KR20170045074A

KR20170045074A - The Productive Method of Fremenatation Beverage and cosmetic ingredients with Gynura Procumbens

Info

Publication number: KR20170045074A
Application number: KR1020150145025A
Authority: KR
Inventors: 전형주; 박세준
Original assignee: 전형주
Priority date: 2015-10-17
Filing date: 2015-10-17
Publication date: 2017-04-26

Abstract

The present invention relates to a method for the treatment and prevention of acne, which is effective for joint health, which is effective for joint health, Hornberry fruit effective for joint health, 10 to 20% (weight) effective for intestinal health, 50 to 70% Mixing the mixture into an extraction device; A step of adding an enzyme liquid phase obtained by fermenting rice bran rice bran extract obtained in a separate process in place of water, heating the extract to 80 to 100 ° C for 6 to 12 hours, Production of a fermented beverage or a cosmetic composition using SJP0808 strain of Saccharomyces sp. Strain deposited with CCC-10966 (KCCM-1096P) and SJP6723L4 strain of Lactobacillus sp. Deposited with KCM-10731P &Lt; / RTI >

Description

Technical Field [0001] The present invention relates to a fermented beverage and a cosmetic composition,

Recent improvements in living standards have increased interest in LOHAS (lifestyle of health substantiality), and the purpose and use of cosmetics have become more fragmented and specialized. The interest in the hot air and anti-aging of LOHAS has increased the consumers' demand for natural materials and environmentally friendly cosmetics. Gynura procumbens is a medicinal crop of Tropical Asteraceae (Compositae) originating in southern Asia, Indonesia, Thailand and Malaysia. This tropical herb medicinal plant is a 10 to 25cm size and grows in the size of 10 ~ 25cm. It has a fuzzy pattern on the green additional leaf and purple stem. In Korea, diabetes mellitus is known as a plant, and ethanol extracts have been shown to increase the glucose uptake of 3T3 adipocytes (Bohari et al., 2006). Aging is a phenomenon in which the physiological changes in the body progressively occur as the age increases, and the function and biological adaptability of metabolism is reduced. It refers to all body changes including biochemical and behavioral aspects. (See Table 1). As the age increases in the skin, the physiological order is destroyed by internal factors and external factors. In other words, external environmental factors such as ultraviolet rays generate active oxygen in the living body, and when a large amount of active oxygen is generated or chronic active oxygen is generated, the antioxidant defense system is collapsed and causes various diseases. The skin damages cellular components as well as cell and tissue damage, including lipid peroxidation, degeneration of dermal fibroblasts and elastic cells, promotion of melanin production, skin dryness, and DNA changes. As a result, skin wrinkles, pigmentation, deterioration of moisturizing function and inflammation of skin appear, and skin aging is accelerated. Therefore, in order to prevent and inhibit skin aging, there is a need for an antioxidant system capable of suppressing excessive active oxygen species in the skin as well as in vivo and removing active oxygen. Antioxidants that can prevent skin aging are widely distributed in the vegetable field. Fruits and vegetables contain many phenolic compounds, flavonoids, vitamin E, carotenoids, vitamin C, phytochemicals and selenium, It is also important for delay. Cosmetics are used to prevent skin damage due to these physiological and environmental factors, but synthetic antioxidants have been reported to cause toxic effects when overdosed. Therefore, research on finding antioxidants that inhibit aging and oxidation reactions from natural materials is continuously required, and therefore, research on medicinal materials having excellent physiological activity in natural products and oriental medicine is underway. Korean patent 501791 (High blood pressure and three bongnia and extract having diabetic or chronic kidney disease prevention and relieving effect) Korean Patent 1360223 (Method of manufacturing functional food) Application No. 10-2014-0096437 10-2015-0045970 (cosmetic composition containing vanadium fortified name), and the like. Also, Opuntia humifusa is a Korean native cactus, commonly called the palm cactus, which is similar to the palm of the hand and is also called the palm cactus. If the stem, fruit and root of the perennial plant contain flavonoids, dietary fiber, vitamin X, calcium, minerals, amino acids, complex polysaccharides and important nutrients of the human body, dietary fiber and calcium are contained in a larger amount than other plants have. Studies on the physiological activity of T. meningitidis have been reported on the anticancer effect and liver protective effect on cervical cancer and breast cancer. Korean Patent No. 435855 (composition for external application for skin containing palm cactus extract) Patent No. 698533 (Method for preparing antibiotic infectious disease and anticancer composition using fermented liquid of winter cactus) Korean Patent 641699 Korean Patent No. 963161 (Fermentation product of cactus of the genus Cernobus that can prevent and treat contact dermatitis) Korean Patent No. 1158409 (Title of the invention: composition for learning and memory enhancement containing extract of Opuntia ficus-indica) Korean Patent 1505599 (Cream for prevention and improvement of environmentally friendly atopic dermatitis and its preparation method) Korean Patent 1164020 (Pharmaceutical composition containing extract of Opuntia ficus-indica) Korean patent 549506 (Method for producing beverage containing extract of cactus plant) Korean Patent No. 666283 Cactus fermented liquid and food using the same) Korean Patent 1528557 U is a pharmaceutical composition), many of which are published Korea Patent 961 217 (allergic contact dermatitis to prevent and to treat cheonnyeoncho cactus fermentation) A cosmetic composition having a Korea Patent Application 641 699 (antioxidant and antimicrobial activity), and the like.

