KR102843808B1 - The cosmetic composition containing Vicia faba-derived PDRN - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising PDRN derived from faba bean ( Vicia faba ) as an active ingredient. The PDRN derived from faba bean according to the present invention has excellent effects of preventing pigmentation, whitening, improving wrinkles, improving skin elasticity, and anti-inflammation, and thus can be usefully used as a cosmetic composition.

Description

Cosmetic composition containing Vicia faba-derived PDRN as an active ingredient {The cosmetic composition containing Vicia faba-derived PDRN}

The present invention relates to a cosmetic composition comprising PDRN derived from faba bean ( Vicia faba ) as an active ingredient. The PDRN derived from faba bean according to the present invention has excellent effects of preventing pigmentation, whitening, improving wrinkles, improving skin elasticity, and anti-inflammation, and thus can be usefully used as a cosmetic composition.

PDRN (Polydeoxyribonucleotide) is a tissue regeneration-activating substance that promotes the self-regeneration of damaged tissues and cells. It is a polymer manufactured from DNA with a molecular weight ranging from 50 to 1,500 kDa. PDRN possesses diverse efficacies, including tissue repair, anti-ischemia, and anti-inflammation, suggesting its potential applications in regenerative medicine and the treatment of diabetic foot ulcers.

PDRN is typically extracted from the semen and testes of large fish, such as salmon, and then purified in large quantities. However, several issues arise, including the risk of mass fish mortality, the potential for infection, and limited extraction times. Consequently, interest is focused on securing and utilizing plant-based PDRN as an alternative.

Plant-derived PDRN, as used herein, refers to a polynucleotide material extracted from plants instead of animal-derived PDRN. Conventional PDRN is primarily manufactured by extracting DNA from the semen or testes of large fish, such as salmon. Plant-derived PDRN is manufactured based on plant-derived DNA. Plant-derived PDRN can be mass-produced and harvested year-round, making it an effective alternative to fish-derived PDRN, as it addresses the limitations of seasonality, mass mortality, and the potential for infection.

In particular, plant-derived PDRN minimizes the risk of viral infections, including zoonotic infections, because it is free from the risk of contamination by viruses derived from animal tissues. For these reasons, plant PDRN is attracting attention as an alternative agent in regenerative medicine and related medical fields, due to its tissue regeneration, anti-inflammatory, and anti-ischemic properties.

The beauty industry has recently been garnering attention for its skin regeneration properties, with new products utilizing PDRN being released one after another. PDRN, an ingredient that improves skin condition by promoting fibroblast regeneration and metabolic activity, is particularly popular in skin booster products. Skin boosters, which inject effective ingredients into the skin to promote skin regeneration, are expected to have a market size exceeding 2 trillion won. Leading companies such as Amorepacific and APR are currently releasing products containing PDRN. Amorepacific's Innisfree launched the "Retinol Green Tea PDRN Skin Booster Ampoule," while AP Beauty launched the "Dual Repair Lift Cream." APR plans to produce its own PDRN-containing ampoules and creams. With increasing demand for PDRN products, the beauty industry anticipates the growth of the skin booster market.

Fava beans ( Vicia faba ), which the inventor of the present invention focused on, are known by various names, including broad beans, sword beans, and mama beans. Rich in protein and dietary fiber, they are a highly nutritious food rich in various vitamins and minerals. They also help alleviate inflammation and lower blood cholesterol.

Native to North Africa and southwestern Asia, it is a legume with strong cold tolerance but is highly susceptible to drought. It requires hot, humid conditions from the time it flowers, so it is primarily produced in China, Ethiopia, and Egypt. China accounts for approximately 70% of global production.

In Korea, it began to be cultivated in Jeju Island and the southern coastal region of Jeollanam-do in the late 1970s, and has gradually become known since 1994 through contract cultivation with Japan in the Namhae and Sacheon regions.

Broad beans are rich in protein and dietary fiber, low in fat, and are a highly nutritious food containing vitamins A, B1, B2, and C, as well as various minerals. They are also a good source of vitamin B1, thiamine, iron, copper, phosphorus, calcium, and magnesium. Vitamin B1, found in broad beans, plays a crucial role in nervous system function and energy metabolism. Iron and copper help form red blood cells, while phosphorus and magnesium help maintain strong bones. Broad beans also help alleviate inflammation, such as hemorrhoids, sinusitis, otitis media, and gastritis, and have the effect of lowering blood cholesterol. In oriental medicine, they are also used medicinally, with mature seeds and leaves used as a laxative, diuretic, and detoxifier.

