KR102481945B1 - Cosmetic composition for scalp and hair containing natural complex extract as an active ingredient - Google Patents

Abstract

The present invention relates to a cosmetic composition containing extracts of Indian gooseberry fruit, bramble root, and saw palmetto fruit as active ingredients. The composition containing extracts of the Indian gooseberry fruit, bramble root, and saw palmetto fruit as active ingredients has superior antioxidant activity compared to the respective extracts and increases tyrosinase activity and melanin production. As no cytotoxicity is observed in human hair papilla cells, the cell growth is excellent and hair growth factor protein expression is effective. Thus, the composition is to be provided as a cosmetic composition for preventing hair loss, promoting hair growth, darkening hair, cleaning the scalp, or antioxidant purposes.

Description

Cosmetic composition for scalp and hair containing natural complex extract as an active ingredient {Cosmetic composition for scalp and hair containing natural complex extract as an active ingredient}

The present invention relates to a cosmetic composition for scalp and hair containing a natural complex extract as an active ingredient.

In order for hair to grow well, the role of the papilla is important. However, dermal papillae are active throughout life and do not produce hair continuously. If activity continues to a certain extent, activity temporarily ceases. Hair repeats the growth cycle of anagen, catagen, and telogen. In normal scalp, 90 to 95% is in the anagen phase, less than 1% is in the catagen phase, and 5 to 10% is in the telogen phase. 60 to 80 or more hairs enter the telogen phase every day, and the same number of hair follicles enter the anagen phase again. Therefore, 60 to 80 or more hairs fall out and are newly generated every day, and a symptom in which more hairs are abnormally dropped or not regenerated is called alopecia.

As a treatment method for the alopecia, Minoxidil as a transdermal application agent and Propecia as an oral agent are widely used as the only pharmaceuticals approved by the FDA.

In the case of the minoxidil, it has been reported that it promotes hair growth by delaying the anagen phase of hair, and induces hair growth by increasing nutrient supply and potassium channel opening effect due to vasodilation, but still The mechanism has not been clearly elucidated. Continuous use of minoxidil is required to maintain the effect, and weight gain, edema, angina, dermatitis and pruritus have been reported from clinical cases, and dermatitis accompanied by erythema, itching, epidermal peeling and dryness, and rapid application in case of excessive application. It can cause side effects such as lowering blood pressure.

Propecia has the effect of delaying the progress of hair loss and hair growth by inhibiting the activity of 5α-reductase, but long-term administration for maintaining the effect may cause serious side effects such as male sexual dysfunction and birth defects in females.

These side effects can be seen as having significant limitations in active treatment for hair loss. Therefore, in recent years, alternative medicine that compensates for these disadvantages and uses various natural extracts and herbal medicine materials that are safe for continuous use is attracting attention.

On the other hand, in order to improve gray hair, it is common to use hair dyes mainly, but hair dyes using natural plants have a disadvantage in that the color is limited and the color durability is short. Chemical hair dyes dye hair in various colors and have excellent durability, but they also cause side effects such as scalp irritation and hair damage. In addition, existing hair dyes have a problem in that as the hair grows, the existing white hair is revealed again, requiring repeated dyeing. Therefore, it is necessary to develop a natural material that is safe and less irritating to the scalp, which can go beyond simply dyeing hair black and increase melanin production by stimulating scalp melanocytes to continuously blacken hair.

Republic of Korea Patent Publication No. 10-1780692 (registered on September 15, 2017)

The present invention is a natural complex extract that is safe and less irritating to the scalp by preventing the side effects of existing chemical compositions by using a cosmetic composition for preventing hair loss, promoting hair growth, blackening hair, and antioxidation containing a natural complex extract as an active ingredient. want to provide

The present invention provides a cosmetic composition for preventing hair loss, promoting hair growth, blackening hair or cleaning the scalp, containing extracts of Indian gooseberry fruit, bramble root, and saw palmetto fruit as active ingredients.

In addition, the present invention provides a cosmetic composition for antioxidant containing extracts of Indian gooseberry fruit, bramble root and saw palmetto fruit as active ingredients.