It is an object of the present invention to provide a cosmetic composition which is effective for prevention and treatment of diabetes, prevention of allergic dermatitis and skin active oxygen or removal of active oxygen to prevent skin aging.

Gynura procumbens is a plant of the Asteraceae family called "sambungnyawa" in Indonesia, Thailand and Malaysia. According to literature reports, Gynura procumbens is not only antioxidant but also physiologically diverse Lt; / RTI > Studies on the regenerating effect of skin aged by ultraviolet irradiation to date have shown that there is anti-aging ability of plants containing a lot of flavonoids and that flavonoid-based components inhibit the synthesis of MMP enzymes. Respectively. In addition, we confirmed the antioxidant function of the stem in order to confirm the possibility of utilizing the discarded stem of the early May. In addition, the antioxidant activity was confirmed by analyzing the contents of Quercetin, Vitamin E and β-carotene in leaves. It is confirmed that there is a pharmaceutical composition which can prevent and treat diabetes, brain health promotion, osteoporosis and allergic contact dermatitis through diverse literatures such as patent search in the case of the next most commonly used milnacil. It is effective for joint health through many medical books and clinics such as Dongbokgop, and Hwanggi is effective for liver health and Saururus chinensis is effective for intestinal health. The strain SJP0808 strain of Saccharomyces cerevisiae strain KCCM-10966P and the strain Lactobacillus strain SJP6723L4 deposited with KCCM-10731P deposited with the native super lactic acid bacteria fermenting the material described above are superior to antibiotics in the ability to inhibit harmful bacteria, It is not only effective in relieving discomfort such as diarrhea and constipation but also has excellent ability to suppress skin harmful bacteria when applied to skin. Therefore, the nutrients are absorbed into the skin that is ingested as a drink or applied to the skin, resulting in allergic dermatitis or aging of the skin. The strains SJP0808 and SJP6723L4 deposited with KJMM-10966P and SJP6723L4 deposited with KCCM-10731P and SJP6723L4, respectively, deposited at KJCM-10966P and KJCM-10766, , The research institute was evaluated as superior to the antibiotics in the antibiotics comparison evaluation experiment with the national research fund and the double effect of the increase in the human immunity enhancement evaluation was verified in the domestic journals at Chungbuk National University . In addition, the Chungcheong National University Industry and University Cooperation Center has improved the food poisoning diarrhea and hangover diarrhea in 164 patients with irritable bowel syndrome in 3 to 4 hours and reported the 100% . Extracting the nutrients by heating the mixture in an extraction apparatus at a rate of 1 liter of rice bran and 2 liters of water, which can be evaluated as a report of necessary nutrients required for human body, at 80 to 100 ° C for 5 to 10 hours, After the temperature was dropped to 30 to 40 ° C, the strain SJP0808 strain of Saccharomyces sp. And the strain SJP6723L4 of the genus Lactobacillus deposited with KCM-10731P (deposited with KCCM-10966P) A step of fermenting the fermented liquid phase by inoculation to prepare a fermented liquid phase, and a step of mixing 10 to 20% of 50 to 70% of sunflower seeds, 30 to 50% of sunflower seeds, 10 to 30% of sunflower seeds, Adding the nutrient solution to the nutrient extraction device of the mixed material and replacing the water to add the fermentation liquid phase produced in the first step and adding SACP0808 strain Saccharomyces cerevisiae strain deposited with KCCM-10966P (KCCM-10966P) KCCM-10731P (KCCM-10731P), and fermented for 72 hours to inoculate the lactobacillus strain SJP6723L4, which is deposited with KCCM-10731P, to provide a fermented beverage and a cosmetic effective for preventing diabetes and preventing skin aging.