1. Patent No. 10-2707680 (Method for producing high-purity plant-derived PDRN with excellent preservation stability and composition for anti-inflammation, skin regeneration, and wound healing containing the same as an effective ingredient) 2. Patent No. 10-2237543 (Method for producing DNA derived from Aloe plants, and anti-aging and anti-inflammatory composition containing DNA derived from Aloe plants as an active ingredient)

The present invention was conceived under the above background, and aims to experimentally prove the possibility of using PDRN as a cosmetic composition by producing PDRN derived from faba bean as a plant rather than an animal tissue and confirming its efficacy.

The present invention is characterized by being a cosmetic composition comprising PDRN derived from faba bean as an active ingredient.

At this time, the cosmetic composition is characterized in that it has at least one use among anti-pigmentation, whitening, wrinkle improvement, skin elasticity improvement, and anti-inflammation.

In addition, the above cosmetic composition is characterized by having the combined uses of anti-pigmentation, whitening, wrinkle improvement, skin elasticity improvement, and anti-inflammation.

PDRN derived from faba bean according to the present invention has excellent effects of preventing pigmentation, whitening, improving wrinkles, improving skin elasticity, and anti-inflammation, and thus can be usefully used as a cosmetic composition.

The present invention is described in detail below with specific examples. However, the following examples are intended to aid understanding of the present invention and are not intended to limit the scope of the present invention.

A. Preparation of samples

1. DNA sample preparation method

(1) Plant tissue preparation

Frozen or dried plants are finely ground using a mortar and pestle and a mixer, then CTAB Extraction Solution and distilled water are added and mixed. The mixture is left at 65°C for 2 hours.

(2) SDS processing

Add 10% of the mixture to a 20% SDS solution and react at 65°C for 1 hour.

(3) Potassium acetate treatment

Add 5M potassium acetate to the reacted mixture, shake well to mix, and precipitate overnight at -20°C.

(4) Centrifugation

Centrifuge the mixture at 3,000 rpm at 4°C for 30 minutes to separate the supernatant.

(5) DNA precipitation

Transfer the supernatant to a new tube, add isopropanol equivalent to 30% of the supernatant volume, and precipitate overnight at -20°C.

(6) Centrifugation and ethanol washing

Centrifuge the solution containing the precipitate at 3,000 rpm at 4°C for 30 minutes to precipitate the DNA.

After removing the supernatant, add 70% ethanol to the sediment, centrifuge at 3,000 rpm for 30 minutes, and repeat this process to wash the sediment.

(7) Ethanol removal and drying

After removing all supernatant, dry the sediment at room temperature for 3 hours to completely remove any residual ethanol.

(8) DNA dissolution

The dried DNA precipitate is dissolved in an appropriate amount of distilled water (DW) to obtain the final dissolved DNA.

2. Extract sample preparation method

① 10g of raw material was mixed with 990g of DW (extraction ratio 10%).

② The extraction mixture was placed in a water bath and extracted at 55℃ for a certain period of time.

③ The extract was filtered and then autoclaved for sterilization.

④ Prepare the sterilized sample by placing it in a sample bottle.

B. In vitro experiments

1. DPPH activity inhibition experiment

(1) Purpose of the experiment

The purpose of this study is to evaluate the DPPH radical scavenging activity of a specific substance to confirm its antioxidant effect.

(2) Preparation of experimental and control groups

Experimental group: 100 ppm solution of a single DNA sample

Control: Reaction solution containing no sample

Positive Control: 500 ppm Ascorbic acid solution

(3) Experimental process

Using a 96-well plate, dispense into each well in the following order.

① Prepare a sample diluted to a certain concentration.

② Prepare DPPH diluted to 0.2 mM (use 99% methanol as the solvent).

③ Prepare a 96-well plate.

④ First, add 100㎕ of the solvent in which the sample was diluted and the sample diluted by concentration to 4 locations.

⑤ Add 100㎕ of 99% methanol to each of the four locations in ④.

⑥ Add 100㎕ of DPPH diluted to 0.2 mM to 3 locations in ④.

⑦ Immediately after adding DPPH, store in a dark place for 30 minutes.

⑧ Measure the absorbance at 517 nm after 30 minutes.

⑨ Calculate by substituting into the following equation.

