatica has a weight ratio of 3:1:1:1:1:1:1, and the mixture is dried and pulverized mulberry 3 It is prepared by mixing 1 part by weight, 1 part by weight of peony flower, 1 part by weight of peony leaf, 1 part by weight of peony stem and 1 part by weight of dried vitamin leaves, 1 part by weight of camphor leaf and 1 part by weight of Centella asiatica.

(2) Mixture sterilization step (B step, S120)

In this step, after adding the mixture prepared through the natural plant mixing step (S110) performed above to the Broth-type medium, sterilization is performed for 15 minutes at a temperature of 120 degrees to 122 degrees and 1.5 atmospheres in an environment.

Here, in the mixture sterilization step (S120), 10 parts by weight of the mixture prepared through the natural plant mixing step (S110) was added to 1000 parts by volume of Difco's Lactobacilli MRS Broth medium, and then sterilized for 15 minutes in an environment of 121 degrees Celsius and 1.5 atmospheres. do.

Most preferably, 10 g of the mixture prepared through the natural plant mixing step (S110) is added to 1 L of Lactobacilli MRS Broth medium, and then sterilization is performed for 15 minutes at a temperature of 21 degrees and an environment of 1.5 atmospheres.

(3) Lactic acid bacteria inoculation and fermentation step (C step, S130)

In this step, the pre-cultured lactic acid bacteria are inoculated into the medium to which the sterilized mixture has been added through the mixture sterilization step (S120) performed above, and then shaken at 50 rpm to 250 rpm using a shaking incubator in a 37 degree temperature environment. Fermentation proceeds through the main culture for 5 days at agitation speed.

Here, the inoculated lactic acid bacteria are prepared in advance through pre-cultivation, and the lactic acid bacteria of Lactobacillus casei KCTC 3109 distributed from the Korea Institute of Biotechnology and Biotechnology Biological Resources Center are mixed with Difco's Lactobacilli MRS Broth medium 400 parts by volume ( Most preferably, the lactic acid bacteria obtained by inoculating 400 mL) and pre-culturing in a temperature environment of 37 degrees for 12 hours will be used for the main culture.

In addition, the lactic acid bacteria obtained through pre-culture were inoculated into Difco's Lactobacilli MRS Broth medium by 50 parts by volume (most preferably 50 mL) based on the bacterial ^{inoculation amount of 1×10 7 CFU/ml, followed by 12 in a temperature environment of 37°C.} Continue this incubation for a period of time.

Through the inoculation and fermentation process of these lactic acid bacteria, the production content of aglycone flavonoid and phenol (Paeonol) is increased.

(4) Fermentation broth sterilization step (D step, S140)

In this step, the process of sterilizing the medium fermented through the previously performed lactic acid bacteria inoculation and fermentation step (S130) is sterilized using a high-pressure sterilizer at a temperature of 120 degrees and a pressure of 1.5 atmospheres for 15 minutes.

(5) centrifugation and supernatant recovery step (E step, S150)

In this step, the medium sterilized through the fermentation broth sterilization step (S140) performed above was centrifuged for 30 minutes at a rotation speed of 8000 rpm using a high-speed centrifuge (Beckman Coulter Life Science, USA) at a temperature of 4 degrees. The process proceeds.

Here, as a result of centrifugation, lactic acid bacteria are precipitated, and the supernatant containing the ferment extract is recovered and used as an active ingredient in a cosmetic composition.

That is, as described above, the cosmetic composition of the present invention having the composition, content level for each composition and step-by-step process conditions as described above contains fermented extract using peony, camphor leaf, vitamin leaf, and Centella asiatica as an active ingredient as an active ingredient for skin regeneration, It can highly provide anti-inflammatory and antioxidant effects.

2. Description of the physical property test results of the cosmetic composition

For a cosmetic composition comprising a fermented extract using peony, camphor leaf, vitamin leaf, and Centella asiatica as an active ingredient according to the present invention, antioxidant, whitening, free radical scavenging power, collagen synthesis amount, elasto The level of physical properties related to the enzyme inhibition rate and the tyrosinase inhibition rate were measured, and the following experimental methods were used for the purpose of defining the properties by means that are obvious to those skilled in the art.

First, the following Examples and Comparative Examples were prepared for comparison of physical properties according to the composition and content level of a cosmetic composition comprising a fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica as an active ingredient.