It is expected that the fermentation will contribute to prevention of diabetes and allergic skin aging which is the origin of panic disease.

(Weight) 50 to 70% (weight) 30 to 50% (weight) of Ganoderma lucidum, Hovenia dulcis, 3 to 100% (weight) Replacing the water with an enzyme liquid phase; Heating the solution to 80 to 100 캜 and extracting the solution by hot water for 6 to 12 hours; The above extract is fermented with Saccharomyces sanguinus SJP0808 strain deposited with KCCM-10966P and Lactobacillus casei SJP6723L4 strain deposited with KCCM-10731P to provide a fermented beverage or a cosmetic composition.

Claims

The strain SJP0808 strain of Saccharomyces sp. And the strain SJP6723L4 of lactobacillus strain deposited with KSM-10731P (KCCM-10731P) were inoculated with 1 liter of rice bran and 2 liters of water with KCSM-10966P Adding the extract to fermentation apparatus and extracting the nutrients by heating at 80 ° C to 100 ° C for 5 hours to 10 hours, dropping the temperature of the extract to 30 to 40 ° C, and then adding KCCM-10966P to KCCM-10966P A primary process for preparing a fermented liquid phase fermented by inoculating Sactobacillus sp. Lactobacillus strain SJP6723L4 deposited with Saccharomyces cerevisiae strain SJP0808 and KCSM-10731P (KCCM-10731P) % 30 to 50% of the millennia early stage, 10 to 20% of the nutshell, 30% of the third millet; Adding the nutrient to the nutrient extraction device of the mixed material and replacing the water to add the fermentation liquid phase produced in the first step and adding SACP0808 strain Saccharomyces sp. Strain deposited with KCCM-10966P (KCCM-10966P) A fermented beverage and a cosmetic composition produced by inoculating a strain of Lactobacillus sp. SJP6723L4 deposited with KSM-10731P (KCCM-10731P) and fermenting it for 72 hours.

How to make a beverage in early May
The first day of incubation for 24 hours was inoculated with Lactobacillus acidophilus after heating at 37 ℃ for 4 hours with purified water at 120 ℃. The cultured product was further extracted with water for 2 hours at 120 ° C. and then cooled to 37 ° C., followed by inoculation with lactic acid bacteria, followed by secondary culture for 24 hours. Oligosaccharide is added, sugar is mixed, sterilized and packaged to prepare drinks.

Figure 1. Beverage production process
Table 1. Percentage of mixed beverages added with menstrual flow

Sensory Evaluation of Lactic Acid Brewer's Beverage
After preparing lactic acid beverages at the beginning of the month, 50 women in their 50s were given 30 ml of sample in a clear glass cup, After drinking one sample, make sure to rinse the mouth with bottled water. The taste, color, flavor, and overall acceptability of the health drink were evaluated, and a very good score was evaluated as 9 points and a very bad score as 0 score. The mean and standard deviation were calculated using SPSS 20.0 program and the difference between A, B and C was analyzed by ANOVA.
Production and Sensory Evaluation of Lactic Acid Bacteria from Korean Beef
The average value of taste and flavor were different according to the increase and decrease of the beginning and the beginning of the season. The average value of beverage A did not show any significant difference from other recipes, but the higher the content of oregano, the lower the sensory score of taste and taste. The evaluators in their fifties showed that the taste and aroma, which contained 13% of the beginning of the month, were very good because they could think of the concept of health as 8.9 ± 1.9. The sensory score according to the content of the first day of the first month did not show a significant difference, but the taste, flavor, and preference increased when the content of the first day of the first month was 13%.