Sample OD = absorbance of the reaction solution containing the sample

Sample X OD = Absorbance of the reaction solution without sample

2. Nitrite scavenging ability test

(1) Experimental reagents and conditions

- Extract: 1 mL of sample used in the experiment

- NaNO₂ solution: 1 mL

- HCl: 0.1 M solution, pH adjusted to 1.2

- Distilled water (DW): Adjust the total solution volume to 10 mL

- Griess reagent:

- 1% sulfanilic acid

- 1% naphthylamine (mixing ratio 1:1)

- 2% acetic acid

(2) Experimental process

① Mixing and reaction

Add 1 mL of NaNO₂ solution and 0.1 M HCl to 1 mL of the extract to adjust the pH to 1.3, then add distilled water to make the final volume 10 mL.

This solution is reacted at 37°C for 1 hour.

② Griess reagent reaction

Take 1 mL of the reaction solution and add the following reagents in order.

After mixing 5 mL of 2% acetic acid and 0.4 mL of Griess reagent (a mixture of 1% sulfanilicacid and 1% naphthylamine), react at room temperature for 15 minutes.

③ Absorbance measurement

After the reaction is complete, the absorbance is measured at a wavelength of 520 nm.

④ Public exam

The experiment was performed under the same conditions using 0.4 mL of distilled water instead of Griess reagent.

(3) The calculation method is as follows.

(4) Use of experimental results

The nitrate scavenging capacity calculated by this measurement method can be used as an indicator to evaluate the antioxidant properties of a sample and indirectly evaluate the anti-inflammatory effect, given that oxidative stress associated with nitrite is closely linked to inflammatory responses.

3. Tyrosinase activity inhibition experiment

(1) Purpose of the experiment

The purpose of this study was to evaluate the ability of a specific substance to inhibit tyrosinase enzyme activity and to confirm its anti-pigmentation and skin whitening effects.

(2) Preparation of experimental and control groups

Experimental group: 100 ppm solution of a single DNA sample

Control: Reaction solution containing no sample

Positive Control: 500 ppm Ascorbic acid solution

(3) Experimental process

Using a 96-well plate, mix the following into each well:

Four wells are used horizontally per sample, with three sample wells and one blank well.

① 50 μL of test substance or control

② 80 μL of 1X PBS solution

③ 50 μL of 2 mM L-DOPA

④ 20 μL of Tyrosinase enzyme solution (1000 U/mL). The same amount of 1X PBS solution is added to the sample blank well and control blank well.

After reacting for 10 to 15 minutes, measure the absorbance at 490 nm and compare it with the control group to calculate the enzyme activity inhibition rate of the test substance.

(4) Result calculation

Tyrosinase activity inhibition rate (%): Calculate the tyrosinase activity inhibition rate of each sample using the formula below:

4. Collagenase activity inhibition experiment

(1) Purpose of the experiment

The purpose of this study was to evaluate the ability of a specific substance to inhibit collagenase enzyme activity and to confirm its wrinkle improvement and anti-aging effects.

(2) Preparation of samples and controls

Sample: Prepare 100 μL of the sample you want to measure.

(-) Control: Reaction solution without sample

(+) Control: Use of EGCG (Epigallocatechin gallate), a positive control substance that has already been proven to inhibit collagenase activity.

(3) Experimental process

① Experimental composition

Sample (or Control): 100 μL

Substrate Buffer: 250 μL

Collagenase: 150 μL

Total reaction volume: 500 μL

② The reaction process is as follows.

Combine sample or control with substrate and collagenase in an e-tube.

Reaction for 20 minutes at room temperature or 30 minutes at 37°C.

Stop the reaction by adding 500 μL of 6% Citric Acid.

Add 1,500 μL of ethyl acetate and dissolve the crystallized collagen after 10 minutes of reaction.

Take the supernatant and measure the absorbance at 320 nm.

(4) Calculating results

The collagenase inhibitory activity of the sample is quantitatively evaluated using the formula below.

Inhibition rate (%) = (1-absorbance of sample/absorbance of control) x 100

5. Elastase activity inhibition experiment

(1) Purpose of the experiment

The ability of a specific substance to inhibit elastase enzyme activity is evaluated to determine its effect on improving skin elasticity and wrinkles.

(2) Preparation of experimental and control groups

Experimental group: 100 ppm solution of a single DNA sample

Control: Reaction solution containing no sample

Positive Control: 500 ppm Ascorbic acid solution

(3) Experimental process

Using a 96-well plate, mix the following into each well:

Four wells are used horizontally per sample, with three sample wells and one blank well.