Additionally, the present invention includes the steps of 1) drying, pulverizing and freezing the dry powder raw material; 2) extracting the frozen dry powder raw material; 3) filtering to separate the residue and obtain a filtrate; 4) concentrating the filtrate under rotary reduced pressure; and 5) obtaining a natural mixed raw material by freeze-drying the concentrated extract under reduced pressure.

According to the present invention, a composition containing, as an active ingredient, a complex extract in which extracts of Indian gooseberry fruit, bramble root, and saw palmetto fruit are mixed in a weight ratio of (1 to 5):(1 to 3):1 It has excellent antioxidant performance compared to the extract of tyrosinase, increases the activity of tyrosinase and melanin production, and has no cytotoxicity in human dermal papilla cells (HFDPC), so it has excellent cell growth and shows the expression effect of hair growth factor protein. As it is confirmed, it is intended to provide the composite extract as a cosmetic composition for preventing hair loss, promoting hair growth, blackening hair, cleaning the scalp and antioxidant.

1 is a graph showing the moisturizing test data of the composite extract and comparative control group of the present invention.
Figure 2 is a graph showing the DPPH radical scavenging ability of the composite extract and each extract of the present invention.
Figure 3 is a graph showing the nitrite scavenging ability of the composite extract and each extract of the present invention.
Figure 4 is a graph showing the results of performing a cellular tyrosinase assay of a 5: 1: 1 natural composite extract.
5 is a graph showing the results of calculating the melanin production increasing efficacy of a 5: 1: 1 natural complex extract.
Figure 6 is a graph showing the degree of cell viability by treating human dermal papilla cells with a 5: 1: 1 natural composite extract by concentration and confirming cytotoxicity by MTT assay.
Figure 7 is a graph measuring the effect of a 5: 1: 1 natural composite extract on the expression level of hair growth factors in human dermal papilla cells through western blot.

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

The present inventors extracted active substances from Indian gooseberry fruit, wild rose root, and saw palmetto fruit to test antioxidant capacity, promote the expression of growth factors that play a role in hair growth in hair follicles by testing the cell proliferation effect of human hair papilla cells, and Western blot. was studied to confirm the possibility of developing a natural composite material for preventing hair loss, promoting hair growth, and blackening hair using natural ingredients that can replace drug therapy, thereby completing the present invention.

The present invention can provide a cosmetic composition for preventing hair loss, purifying the scalp, or blackening hair, containing extracts of Indian gooseberry fruit, bramble root, and saw palmetto fruit as active ingredients.

The Indian gooseberry, also known as Amla, is a fruit of a deciduous tree that grows in India, the Middle East and some Southeast Asian countries, and has been used as an important symbol for health since ancient times in Ayurvedic medicine in India, and has been used to prevent various diseases and aging. It has been introduced as a substance that is highly effective for beauty and health, and contains various antioxidant substances such as vitamin C, phenolic compound, tannins, rutin, curcuminoids, and emlicol, which are known to have antioxidant activity or lipid peroxidation inhibitory effects. can

The root of the briar tree (Rosa multiflora Thunberg) belongs to the Rosaceae family and is called stone coral in oriental medicine, and the fruit may be used as a medicine called Youngsil or Saekmija. The root may be used in the private sector for the treatment of diabetes, dysentery, arthritis, hematemesis, enuresis, menstrual irregularity, and bruises because it is effective in clearing heat, dampness, wind, or blood. Ingredients of brier tree include multiflorin A, multiflorin B, Kaempferol 3-O-α-L-rhamnoside, multinoside A, multinoside A acetate, quercitrin, isoquercitrin, quercetin 3-O-xyloside, hyperin, etc. of flavonoids, scoparone, salicyclic acid, and sterol.

In addition, the bramble tree has been used as a very precious medicinal material in the private sector since ancient times, and washing the head with boiled water of branches and stems helps to prevent hair loss and eliminates dandruff, and sterols and triterpenoids components exhibiting anti-inflammatory activity are known, It may have effects such as anti-inflammatory or antioxidant effects related to skin disease improvement and wound healing.