(1) Preparation of Examples and Comparative Examples

- Example 1

After adding 10 g of a mixture of 3 g dried and pulverized mulberry bark, 1 g peony flower, 1 g peony leaf, 1 g peony stem and 1 g dried vitamin leaf, 1 g camphor leaf, and 1 g centella asiatica to 1 L Lactobacilli MRS Broth medium, the temperature of 21 degrees and 1.5 After sterilization for 15 minutes in an atmospheric pressure environment, the lactic acid bacteria of Lactobacillus casei KCTC 3109 distributed from the Korea Research Institute of Bioscience and Biotechnology's Bioresource Center were inoculated into 400 mL of Difco's Lactobacilli MRS Broth medium containing 0.5% glucose and inoculated at a temperature of 37 degrees. The lactic acid bacteria obtained by pre-culturing in the environment for 12 hours ^{are inoculated into Difco's Lactobacilli MRS Broth medium as much as 50 mL based on an inoculation amount of 1×10 7} CFU/ml, and then the main culture is carried out at a temperature of 37 degrees for 12 hours. .

After that, the medium in which this fermentation was performed was sterilized using a high-pressure sterilizer at a temperature of 120 degrees and a pressure of 1.5 atmospheres for 15 minutes, and then using a high-speed centrifuge (Beckman Coulter Life Science, USA) at a temperature of 4 degrees at a rotation speed of 8000 rpm for 30 minutes. The supernatant recovered by centrifugation is prepared in Example 1.

- Comparative Example 1

In contrast, 6 g of a mixture of 3 g dried and pulverized mulberry bark, 1 g peony flower, 1 g peony leaf, and 1 g peony stem was added to 1 L of Lactobacilli MRS Broth medium and sterilized for 15 minutes at a temperature of 21 degrees and an environment of 1.5 atm. Then, the lactic acid bacteria of Lactobacillus casei KCTC 3109 sold from the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center were inoculated into 400 mL of Difco's Lactobacilli MRS Broth medium with 0.5% glucose added, and pre-cultured at 37°C for 12 hours to obtain. Based on the 1×10 ⁷ CFU/ml of 1×10 7 CFU/ml, 50 mL of Difco's Lactobacilli MRS Broth medium is inoculated, and then the main culture is performed at 37°C for 12 hours.

After that, the medium in which this fermentation was performed was sterilized using a high-pressure sterilizer at a temperature of 120 degrees and a pressure of 1.5 atmospheres for 15 minutes, and then using a high-speed centrifuge (Beckman Coulter Life Science, USA) at a temperature of 4 degrees at a rotation speed of 8000 rpm for 30 minutes. A supernatant recovered by centrifugation was prepared as Comparative Example 1.

- Comparative Example 2

In contrast, 1 g of dried vitamin leaves were added to 1 L of Lactobacilli MRS Broth medium and sterilized for 15 minutes at a temperature of 21 degrees and 1.5 atm. Lactobacilli obtained by inoculating lactic acid bacteria in 400 mL of Difco's Lactobacilli MRS Broth medium with 0.5% glucose added and pre-culturing at 37 degrees for 12 hours at a temperature of 1×10 ⁷ CFU/ml based on a bacterial inoculation amount of 50 mL After inoculating it in Difco's Lactobacilli MRS Broth medium, the main culture is carried out for 12 hours at a temperature of 37°C.

After that, the medium in which this fermentation was performed was sterilized using a high-pressure sterilizer at a temperature of 120 degrees and a pressure of 1.5 atmospheres for 15 minutes, and then using a high-speed centrifuge (Beckman Coulter Life Science, USA) at a temperature of 4 degrees at a rotation speed of 8000 rpm for 30 minutes. A supernatant recovered by centrifugation was prepared as Comparative Example 2.

- Comparative Example 3

In contrast, after adding 1 g of dried camphor leaves to 1 L of Lactobacilli MRS Broth medium, sterilization was carried out for 15 minutes at a temperature of 21 degrees and 1.5 atm. Lactobacilli obtained by inoculating lactic acid bacteria in 400 mL of Difco's Lactobacilli MRS Broth medium with 0.5% glucose added and pre-culturing at 37 degrees for 12 hours at a temperature of 1×10 ⁷ CFU/ml based on a bacterial inoculation amount of 50 mL After inoculating it in Difco's Lactobacilli MRS Broth medium, the main culture is carried out for 12 hours at a temperature of 37°C.