① 20 μL of test substance or control

② 140 μL of 0.2 M Tris-HCl solution

③ 20 μL of 1.0mM elastase substrate (N-Succinyl-Ala-Ala-Ala-p-nitroanilide)

①~③ Let the mixture react for 10 minutes.

④ 20 μL of Elastase enzyme solution (1 unit/mL). The same amount of 0.2 M Tris-HCl solution is added to the sample blank well and control blank well.

After reacting for 20 minutes, the absorbance is measured at 405 nm and the enzyme activity inhibition rate of the test substance is calculated by comparing it with the control group.

(4) Result calculation

Elastase activity inhibition rate (%): Calculate the tyrosinase activity inhibition rate for each sample using the formula below:

C. Candidate Screening

1. Results of the electrophoresis test of fababin PDRN

The concentration of PDRN in the electrophoresis test on Agarose Gel for fababin PDRN was 1697.4 ng/㎕, and the purity was confirmed to be high at 1.925 through absorbance measurement at 260A/280A (the purity was judged to be high in the range of 1.8 to 2.1).

2. Primary Screening Using Collagenase Inhibition Assay (12 Types)

In order to select Phyto-PDRN with excellent cosmetic efficacy by evaluating the efficacy of PDRN extracted from various raw materials, collagenase inhibitory efficacy was evaluated for PDRN extracted from 12 plants (licorice, burdock, yellow ttari mushroom, green tea, paper mulberry, Ophiopogon japonicus, centella asiatica, burdock, elderflower, smilax vine, faba bean, and hemp seed). Each PDRN was compared at the same concentration of 100 ppm, and the results of the collagenase inhibition assay performed on the 12 plants were as follows.

Seven species (licorice, green tea, Ophiopogon japonicus, Centella asiatica, elderflower, Siberian chrysanthemum, and faba bean) with a collagenase inhibition rate of 30% or higher were selected for additional efficacy evaluation.

3. In-vitro efficacy evaluation for secondary screening (7 types)

1) DPPH Radical Scavenging Assay (7 types)

To compare the antioxidant efficacy of the 7 species selected in the first round (licorice, green tea, Ophiopogon japonicus, centella asiatica, elderflower, smilax vine, and faba bean), a DPPH radical scavenging activity test was conducted. As a result, licorice PDRN showed the best efficacy at 82.14%, followed by centella asiatica at 64.26% and Ophiopogon japonicus at 51.64%, showing excellent antioxidant efficacy.

2) Elastase Inhibition Assay (7 types)

To compare the skin elasticity improvement efficacy of the 7 species selected in the first round (licorice, green tea, Ophiopogon japonicus, centella asiatica, elderflower, schisandra chinensis, and faba bean), an Elastase Inhibition Assay was conducted. As a result, Ophiopogon japonicus PDRN showed the best efficacy at 50.36%, followed by schisandra chinensis at 42.32% and elderflower at 30.79%, showing excellent Elastase inhibition efficacy.

3) Tyrosinase Inhibition Assay (7 types)

In order to compare the skin whitening and pigmentation improvement efficacy of the 7 species selected in the first round (licorice, green tea, Ophiopogon japonicus, centella asiatica, elderflower, schisandra chinensis, and faba bean), a Tyrosinase Inhibition Assay was performed. As a result, licorice PDRN showed the best efficacy at 45.24%, followed by faba bean PDRN at 31.039%, but the remaining PDRN did not show excellent tyrosinase inhibition efficacy.

4) NO Assay (7 types)

To determine the anti-inflammatory efficacy of the 7 species selected in the first round (licorice, green tea, Ophiopogon japonicus, centella asiatica, elderflower, Smilax chinensis, and faba bean), a nitrite scavenging activity test was conducted. As a result, centella asiatica and elderflower PDRN were confirmed to have excellent anti-inflammatory efficacy of 66.65% and 60.29%, respectively, followed by Smilax chinensis and licorice to show excellent anti-inflammatory efficacy of 54.27% and 52.58%.

Among the seven initially selected species, faba bean showed relatively excellent efficacy in all evaluation items except antioxidant activity, so it was judged to have high value as a cosmetic material. Therefore, the following was conducted to confirm its potential through comparative evaluation with general extracts of the same plant.