Saw palmetto is a plant native to the Caribbean in the Americas. Native Americans have been using saw palmetto as a food and medicine for centuries, and studies have shown that it can reduce hair loss and promote hair growth when ingested or applied topically. there is. Saw palmetto can inhibit the conversion of the male hormone testosterone to dihydrotestosterone (DHT).

The composition comprises extracts of Indian gooseberry fruit, bramble root and saw palmetto fruit in a weight ratio of (1-5):(1-3):1. Preferably, the weight ratio of extracts of Indian gooseberry fruit, bramble root and saw palmetto fruit is a composite extract having a ratio of 5: 1: 1. The composite extract has a significantly higher moisturizing effect, antioxidant effect (polyphenol content, flavonoid content, DPPH radical scavenging activity, nitrite scavenging activity), hair blackening than the effect of substances consisting of single-ingredient Indian gooseberry fruit, bramble root or saw palmetto fruit. It shows the effect (increased tyrosinase activity, promotion of melanin production) and hair growth factor protein expression (western blot).

The present invention can provide a cosmetic composition for antioxidant containing extracts of Indian gooseberry fruit, bramble root and saw palmetto fruit as active ingredients.

In addition, the present invention comprises: 1) drying, pulverizing and freezing the dry powder raw material (first step) 2) extracting the frozen dry powder raw material (second step); 3) filtering to separate the residue and obtain a filtrate (third step); 4) concentrating the filtrate under reduced pressure by rotation (fourth step); And 5) Freeze-drying the concentrated extract to obtain a natural mixed fuel (step 5) A method for producing a composition containing extracts of Indian gooseberry fruit, bramble root and saw palmetto fruit as active ingredients can provide.

The pulverization of the dried powder raw material may be secondary pulverization to 10 to 40 mesh separately.

Freezing in the first step is for ease of use and unification of raw material supply and extraction methods, and the moisture content of the dry powder raw material may be 1 to 13%.

In order to minimize the loss and destruction of the main components of the base raw material, the second step of extracting the raw material is to select the vacuum low-temperature decompression extraction method among the extraction methods that minimize the transformation and destruction of the components of the vegetable raw material according to many literature reports. It may be that the average value of the extraction yield was shipped through three or more repeated extractions.

The vacuum low-temperature pressure extraction method is a method of extracting using purified water without going through a separate filtration step such as an organic solvent, adjusting the pressure in a vacuum state and lowering the boiling point using a convection phenomenon to lower the natural product at a low temperature of 55 to 65 ° C. It can be a stable extraction method that is easy to extract without transforming or destroying the components of

The filtration is a first filtration process of filtering out impurities from the filtrate using filter paper, and the first filtrate is passed after forming a filtration layer using powdered activated carbon to adsorb contaminants in the filtrate and absorb solvents and plants It may include a secondary filtration process to separate the chlorophyll component within.

The concentration process is a step of concentrating the components of the filtrate in the third step to obtain high-purity components. It may be a step of fractionation.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Preparation of Mixed Extracts of Indian Gooseberry Fruit, Brier Root and Saw Palmetto Fruit

Materials used in this experiment were purchased and used in a dried state from Booyoung Pharmacy in Seoul. The reagents used in the experiment were acetic acid and sodium hydroxide, hydrogen chloride (HCl) was purchased from Duksan (Korea), and sodium chloride (NaCl) was purchased from Daejung (Korea). Tris-HCl was purchased and used from Difco™ (Becton, Dickinson and Company, USA). DPPH, L-ascorbic acid (vitamin C), tannic acid, naringin, sodium nitrite, diethylene glycol, grease reagent, MTT, tryptic soy broth (TSB), yeast extract, malt extract, peptone, dextrose, tryptone, and bacto agar were obtained from Sigma-Aldrich (USA). Purchased and used.

In addition, fetal bovine serum (FBS), antibiotic-antimycotic, and ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco™ (Thermo Fisher Scientific, USA), and human dermal papilla cells (HFDPC) used for cell culture were purchased from PromoCell (Germany). ), and follicle dermal papilla cell growth medium (C-26501; PromoCell), a medium exclusively for human hair papilla cell culture, was used as the cell culture medium. Platelet-derived growth factor B subunit (PDGFB) and HGF, which are primary hair growth factor antibodies, are from Santa Cruz Biotechnology (USA), and KGF, IGF1, VEGF, and epidermal growth factor (EGF) are from PeproTech (USA), and secondary antibodies Phosphorus goat anti-rabbit IgG-horseradish peroxidase (HRP) was purchased and used from Santa Cruz Biotechnology.