After that, the medium in which this fermentation was performed was sterilized using a high-pressure sterilizer at a temperature of 120 degrees and a pressure of 1.5 atmospheres for 15 minutes, and then using a high-speed centrifuge (Beckman Coulter Life Science, USA) at a temperature of 4 degrees at a rotation speed of 8000 rpm for 30 minutes. A supernatant recovered by centrifugation was prepared as Comparative Example 3.

- Comparative Example 4

In contrast, 1 g of dried Centella asiatica was added to 1 L of Lactobacilli MRS Broth medium and sterilized for 15 minutes at a temperature of 21 degrees and 1.5 atm. 0.5% glucose (glucose) was added a by Difco Inc. Lactobacilli MRS Broth medium was inoculated in 400mL at 37-degree temperature environment for 12 hours before incubation with the obtained lactic acid bacteria on the basis of 1 × 10 ⁷ CFU / ml of bacteria inoculum size 50mL After inoculation in Difco's Lactobacilli MRS Broth medium, the main culture is carried out for 12 hours at a temperature of 37°C.

After that, the medium in which this fermentation was performed was sterilized using a high-pressure sterilizer at a temperature of 120 degrees and a pressure of 1.5 atmospheres for 15 minutes, and then using a high-speed centrifuge (Beckman Coulter Life Science, USA) at a temperature of 4 degrees at a rotation speed of 8000 rpm for 30 minutes. A supernatant recovered by centrifugation was prepared as Comparative Example 4.

(2) Antioxidant effect test

In Example 1 and Comparative Examples 1 to 4, the antioxidant effect was measured through a thiobutylate acetic acid (TBA) test method using a lipid peroxidation reaction system by Fenton reaction in order to examine the antioxidant effect of each.

First, in this experiment, each of Examples 1 and Comparative Examples 1 to Comparative Examples was added to 5 ml of 25 mM Tris buffer/0.27 mM potassium buffer containing 10 ml of ethyl linoleate, 0.2% dodecyl sulfate, 10 μmol of ferric chloride, and 2 μmol of hydrogen peroxide. 4 Add 0.1 ml of dodecylmethyl sulfate dissolved in 0.05 g.

Next, after 16 hours of incubation in a temperature environment of 55 degrees, 50 μl of 4% butyl hydroxyl toluene was added, and the absorbance was measured at 535 nm after centrifugation.

At this time, the antioxidant effect (%) was obtained by using a single peony extract as a control, not in Example 1 and Comparative Examples 1 to 4, and the results are shown in Table 1 below.

For reference, antioxidant effect (%) = [1- (Example 1, Comparative Examples 1 to 4 absorbance each / absorbance of the control group)] X 100 corresponds to.

Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Antioxidative effect(%) 96 81 23.5 22.1 28.9

As shown in Table 1, it can be confirmed that Example 1 shows a clear antioxidant effect compared to Comparative Examples 1 to 4.

(3) Whitening effect test

Using Example 1 and Comparative Examples 1 to 4, the tyrosinase inhibition rate was measured to examine the respective whitening effects.

Specifically, in this experiment, L-Tyrosine (L-Tyrosine, 0.3 mg/mL) in 1.0 mL of potassium phosphate buffer solution (Potassium phosphate buffer solution, pH 6.8, 0.1M) and 1.0 mL of the same concentration as Example 1, Comparative Example 1 to Comparative Example 4 0.9 mL was added at room temperature and maintained for 10 to 20 minutes in a temperature environment of 37°C.

After that, 0.1 mL of tyrosinase (Tyrosinase, 1250 units/mL) was added and maintained for 10 minutes.

Here, the control group corresponds to an extract prepared by hot water extraction of Mokdanpi, peony flower, peony leaf, peony stem, vitamin leaf, camphor leaf and centella asiatica, and the tyrosinase inhibition rate measured for Example 1 and Comparative Examples 1 to 4 is shown in Table 2 below.

For reference, tyrosinase (Tyrosinase) inhibition rate = [1 - (Example 1, Comparative Examples 1 to 4, each enzyme activity / control group enzyme activity)] corresponds to X 100.

Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Tyrosinase inhibition rate (%) 96.2 59.6 23.3 28.2 21.4

As such, it can be confirmed that Example 1 can provide a whitening effect by showing a certain tyrosinase inhibition rate compared to Comparative Examples 1 to 4.

(4) Free radical scavenging power test

Using Example 1 and Comparative Examples 1 to 3, each free radical scavenging power was measured.

Specifically, in this experiment, each of Example 1 and Comparative Examples 1 to 3 was reacted in 2 mL of a 60 μM difenylpicryl hydrazyl (DPPH) solution at the same concentration at room temperature for 10 minutes, and then absorbance was measured at 520 nm to free The radical scavenging power was obtained, and the results are shown in Table 3 below.

Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Free radical scavenging rate (%) 95.9 68.6 20.8 19.6

As such, it can be confirmed that Example 1 shows a clear free radical scavenging power compared to Comparative Examples 1 to 3 and can provide an excellent antioxidant effect.

For reference, free radical scavenging rate (%) = [1- (Example 1, Comparative Examples 1 to 4, respectively, the absorbance of the DPPH solution / absorbance of the DPPH solution itself)] X 100.

(5) Collagen synthesis amount test

Each collagen synthesis amount was measured using Example 1 and Comparative Examples 1 to 5.

Specifically, in order to measure the effect of Example 1 and Comparative Examples 1 to 4 on the amount of collagen synthesis in fibroblasts (CCD-986sk) HDFs cells, fibroblasts with 1,000 μg/ml group were placed in a 48-well plate well. The cells were divided into 5 × 104 cells and cultured for 24 hours under cell culture conditions.

Next, the medium was discarded, washed with 10% phosphate buffered saline (PBS), and the sample solution and a new medium were added and cultured for 24 hours. The culture medium was taken and the change in the amount of collagen synthesis was measured by ELISA (Enzyme-Linked ImmunoSorbent Assay).

Here, in Comparative Example 5, in order to measure the effect of retinol on the amount of collagen synthesis in fibroblasts (CCD-986sk) HDFs cells, fibroblasts were aliquoted in a 48-well plate with a group of 1.0 μg/ml and 5 × 104 cells per well. The cells were cultured for 24 hours under the following cell culture conditions.

Next, the medium was discarded, washed with 10% phosphate buffered saline (PBS), and the sample solution and a new medium were added and cultured for 24 hours. The culture medium was taken and the change in the amount of collagen synthesis was measured by ELISA (Enzyme-Linked ImmunoSorbent Assay).

As a result, the numerical values of the amount of collagen synthesis using each of Example 1 and Comparative Examples 1 to 5 are shown in Table 4 below.

Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Collagen synthesis amount (㎍/㎖) 71.6 21.1 19.2 18.2 19.3 99.2

As such, it can be confirmed that Example 1 exhibits a higher collagen synthesis amount compared to Comparative Examples 1 to 4 and has a collagen synthesis level close to that of Comparative Example 5.

(6) Elastase activity test

Using Example 1 and Comparative Examples 1 to 5, the elastase inhibition rate was measured to compare the level of skin wrinkle improvement and skin regeneration effect.

Specifically, to measure the effect of Example 1 and Comparative Examples 1 to 4 on elastase on fibroblast (CCD-986sk) HDFs cells, the elastase solution was quantified by the Lowery method with 1,000 μg/ml group. So, it was put into 96 wells containing 100 ug of protein per well, and 0.2 M Tris-HCl (pH 8.0) was added to 88 μL. 10 μL of the test solution was added to each well. 2 μL of STANA (N-succinyl-tri-alanyl-p-nitroanilide, 50 mM) solution, which is a substrate for elastase, was added to each well and incubated at 37°C.

Then, after 90 minutes, the inhibitory effect on the amount of elastase synthesis measured with a 405 nm ELISA Reader was measured.

In Comparative Example 5, in order to measure the effect on the amount of collagen synthesis in fibroblast (CCD-986sk) HDFs cells as retinol, the elastase solution was quantified by the Lowery method with a 1.0 μg/ml group, and 100 μg per well Put in 96 well containing protein of 0.2 M Tris-HCl (pH 8.0) to 88 μL. 10 μL of the test solution was added to each well. 2 μL of STANA (N-succinyl-tri-alanyl-p-nitroanilide, 50 mM) solution, which is a substrate for elastase, was added to each well and incubated at 37°C.