Table 2. Sensory Evaluation Results of Lactic Acid Bacterial Beverages Containing Sun Beginnings

P-value NS: Non significant by ANOVA at 0.05

How to make essence in early June
For the cosmetics containing the extract from the beginning of October, the phase B phase and phase A phase are separated and heated to 80 ℃ for dissolution. At this time, the H liquid of the mulberry extract was dissolved in the oil phase and dissolved. The flies of C, D, E, F and G phase were dissolved in the mixer at 3000 rpm until the polymer was uniformly dispersed and added to the phase B water phase. The mixture was emulsified at 50 ° C in a homomixer at 3000 rpm for 3 minutes, then triethanolamine was added, and the mixture was emulsified for 2 minutes, cooled to 30 ° C, and defoamed

Table 3. Ingredients Ingredients Ingredients

Evaluation of stability and usability of cosmetics containing extract

5-1. Viscosity change of essence with temperature
The viscosity was measured by Brookfield Viscometer (Brookfield LVDV-II +, Brookfield Laboratories, Inc., Middleboro, MA) after storing 3% essence at 4 ℃ and 35 ℃ for 1 day, 7 days, 14 days and 28 days USA). Select the spindle No. 7 at 25 ° C and measure the viscosity of the essence for 1 minute at 10 rpm. In order to reduce the influence of bubbles, defoaming treatment was performed and the average value was obtained by repeating it three times. Data were analyzed using SPSS 20.0 program and the mean and standard deviation were calculated. The change with temperature was analyzed by t-test.
Result of Essence Viscosity Change with Temperature
The viscosity of the essence containing 3% at the beginning of the first month showed no significant difference from 1 to 13 days, but it showed significant difference (P <0.05) from 181.0 ± 0.02 at 4 ℃ to 169.5 ± 0.06 at 35 ℃ . After 3 days of storage at 4 ° C, the natural cosmetics without significant preservatives showed a significant difference (P <0.05) from 180.2 ± 0.06 at 4 ℃ to 162.5 ± 0.03 at 35 ℃. I think it would be preferable
Table 4. Viscosity change of essence with temperature

P-value: P < 0.05

5-2. PH change of essence with temperature
The pH was measured using a pH meter (Sartorius PB-20, Thermo Scientific, Japan). The pH is measured after storing 3% essence at 4 ℃, 35 ℃ for 1 day, 7 days, 14 days and 28 days. Data were analyzed using SPSS 20.0 program. The mean and standard deviation were calculated. The change with temperature was analyzed by t-test.
Result of pH change of essence according to temperature
The essence containing 3% at the beginning of the month does not show a difference according to the temperature. 4.40 ± 0.01 at 4.degree. C. for 1 day and 4.39. + -. 03 at 35.degree. C. after 28 days, showing no significant difference and can be interpreted as stability.
Table 5. Essence pH change with temperature

NS: Non-significant by t-test = 0.05

Sensory Evaluation of Cosmetics with 5-3 Monthly Extracts Added
50 people in their 50s who are looking for a skin clinic. The 3% essence at the beginning of the month is used for refrigeration for 20 days, and then examined for 7 items: incense, color, viscosity, spreadability, feeling after application and absorption. The subjects were surveyed by self-entry method, and the data were obtained on a scale of 5 points. Data were analyzed using SPSS 20.0 program. The mean and standard deviation were calculated. The change with temperature was analyzed by t-test.
Sensory evaluation results of cosmetics containing extract
The sensory evaluation of 7 items of fragrance, color, viscosity, spreading feeling, absorbency, viscosity, and moisturizing ability were evaluated as 'very good' 68%, moisturizing 62%, feeling 34% and viscous 24%. The items of 'bad' were relatively low at 10% and 2%, respectively. 'Very Poor' did not appear in any item, and it showed high value of 4.5 or more out of 5 out of 5 points, showing 4.80 ± 0.57 in wettability, 4.52 ± 0.69 in absorbency, 4.62 ± 0.33 in moisturizing power and 4.26 ± 0.55 in feeling after application. show.
It is considered to be a result of the fact that the fragrance of the fragrance is reflected in the fragrance of the fragrance. The fragrance and the color of the beginning of the month of the commodity were considered to be adjusted according to the consumer 's preference.
Table 6. Sensory evaluation of cosmetics containing added extracts at the beginning of June