After the secondary drying of the provided outpost and dried raw materials as shown in Table 1 below, the moisture content was adjusted to 13% or less, and the powder ground to 40 mesh or less using a grinder (IKA A11 basic) was stored at -20 ° C and tested. used in

division material name English name Substance control number sample form One Indian gooseberry fruit Phyllanthus emblica fruit 21915-M1-S1 dry sample 2 brier root Rosa multiflora Thunberg root 21915-M2-S2 dry sample 3 saw palmetto fruit Serenoa Serrulate fruit 21915-M3-S3 dry sample

Extraction experiments were conducted using samples of secondary grinding of dried samples of Indian gooseberry fruit, bramble root, and saw palmetto fruit, which are the main raw materials of this experiment, with a separate 40 mesh or less.

In the extraction experiment, a vacuum low-temperature decompression extraction method was used, and 10 times of distilled water was added to 100 g of the pulverized sample, and then the boiling point was lowered in a vacuum state, using a convection phenomenon. It was extracted and the average yield is shown in Table 2 below.

Samples Average Yields (%, dry basis) Test 1 Test 2 Test 3 Indian gooseberry fruit 45.94 45.91 46.23 45.68 brier root 51.17 51.20 53.28 49.04 saw palmetto fruit 39.48 38.56 40.01 39.88

In order to find the optimal active ingredient for the samples, the mixing ratio of each extract was checked and the moisturizing effect of the mixed extract was tested to find the optimal weight ratio condition. Moisturizing rate is a basic characteristic of healthy hair, and it is an experiment to confirm the appropriate mixing ratio as the most basic measure for improving scalp health and hair growth.

<Experimental Examples 1 to 5> Statistical processing

The analysis of all samples was repeated three times, and the experimental results were average ± standard deviation ( M±SD), statistical analysis was analyzed by Student's t-test, and p<0.05 value was interpreted as significant.

<Experimental Example 1> Experiment to confirm the moisturizing effect of the mixed extract

In order to confirm the possibility as a functional cosmetic for preventing hair loss and improving hair growth, and to confirm the optimal mixing ratio (weight ratio) of the three extracts, an experiment on the moisturizing effect of raw materials was conducted. The weight ratio for appropriate mixing of Indian gooseberry fruit extract, wild rose root extract, and saw palmetto fruit extract was based on a weight ratio of 1 to 5: 1 to 5: 1 to 5, respectively.

Using completely dried CaCl ₂ as a desiccator, put it in a desiccator, put 4 g each of the extracts mixed by each ratio on a dish, and put each sample in a desiccator at 50% relative humidity and 35 ℃ for 12 hours. The weight of was measured three times and is shown in Table 3.

Complex extract weight ratio Moisturizing effect by hour 0 hours 6 hours 12 hours 1:1:1 100% 72% 31% 1:5:3 100% 71% 30% 1:3:5 100% 72% 28% 3:1:5 100% 76% 33% 3:5:1 100% 78% 33% 5:3:1 100% 85% 48% 5:1:1 100% 86% 50%

In Table 3, the moisturizing effect of the complex extract over time was the best when the weight ratio was 5: 1: 1, and in the case of the weight ratio 5: 1: 1, an in vitro test was conducted to confirm the moisturizing power, and the results are shown in Table 4 .

exam identification code test product before use after 6 hours of use After 12 hours of use 1-1 100.000 86.012 49.915 2-1 100.000 86.134 49.942 3-1 99.881 86.989 50.121 1set_average 99.960 86.378 49.993 1-2 100.000 86.001 50.495 2-2 100.000 86.128 49.888 3-2 100.000 86.991 50.101 2set_average 100.000 86.373 50.161 1-3 99.965 86.899 50.055 2-3 99.889 86.201 50.201 3-3 100.000 86.019 49.999 3set_average 99.951 86.373 50.085 overall average 99.971 86.375 50.080 Comparative control (purified water) 99.992 52.326 28.329