Then, after 90 minutes, the inhibitory effect on the amount of elastase synthesis measured with a 405 nm ELISA Reader was measured.

As a result, the numerical values of the elastase inhibition rate using each of Example 1 and Comparative Examples 1 to 5 are shown in Table 5 below.

Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 elastase
Inhibition rate (%) 60.1 9.1 6.2 8.2 8.5 69.3

As such, it can be confirmed that Example 1 shows a higher elastase inhibition rate compared to Comparative Examples 1 to 4 and has an elastase inhibition level close to that of Comparative Example 5.

In summary, the cosmetic composition comprising, as an active ingredient, a fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica prepared as in Example 1 can provide excellent skin regeneration, anti-inflammatory and antioxidant effects.

The embodiments disclosed in the present invention are for explanation rather than limiting the technical spirit of the present invention, and the scope of the technical spirit of the present invention is not limited by these embodiments. The scope of protection should be construed by the claims below, and all technical ideas within the equivalent range should be construed as being included in the scope of the present invention.

Claims (6)

Step A of preparing a mixture by mixing mulberry bark, peony flower, peony leaf, peony stem, vitamin leaf, camphor leaf and centella asiatica;
B step of adding the mixture prepared through step A to the Broth-type medium and then sterilizing it for 15 minutes in a temperature of 120 degrees to 122 degrees and 1.5 atmospheres;
After inoculating the pre-cultured lactic acid bacteria in the medium to which the sterilized mixture has been added through step B, using a shaking incubator at a temperature of 37 degrees at a shaking stirring speed of 50 rpm to 250 rpm For 5 days through the main culture Step C to proceed with the fermentation;
D step of sterilizing the medium fermented through step C for 15 minutes in an environment of 120 degrees Celsius and 1.5 atmospheres; and
Step E of recovering the fermented extract supernatant by centrifuging the medium sterilized through the step D for 30 minutes at a rotation speed of 8000 rpm using a high-speed centrifuge in a temperature environment of 4 degrees C.
In step A,
A-1 step of drying mulberry skin, peony flower, peony leaf, peony stem, vitamin leaf, camphor leaf and centella;
A-2 step of pulverizing the dried mulberry bark, peony flower, peony leaf and peony stem through the A-1 step; and
Step A-3 of preparing a mixture by mixing the dried and pulverized wood dandelion skin, peony flower, peony leaf and peony stem through step A-2, and vitamin leaf, camphor leaf and centella asiatica dried through step A-1; includes,
The step A-3 comprises 3 parts by weight of the dried and pulverized mulberry bark, 1 part by weight of peony flower, 1 part by weight of peony leaves, and 1 part by weight of peony stems dried and pulverized through step A-2, and vitamin leaves dried through step A-1 1 part by weight, 1 part by weight of camphor leaf, and 1 part by weight of Centella asiatica to prepare a mixture,
Step B is a step of adding 10 parts by weight of the mixture prepared in step A to 1000 parts by volume of Lactobacilli MRS Broth medium, and then sterilizing for 15 minutes in an environment of 121 degrees Celsius and 1.5 atmospheres,
The step C is a step of inoculating lactic acid bacteria of Lactobacillus casei KCTC 3109 into 400 parts by volume of Lactobacilli MRS Broth medium containing 0.5% glucose and pre-culturing for 12 hours in a temperature environment of 37 ° C. Using the obtained lactic acid bacteria for the main culture characterized by
A method for manufacturing a cosmetic composition having skin regeneration, anti-inflammatory and antioxidant effects by including a fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica as an active ingredient.
delete delete delete The method of claim 1,
In step C, 50 parts by volume of the lactic acid bacteria obtained through pre-culture in the medium to which the sterilized mixture has been added through step B ^{is inoculated based on the bacterial inoculation amount of 1×10 7} CFU/ml, and then the main culture is performed. characterized in that
A method for manufacturing a cosmetic composition having skin regeneration, anti-inflammatory and antioxidant effects by including a fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica as an active ingredient.
A cosmetic composition prepared by a method of manufacturing a cosmetic composition having skin regeneration, anti-inflammatory and antioxidant effects, including a fermented extract using peony, camphor leaf, vitamin leaf and Centella asiatica as an active ingredient according to any one of claims 1 to 5 A cosmetic composition, characterized in that

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