Very good: 5, Good: 4, Moderate: 3, Poor: 2, Very poor: 1

Extraction
Total 6000g at the beginning of the first month, separated from the raw leaves and stems, and lyophilized at -20 ℃.
Straw dry weight 164g, leaf dry weight 240g, seed dry weight 404g, 6.7% dry weight. 92 g of dry powder was added to conical tibe at 1 g per day, and 92 of them were extracted by 3 times ultrasonic vibration method. 3.3 L of extract was concentrated under reduced pressure.
After adjusting the volume to 240 mL with water and dividing into 10 mL of 50 mL conical tube, lyophilized to obtain 16.77 g
Table 7.

Antioxidant extraction
7-1 Tyrosinase inhibition assay method
Measured by modifying the method of Momtaz (2008).
The inhibitory activity was measured by mixing mushroom tyrosinase (T3824, Sigma) with substrate and tyrosine. The total amount was 1 mL, mixed with 500 unit / mL tyrosinase and 10 mg / L tyrosine mixture. After incubation at 37 ° C for 30 min, the reaction was stopped by heating at 80 ° C for 3 min and absorbance was measured at 480 nm.
The degree of inhibition of tyrosinase activity was calculated as a percentage of the absorbance of 4 samples of leaf ethanol extract, leaf water extract, stem ethanol extract, stem water extract and distilled water
Calculation.

A: Absorbance of control group, B: Absorbance of sample group

Tyrosinase inhibition assay
Tyrosinase contains copper and is an important enzyme in the stage of melanin synthesis, resulting in hyperpigmentation due to excessive melanin production. Therefore, tyrosinase inhibitors can be used to control skin melanin pigment production.
The inhibition of tyrosinase inhibition at the beginning of early summer was 45.3% for leaf ethanol extract, 46.1% for leaf water extract, 39.9% for stem ethanol extract and 39.4% dml for stem water extract at 1 mg / mL concentration.

7-2 How to measure Vitamin C
The concentration of Vitamin C was measured by HPLC. Revesed phase column (Restek, USA) filled with Ultra C18-coated silica was used as stationary phase and 40% methanol as mobile phase. The sample separated through the C18 column was detected using a UV detector (Younglin, UV730D). Samples were filtered using a 0.22 mm-pored membrane filter (Sartorius, Hannover, Germany) and 20 μL was injected into the HPLC injection port.
The temperature of the column and detector was adjusted to 35 ° C and the flow rate was 0.3 ml / min.
The concentration of vitamin C in the extract was determined by using the chromatograph peak area of the aqueous solution of Vitamin C (0.1 mg / mL) used as a standard.
Vitamin C measurement result
Vitamin C promotes the synthesis of hydroxyproline and is involved in the synthesis of collagen. Therefore, it has been reported that when melanin and erythema index are decreased when the strawberry plant extract containing vitamin C is applied to the skin, , 2013), vitamin C contained in the beginning of the first half of the menstrual cycle is expected to promote skin whitening and collagen synthesis.
The content of vitamin C in dandelion leaves was 67.4 mg / 100g, and the vitamin C content of Korean vegetables was 64.3 mg / 100g of sesame leaf, 60.2 mg / 100g of spinach, and 30.2 mg / 100g of lettuce.
Vitamin C of leaves contained 291.2 mg / 100 g of leaf ethanol extract, 185.6 mg / 100 g of leaf water extract, 51.4 mg / 100 g of stem ethanol extract and 26.4 mg / 100 g of stem water extract.

Cytotoxicity experiment
The effect of reactive oxygen species on skin tissue can be largely reduced by cell death, pigmentation, and inflammation. Therefore, in this study, MTT cytotoxicity tests were performed on the cell death, pigmentation, and inflammation factors in the stomach, and MMP activity test, whitening effect test, anti-inflammation test In the process.

MTT
Human dermal fibroblast (HDF) is a cell in the dermal layer of the skin that produces connective tissue and plays an important role in restoring skin from scratches and the like.
In the present study, when the ethanol extracts were exposed to HDF at a concentration of 20 μg / mL or lower, the survival rate was 90% or higher, and the experiment was conducted at a concentration below 20 μg / mL .