Figure 1 is a graph showing the data of the overall average and comparative control group in Table 4, which is the result of the moisturizing power test. Referring to FIG. 1, it was confirmed that the vaporization rate in the air was slower than that of the general aqueous phase series due to the oil phase effect of the extra due to the essential amino acid series component content and pressure. In addition, through the desiccator moisture content test, the effectiveness of the moisturizing component of the raw material steel could be confirmed. Therefore, the mixing ratio of the complex extracts per 100 g of total weight in terms of the weight ratio of the raw materials in the liquid extract is Indian gooseberry fruit extract: Wildflower root extract: Saw Palmetto fruit extract mixed at a ratio of 5: 1: 1 (5: 1: 1 It can be seen that when it is a natural complex extract), the moisturizing effect is confirmed to be the best.

<Experimental Example 2> Antioxidant effect analysis of mixed extracts

2-1. Measurement of content of polyphenol compounds

First, 100 μl of Folin-Dennis reagent (Fluka, Buchs, Switzerland) was added to 100 μl of the prepared sample, mixed, and reacted at room temperature for 3 minutes. After 3 minutes, 100 μl of 10% sodium carbonate solution was added and mixed, and after reacting for 1 hour, the supernatant was taken and the absorbance at 760 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). was measured. The content of phenolic compounds was obtained using a standard curve of 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, and 50 μg/mL gallic acid dissolved in methanol, and it was tabulated. 5.

2-2. Measurement of content of flavonoid and tannin phenolic compounds

The content of flavonoid-based and tannin-based phenolic compounds was measured by the method of Kim et al. (2012), and diethylene glycol ( diethylene glycol) was added and mixed by 1 mL. After mixing, 100 μl of 1 N sodium hydroxide was added, mixed well, and reacted in a 37° C. water bath for 1 hour. After 1 hour, absorbance was measured at 420 nm using a microplate reader. Naringin was used as a standard material, and standard calibration curves were prepared at concentrations of 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, and 50 μg/mL to determine the concentration of flavonoids and tannins. The contents of phenolic compounds were converted and shown in Table 5.

Extracts Condition Sample Total phenol content (mg GAE/%) Total flavonoid and tannin content (mg QE/%) Indian Gooseberry Fruit Extract 43.232±3.329 4.046±0.303 Brier Root Extract 9.448±0.831 0.000±0.000 Saw Palmetto Fruit Extract 14.522±1.597 0.051±0.002 5:1:1 natural complex extract 58.046±3.251 9.365±1.508

Table 5 is an analysis of the content of phenolic compounds in each of the extracts and the 5: 1: 1 natural composite extract. Referring to Table 5, the content of total phenolic compounds of the 5: 1: 1 natural composite extract was 58.046 ± 3.251 mg GAE / g, and the content of total flavonoid and tannin phenolic compounds was 9.365 ± It can be seen that it is 1.508 mg QE / g, indicating a significantly higher value compared to each extraction amount. Therefore, it can be seen that the content of polyphenol, which is excellent in antioxidant effect, is high, and through this, the excellent antioxidant ability of the 5: 1: 1 natural composite extract can be confirmed.

2-3. Measurement of DPPH radical scavenging activity

DPPH radical scavenging ability was measured by the method of Kim et al. (2012). The sample was prepared by dissolving in methanol at a concentration of 1 mg/mL, and the DPPH reagent was prepared by dissolving in methanol to a concentration of 0.1 mM in a light-blocking state. After adding 100 μl of the sample and 0.5 mL of DPPH reagent to a final concentration of 0.2 mg/mL, the mixture was reacted under light-blocking conditions for 20 minutes, and the absorbance was measured at 517 nm using a microplate reader. As a negative control group, methanol was used instead of the sample, and as a positive control group, ascorbic acid at a concentration of 1 mg/mL was added instead of the sample, and the experiment was performed under the same conditions. DPPH radical scavenging ability was calculated by DPPH inhibition rate using Equation 1 below.