D. Comparative evaluation with extracts

The efficacy of a general extract of fava bean and PDRN was compared and evaluated. The PDRN concentration was evaluated at 100 ppm. For example, if the concentration is higher than 1,000 ppm, the manufacturing process and manufacturing costs increase to increase yield and purity. Conversely, if the concentration is too low, it is difficult to expect significant efficacy when used in cosmetics. Therefore, considering manufacturing costs and effective concentration, the experiment was conducted at 100 ppm.

100 ppm is equivalent to 0.01% when converted to a percentage. This is the result of comparing a very small amount compared to the content of a general extract in the experimental results below. As such, it is judged to have technological value because it exhibits relatively superior or similar efficacy even in very small amounts, as described below.

1. Nitrite scavenging activity

Nitrite scavenging activity is the most common and widely used evaluation method to evaluate anti-inflammatory efficacy. Nitrite scavenging activity was tested on 0.1%, 1%, and 3% faba bean extracts and 100 ppm faba bean PDRN. As a result, while the nitrite scavenging activity of the general extract was 4.43%, 24.41%, and 35.86%, respectively, faba bean PDRN showed approximately 40% scavenging efficacy, confirming that the scavenging efficacy was superior to the extract by at least 9.98% to 790.29% within the test concentration range. This confirmed that faba bean DNA has excellent anti-inflammatory efficacy, and has skin soothing effects and effects to protect the skin from skin troubles.

2. Tyrosinase Inhibition Assay

Tyrosinase Inhibition Assay was performed on faba bean extract and faba bean DNA. In the case of the extract, the tyrosinase inhibition rate showed 4.51%, 23.57%, and 27.6% inhibition ability at concentrations of 0.1%, 1%, and 3%, respectively, and showed significant results at concentrations above 1%. Faba bean PDRN at 100 ppm showed an inhibition efficacy of 31.03%, confirming superior efficacy compared to the general extract. Within the test range of the extract, PDRN showed a superior inhibition efficacy ranging from 12.43% to 588.03% depending on the concentration, and based on this, it was confirmed that faba bean DNA can be used as a cosmetic material that prevents pigmentation or inhibits melanin synthesis to impart whitening effects.

3. Collagenase Inhibition Assay

Collagen is the most important component of skin, playing a crucial role in maintaining its structure, hydration, and elasticity. A collagen deficiency makes it difficult for skin to maintain moisture, reduces elasticity, and leads to wrinkles. Therefore, collagen is the most crucial component in anti-aging and wrinkle improvement. Collagenase, an enzyme that breaks down collagen, is a key target ingredient in wrinkle improvement and anti-aging research. When comparing the collagenase inhibitory efficacy of the extract and DNA using faba bean as the material, the collagenase inhibitory efficacy was 6.97%, 20.57%, and 24.58% at extract concentrations of 0.1%, 1%, and 3%, respectively, confirming that significant efficacy was present in extracts of 1% or more. However, in the case of faba bean PDRN, the inhibitory efficacy was 35.03% at a concentration of 100 ppm, confirming that the efficacy was superior to the extract, ranging from 42.51% to 402.58% over the tested concentration range. This shows that using DNA extracted from faba bean rather than extracting it as is can be a better choice for developing products for wrinkle improvement or wrinkle prevention.

4 Elastase Inhibition Assay

Elastin is a protein involved in skin elasticity, and elastase is an enzyme that breaks down elastin. When elastin breaks down, skin elasticity decreases and wrinkles form. Fava bean extract is expected to be rich in protein, which is expected to have an elastase expression inhibition effect. The test results confirmed that 0.1%, 1%, and 3% extracts had an elastase inhibition effect of 7.51%, 23.53%, and 25.36%, respectively. In contrast, 100 ppm fava bean PDRN confirmed an elastase inhibition effect of 31.11%, confirming an inhibition effect of 22.67% to 314.25% superior to the extract. This shows that fava bean DNA has the potential to be a cosmetic that improves elasticity and wrinkles.

Claims (3)

A cosmetic composition comprising PDRN derived from faba bean as an active ingredient. In the first paragraph,
A cosmetic composition characterized in that the use of the cosmetic composition is at least one of anti-pigmentation, whitening, wrinkle improvement, skin elasticity improvement, and anti-inflammation. In the second paragraph,
The cosmetic composition is characterized in that the above cosmetic composition has a combined use of preventing pigmentation, whitening, improving wrinkles, improving skin elasticity, and anti-inflammation.