Cytotoxicity test (MTT assay)
Dissolve HDF (Human Dermal Fibroblast) cells into a 96-well plate at 5 × 10 3/200 μL per well. Dissolve ethanol extract in early stage of early growth in DMSO (Dimethyl Sulfoxide).
Cells were cultured in 96-well plate and 100 μL of ethanol extract was added to each well. After 48 hours, 10 μl of 10-fold concentrated MTT (tetrazolium dye) solution was added to the wells. After 2 hours, the cells were removed by suction. 100 μL of DMSO was added and shaken to dissolve the MTT well and measured at 595 nm using an enzyme-linked immunosorbent assay (ELISA) reader.
Cytotoxicity test results
Melanin synthesis enzymes include tyrosinase, tyrosinase related protein 1 (TRP1), and tyrosinase related protein 2 (TRP2). MITF is a typical transcription regulator that regulates three proteins. HDF (Human dermal fibroblast) cells were treated with ethanol extracts at the beginning of each month for each of the concentrations indicated in the graphs.
The expression of MITF (tyrosinase-associated transcription factor) and tyrosinase was markedly decreased when α-MSH 200 nM, a tyrosinase-producing stimulatory hormone, was added and ethanol extracts were added. This suggests that the inhibition of the activity of MITF transcription factors inhibited the expression of tyrosinase, which is regulated by MITF.
The cell viability at 24 or 48 hours was not more than 80 μg / mL at the concentration of 100 μg / mL of the ethanol extract of early summer. The survival rate was 90% or more at a concentration of 20 μg / mL.

Figure 2. HDF cell survival according to ethanol extract concentration

Whitening effect experiment
9-1 Measurement of tyrosinase expression level
Melanin production inhibition of ethanol extracts from early May was modified by the method of Hosoi (1985). Dilute the cultured B16F10 cells to 1 × 10 ⁵ / well and dispense 200 μL into 6 well plates. After incubation for 6 hours, add 100 μL of 100 nM α-MSH to one well, 200 μL of 100 μM of α-MSH and 100 nM of diluted mulberry leaf extract in two wells.
After culturing for 48 hours, the dendritic cells and changes in cell proliferation of melanocytes were observed with a phase contrast microscope. The cells were recovered by treatment with trypsin, and then dried at 60 ° C to add a cell lysis buffer. Then, intracellular melanin was extracted from the cells at 60 ° C Calculate the amount of melanin by measuring the absorbance at 490 nm with a microplate reader.

9-1 Tyrosinase expression level
The expression level of tyrosinase, an enzyme producing melanin pigment, was determined at the protein level using Western blotting.
The expression level of MITF and tyrosinase, a transcription factor that promotes tyrosinase expression, was significantly increased by adding 200 nM of α-MSH, a hormone that increases tyrosinase expression (lane 2).
The expression level of MITF and tyrosinase was measured again after addition of 10 μg / mL of ethanol extract from early May (lane 4). The expression level of MITF and tyrosinase was significantly decreased by α-MSH. It is also confirmed that the expression of MITF and tyrosinase can be inhibited even in the absence of α-MSH (lane 1 and 3).
These results suggest that the ethanol extract of early mongolian early suppresses the activation of tyrosinase-expressing MITF transcription factor and ultimately inhibits the expression of tyrosinase. It inhibits the expression of tyrosinase and ultimately inhibits the production of melanin pigment. .

Figure 3. Inhibition of Tyrosinase, TRP1, and TRP2 by Addition of a-MSH to Ethanol Extracts in Early May.

9-2 Relative tyrosinase activity measurement
In order to further investigate whether the ethanol extracts of early-flowering plants affect the activity of tyrosinase, the activity of tyrosinase was measured by adding α-MSH and ethanol extracts of early summer.
Cells were harvested 48 hours after each treatment to determine the tyrosinase activity of the cells. The same number of cells were lysed using NP-40 lysis buffer. The tyrosinase activity was measured by L-DOPA. 5 mM of L-DOPA was completely dissolved in sodium phospate buffer (0.1 M, Ph 6.0). L-DOPA stock 20 μL + buffer up to 100 μL, react at 37 ° C for 3-4 hours, and measure the absorbance at 405 nm.