[Equation 1]

DPPH radical scavenger activity (DPPH radical scavenging activity, %)

=[1-(absorbance of sample treated group/absorbance of untreated sample group)]X100

The DPPH inhibition rate was calculated using Equation 1 and shown in FIG. 2 . As a result of analyzing the DPPH radical scavenging activity with reference to FIG. 2, the 5: 1: 1 natural composite extract showed the highest DPPH radical scavenging activity of 98.24%. The DPPH radical scavenging activity of each extract was 76.87% for the Indian gooseberry fruit extract, 35.85% for the wild rose root extract, and 55.80% for the saw palmetto fruit extract.

The DPPH radical scavenging ability is a function of providing protons having a reducing function to unstable free radicals to induce them to be stabilized, and can play a role in stabilizing unstable and harmful free radicals generated in vivo. Therefore, it is an indicator used to evaluate the antioxidant ability of an unknown specific substance to remove hydroxyl radicals or superoxide radicals generated by physiological or oxidative actions of the living body. The higher the value, the more antioxidant. performance may be excellent.

2-4. Nitrite scavenging ability measurement

Nitrite scavenging ability was measured by the method of Gray & Dugan (1975). 50 μl of 1 mg/mL sample was added to 50 μl of 1 mM sodium nitrite solution, and 300 μl of 0.1 N hydrochloric acid (HCl) solution (pH 1.2) was added thereto to make the final volume of the reaction solution 0.4 mL. and reacted at 37°C for 1 hour. After the reaction, add 2 mL of 2% acetic acid solution and 0.2 mL of grease reagent, mix well to a final concentration of 0.02 mg/mL, react at room temperature for 15 minutes, and measure the absorbance at 520 nm using a microplate reader to determine the amount of remaining nitrite. quantity was calculated. Distilled water was used instead of the sample as a control group, distilled water was used instead of the grease reagent as a negative control group, and ascorbic acid at a concentration of 1 mg/mL was added instead of the sample as a positive control group, and the experiment was performed under the same conditions. For the nitrite scavenging ability, the inhibition rate was calculated using Equation 2 below, and the results are shown in FIG. 3 .

[Equation 2]

Nitrite scavenging activity (%)

=[1-(absorbance of sample treated group/absorbance of untreated sample group)]X100

Figure 3 is a graph analyzing the nitrite scavenging ability of each extract and natural composite extract. As a result of analyzing the nitrite scavenging ability, the 5: 1: 1 natural composite extract showed the highest nitrite scavenging ability of 75.86%, and the DPPH radical scavenging ability of each extract was 49.86% for Indian gooseberry fruit extract, 18.28% for Brier root extract, and 18.28% for Indian gooseberry fruit extract. It can be seen that the saw palmetto fruit extract is 22.76%.

The nitrite acts as an oxidizing agent and a reducing agent, and is a highly reactive free radical that is converted into L-arginine through the catalysis of an enzyme called nitric oxide synthase in vivo. It is known that silver is reduced to nitrite and reacts with amines to produce nitrosamine, which is converted into diazoalkane (C _n H _2n N ₂ ) in the body. It may cause cancer by alkylating nucleic acids, proteins, or intracellular components, and may itself be toxic. Therefore, a natural product that is safe for the human body capable of suppressing the production of nitrosamine by scavenging nitrite may be used for functional cosmetics or food preservation and as a natural anti-oxidation agent.

<Experimental Example 3> Analysis of hair blackening effect of composite extract

3-1. Measurement of tyrosinase activity increasing efficacy

In order to confirm the activity promoting effect of tyrosinase, a major enzyme involved in melanin production, the degree of activity increase was measured using mushroom tyrosinase (sigma, USA). L-3,4-dihydroxyphenyl alanine (L-DOPA) prepared by dissolving at a concentration of 8.3 mM in 80 mM phosphate buffer (pH 6.8) in a 96-well plate ) 100 μl of the solution was added, and 50 μl of 5: 1: 1 natural composite extract was added to the final 1% and 2%, respectively. Thereafter, 100 μl of mushroom tyrosinase prepared by dissolving 250 U in 80 mM phosphate buffer was added to each well plate. After reacting in a shading incubator at 37 ° C. for 30 minutes, the absorbance was measured at 475 mm using a multimicroplate reader to calculate the enzyme activity increase efficiency as shown in Equation 3 below, and the results are shown in FIG. 4 .