9-2 Relative tyrosinase activity measurement result
It was confirmed that the addition of the ethanol extract of early summer to the beginning of day significantly reduced the activity of tyrosinase (166.46%) increased by α-MSH (138.75%). As a result, the ethanol extracts of early early May showed inhibitory activity of tyrosinase.
Table 8. Relative tyrosinase activity measurement

Anti-inflammatory experiment
10-1 Separation of Cytosol and Nucleus
The buffers A and C were prepared, and the composition of each buffer is shown in Table 9.
Allow to dissolve in cold condition for 15 minutes, shaking once every 5 minutes.
After 15 minutes, 10% NP-40 was added to 0.1%, followed by inverting for 3 minutes, followed by centrifugation at 12000 rpm for 5 minutes. Transfer the supernatant to a new tube (this supernatant is cytosol).
Add 100 μL of buffer A to the remaining pellet and wash to remove cytosol extract completely. After that, add buffer C and dissolve the pellet. After cooling, dissolve for about 1 hour. After centrifugation at 12000 rpm for 7 minutes, transfer the supernatant to a new tube (this supernatant is nucleus).
Table 9. Components of Buffer

10-2 Measurement of NF-kB concentration
Inflammation in the skin is expressed before the in vivo defense against internal and external stimuli such as external infection through various pathways and metabolites in vivo, and various inflammation regulators in the cell are involved as mediators. NF-kB is a prominent inflammatory signaling pathway that is activated by the inflammatory cytokines IL-1 and TNF-a (tumor necrosis facto-a).
1 × 10 ⁵ to the frequency divider / 2 mL per the HDF cell on 6-well plate well.
Dissolve the extract at the beginning of the next day in DMSO, and dissolve it in the culture medium for each concentration to be tested. After removing the culture medium from the 6-well plate in which cells were dispensed, add 2 ml of the prepared extract to each well. After 24 hours, additional TNF-α (10 mg / mL) was added.
After 1 hour, the culture solution was removed, and the cells were collected and centrifuged at 8000 rpm. The supernatant was removed and the cytosol and nucleus were separated. The separated cell lysate was quantitated by the bradford method, and the amount of protein was calculated, mixed with 5 times of sample buffer and lysis buffer, and heated at 100 ° C for 10 minutes.
Western blot kit was assembled to make 10% gel, and after hardening properly, each sample was loaded and run at 80 V. For transfer, 3M paper and membrane were placed for transfer kit and transferred at 90 V for 2 hours. The membrane was then removed and blocked in 5% skim milk for 1 hour. After removing the blocking buffer, primary antibody is added and incubated at room temperature.
The next day, the cells were washed with 0.1% TBST for 30 min and incubated for 2 h. The cells were then washed with 0.1% TBST for 30 min. ECL solution is mixed and spread on the membrane, then developed using film.

10-2 Protein expression results of NF-kB
NF-kB regulates the genes of other inflammatory cytokines, chemokines and adhesion molecules. NF-kB is a transcription factor that induces an inflammatory reaction. It is located in the cytoplasm in the inactivation phase and moves into the nucleus when activated. Using these characteristics, the degree of activation of NF-kB was analyzed by Western blotting of the amount of NF-kB protein in the cytoplasm and nucleus by fractionation of cytoplasm and nucleus.
To determine whether cytoplasm and nuclei are well fractionated, α-tubulin, which is present only in the cytoplasm, and Lamin B, a nuclear protein component, are used.
After the treatment of NF-kB with TNF-α, we observed the migration of NF-kB into the nucleus. As a result, the cytoplasm (lane 2), as seen in lane 2 and lane 5, , The band size of NF-kB decreases and the band size in the nucleus (lane 5) increases.
The band size of lane 3 was relatively increased compared to that of lane 2 and the band size of lane 6 was decreased compared to lane 5, .

Figure 4. Activity of NF-kB and Lamin B
These results indicate that NF-kB is activated by TNF-α, and that the addition of ethanol extracts from the beginning of menstruation inhibits NF-kB activation by TNF-α