[Equation 3]

Tyrosinase activity increase rate (%) = absorbance of sample added group / absorbance of control group X 100

Figure 4 is a graph showing the results of performing a cellular tyrosinase assay to determine whether the 5: 1: 1 natural composite extract has an effect of promoting melanin production by promoting tyrosinase activity. . Cells treated with natural complex extracts at concentrations of 0, 10, 20, and 50 μg/mL were collected and tyrosinase activity was measured. It was confirmed that an increase rate of 95% was observed when treated with the furnace. Therefore, through the increase in the activity of tyrosinase, a major enzyme in the melanin synthesis process, it can be confirmed that the natural complex extract has excellent hair blackening efficacy during high concentration treatment.

3-2. Measurement of melanin production promoting efficacy

Measurement of melanin content was used by modifying the Yasunobu method. In order to confirm the effect of promoting melanin production, B16 melanoma cells were inoculated into a 24-well plate at a cell concentration of 4 × 10 ⁵ , respectively, and cultured for 24 hours at 37° C. in a 5% CO ₂ incubator. After culturing, the medium was removed, and the 5: 1: 1 natural composite extract of this study was added to the medium at 0.1% and 1%, respectively, and the cells were treated and cultured for 72 hours. At this time, the sample was replaced once daily. To check the melanin production rate, the medium was removed and the cells were washed once with PBS. Thereafter, 500 μl of 1 N sodium hydroxide (NaOH) was added to each well, shaken for 30 minutes to dissolve the generated melanin, and the absorbance was measured at 405 nm using a multi-microplate reader to generate melanin as shown in Equation 4 below The increased efficacy was calculated, and the results are shown in FIG. 5 .

[Equation 4]

Increase rate of melanin production (%) = absorbance of sample added group/absorbance of control group X 100

Figure 5 is a graph measuring the amount of melanin using B16 melanoma cells to determine the effect on melanin synthesis of natural composite extracts. Cells treated with 5: 1: 1 natural complex extract at concentrations of 0, 10, 20, and 50 μg/mL were collected and the amount of melanin was measured. It can be seen that an increase rate of 104% is shown. Therefore, in the melanin production promoting efficacy test, it can be confirmed that the hair blackening effect is excellent through the increase in melanin production of the high-concentration natural composite extract.

<Experimental Example 4> Evaluation of in vitro cytotoxicity of mixed extracts on human dermal papilla cells (HFDPC)

In the cytotoxicity test (MTT assay) on human dermal papilla cells, cell culture, subculture, and preparation of cells for experiments in this experiment were performed according to the test method provided by PromoCell. The survival rate on human dermal papilla cells was measured by applying the method of Mosmann (1983). The test extract, 5: 1: 1 natural composite extract, was treated with a control group, and the same amount of phosphate buffered saline (PBS, Thermo Fisher Scientific) used to dissolve the sample was treated as a control group. The natural complex extract was treated to a final concentration of 10, 100, or 500 μg/mL after 50 μl treatment with respect to 1 mL of the culture medium. After completion of the incubation, 100 μl of MTT solution was added to each well and cultured in a cell incubator for an additional 3 hours to reduce MTT. After completely removing the culture medium, absorbance was measured at 550 nm using a microplate reader.

Figure 6 is a graph showing the degree of cell viability by treating 5: 1: 1 natural composite extract with respect to human dermal papilla cells by concentration and confirming cytotoxicity by MTT assay. Referring to Figure 6, compared to the control group treated with PBS, cell growth of 94.4 ± 3% in the 10 μg / mL treatment, 95.5 ± 5% in the 100 μg / mL treatment, and 113.1 ± 6% in the 500 μg / mL treatment It can be seen that the figure represents Therefore, the 5: 1: 1 natural composite extract did not show significant cytotoxicity to human dermal papilla cells within the concentration range of 10, 100, and 500 μg/mL, and when treated with 500 μg/mL, compared to the control group treated with PBS. It shows a cell growth rate of 113.1%, indicating that it has a high potential for development as a hair restorer material.

<Experimental Example 5> Measurement of hair growth factor protein expression of mixed extracts through Western blot

After subculture of human dermal papilla cells, 5: 1: 1 natural composite extract was treated at a concentration of 100 μg/mL, and 48 hours later, six growth factors (PDGFB, KGF, IGF1, VEGF, HGF) expressed in cells , EGF) proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and measured by Western blotting. After culturing human dermal papilla cells for 48 hours, wash them twice with phosphate buffered saline and lyse the cells using 2% NET buffer (150 mM NaCl; 5 mM EDTA, pH 8; 50 mM Tris-HCl, pH 7.4). And a cell extract was prepared by sonication. Protein quantification of the cell extract was quantified using bovine serum albumin as a standard material simply by Quick Start ^TM Bradford Protein Assay (Bio-Rad Laboratories, USA). Protein quantified samples were loaded with 20 μg of protein per well, electrophoresed on 10% SDS-PAGE at 150 V for 1 hour, and skim milk solution ( A filter was immersed in skim milk solution and incubated for 12 hours. Afterwards, the cells were washed 3 times for 10 minutes with phosphate-buffered saline washing solution and reacted for 1.5 hours using 2,500-fold diluted primary antibodies (PDGFB, KGF, IGF1, VEGF, HGF, EGF), and washed 3 times with washing solution. A method of reacting with goat anti-rabbit IgG-HRP, a secondary antibody diluted 2,500 times for 1 hour, washing three times with a washing solution, and then photosensitizing the film using an ECL kit (RPN 2108; Amersham ^TM , GE Healthcare Life Sciences) was used to investigate protein expression. Band density was confirmed by an electrophoretic image analysis system (gel imaging system, Gel Doc ^TM EZ system 1708270; Bio-Rad Laboratories).

Figure 7 is a Western blot showing the effect on the expression level of the six growth factors, which are hair growth factor proteins, after treating human dermal papilla cells with a 5: 1: 1 natural composite extract at a concentration of 100 μg/mL for 48 hours. It is a graph measured through Regarding the effect of the above growth factors on hair growth, it has been previously revealed that growth factors such as EGF, IGF, FGF, and VEGF are involved in hair growth by acting on a specific site of the hair follicle through various experiments (Tsuboi, 1997).

Referring to Figure 7, when treated with a 5: 1: 1 natural composite extract, compared to the control group using PBS, IGF1 and PDGFB showed expression promoting effects of 135 ± 8% and 126 ± 11%, respectively, and the rest of the growth factors HGF was 106±10%, KGF was 100±6%, VEGF was 99±11%, and EGF was 97±10%. there is.

As above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

A cosmetic composition for preventing hair loss, promoting hair growth, blackening hair or cleaning the scalp, containing a composition containing extracts of Indian gooseberry fruit, bramble root and saw palmetto fruit in a weight ratio of 5:1:1 as an active ingredient. delete The cosmetic composition according to claim 1, wherein the composition has no cytotoxicity to human dermal papilla cells and increases the number of cells. An antioxidant cosmetic composition comprising extracts of Indian gooseberry fruit, bramble root and saw palmetto fruit in a weight ratio of 5:1:1 as an active ingredient. The cosmetic composition according to claim 4, wherein the composition has a high polyphenol content. The cosmetic composition according to claim 4, wherein the composition has high DPPH radical scavenging activity and nitrite scavenging activity. 1) drying, pulverizing and freezing the dry powder raw material (first step);
2) vacuum low-temperature and reduced pressure extraction of the frozen dry powder raw material at 55 to 65 ° C. (second step);
3) filtering to separate the residue and obtain a filtrate (third step);
4) concentrating the filtrate under reduced pressure by rotation (fourth step); and
5) Extracts of Indian gooseberry fruit, bramble root and saw palmetto fruit, including the step of obtaining a natural mixed raw material by freeze-drying the concentrated extract under reduced pressure (step 5), included in a weight ratio of 5: 1: 1 A method for producing a composition containing the composition as an active ingredient. delete delete The method for preparing a composition according to claim 7, wherein the filtration comprises a step of using filter paper (first) and powdered activated carbon (second).

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