KR20210141271A - Composition for recylce a skin cell comprising of limonium tetragonum - Google Patents

Abstract

The present invention relates to a composition for regenerating skin cells, which includes Limonium tetragonum as an active ingredient.

Description

A composition for skin cell regeneration, comprising chives as an active ingredient {COMPOSITION FOR RECYLCE A SKIN CELL COMPRISING OF LIMONIUM TETRAGONUM}

The present invention relates to a composition for regenerating skin cells, which includes P.

The dicotyledonous plant is a biennial plant of the dicotyledonous plant Chrysanthemum, which grows on the seashore and has thick and straight roots. The leaves are clustered at the root, spread in all directions, have the shape of a long oval spatula, have no hairs, and have a flat edge.

The flower stalk is 30-60cm, and many branches are divided, and the spike of the stigma hangs at the end, and the length of the spike is 2-4cm. The bract is green, oval, and contains several flowers. The calyx is cylindrical and the tip is divided into 5 parts. The corolla is yellow, deeply divided into 5 parts and longer than the calyx. There are 5 stamens, 1 pistil, and 5 styles.

The fruit is fusiform with a length of 2.5 mm. The roots can be eaten raw, but the hair smells like burning. It is distributed in Korea, Japan (southern), and northeastern China.

Psyllium is a dyed plant that grows in tidal flats and salty soils where seawater flows. The leaves and roots contain catechin, a powerful antioxidant, and myricetin, a kind of flavonoid, in high concentrations.

According to the March 2015 issue of Natural product sciendes published by the Rural Development Administration, the blood alcohol concentration of rats fed psyllium was reduced by 55% faster from 5.34±0.38nM to 2.41±0.36nM in rats fed only alcohol. It is known to be effective in normalizing blood cholesterol and reducing triglycerides in liver tissue.

On the other hand, there are various compositions as compositions having antioxidant and anti-inflammatory effects, and among them, we would like to introduce Jichae, wormwood, and red ginseng.

Jichae ( Triglochin maritimum ) is a monocotyledonous prophylactic plant, a perennial herb of the family Brassicaaceae, which grows in groups on the tidal flats or on the seashore. The rhizome is thick, short, and has strong roots, and the fibers remaining from the old petiole cover the outside. Flower stalks stand upright, do not branch, and are 15-40 cm in height.

The leaves are gathered from the root and are line-shaped, 10-30cm long and 2-4mm wide, with a dull tip, and the lower part becomes the sheath. The sheath is white membranous (膜質: translucent like thin paper) and has a leaf tongue with a length of 3~5mm at the tip.

Flowers bloom in August-September in purplish-green, and run in stellate inflorescences at the end of stalks that emerge from between the leaves. Small peduncle is 2~4mm long, and has 6 petals, oval shape, and runs in 2 lines. There are 6 stamens and there is no operating table, and the pistil has a very short style and has 6 stigma heads.

In oriental medicine, dried outplants including the rhizomes of ajichae are called ajichae, and the fruits are used together. It treats neck pain, and the outpost including roots cools the heat and replenishes and restores the yin liquid depleted due to heat.

Artemisia scoparia Waldst. et Kit. ) is a plant of the genus Artemisia, and the one collected in spring is called 'Myeoninjin (綿茵陳)', and the one collected in autumn is called 'Injinho (茵陳蒿)'. call it

The abbreviation of 'Injinho' of bi-wormwood was first described as a product in the 『Shinnong Bonchogyeong (神農本草經)』, and most of the history books were recorded. Traditionally used in China is this species and Artemisia capillaris Thunb. In the Chinese Pharmacopoeia (Chinese Pharmacopoeia (Chinese Pharmacopoeia) (2015 edition)), this species was listed as a plant of legal origin for Chinese medicine Injin. The main production areas are Shaanxi and Hebei in China. The one from Shaanxi is called Seoinjin (西茵陳), and the quality is the best. In the <Korean Pharmacopoeia and Other Herbal Medicines Specifications> (4th edition), Injinho is referred to as “the above-ground part of Artemisia capillaris Thunberg (Asteraceae). Those harvested in spring are called 'Myeoninjin', and those harvested in autumn are called 'injinho'.”

As the active ingredients of wormwood, it mainly contains essential oils, coumarins, flavonoids, and chromone compounds. Among them, several components have the activity of interpolation idam (保肝利膽). In "Chinese Pharmacopoeia", using high-performance liquid chromatography, the content of chlorogenic acid is 0.50% or more, and the content of scoparone is 0.20% or more, and the specifications of medicinal materials are set.

Through pharmacological studies, it is known that wormwood has anti-inflammatory, anti-inflammatory, analgesic, diuretic, anti-hypertensive, and anti-tumor effects.

In oriental medicine, ginseng has effects such as relieving fever and relieving fever.

Red ginseng is a type of herbal medicine that is steamed from the roots of Panax ginseng CA Meyer belonging to the Araliaceae family. It contains glycosides, ginseng flavoring ingredients, antioxidants, organic acids, etc. It is effective in preventing aging, recovering from fatigue, removing hangovers, and improving blood circulation. In addition, the saponin component contained in red ginseng is known to be effective in diabetes by removing alloxane and streptozotocin, which are substances that cause diabetes.

Saponin is contained in small amounts in various plants such as soybean, green onion, deodeok, buckwheat, and bellflower. The chemical structure is completely different. Therefore, the saponins contained in red ginseng are called ginsenosides to distinguish them from other plant-based saponins.

It is reported that ginsenosides show strong pharmacological effects even in small amounts, and the main pharmacological effects are known to exert various effects on body control functions by affecting the central nervous system, endocrine system, immune system, and metabolic system.

The present applicant intends to propose a composition capable of regenerating damaged skin cells such as collagen or elastin by UV-B (ultraviolet-B), etc., based on the above-described efficacy.

With respect to skin cell regeneration, Patent Publication No. 10-1652956 discloses a human epithelial cell regeneration factor fusion protein with increased antioxidant function and cell proliferation effect and a skin wrinkle improvement and anti-aging cosmetic composition containing the same as an active ingredient. have.

Registered Patent Publication No. 10-1652956 (2016.08.31. Announcement)

It is an object of the present invention to provide a composition for skin cell regeneration comprising phyllotaxis as an active ingredient.

The present invention relates to a composition for skin cell regeneration containing Psyllium as an active ingredient,

The composition is

It is an extract using sagebrush, lichen, wormwood, and red ginseng.

The ratio of gaejigyeong: jichae: mugwort: red ginseng is 2 : 1 : 1 : 2,

The ratio is quantified as 100 g, and a mixed extract is obtained by adding 2 L of 50% (v/v) ethanol and extracting at room temperature for 5 days,

After filtering the mixed extract, it is characterized in that it is a complex extract obtained by concentrating through a vacuum concentrator, drying through a freeze dryer to recover, and storing at -20°C.

At this time, the composition,

At a concentration of 100 μg/ml or less, it is characterized in that it does not affect the viability of human-derived HaCat cells,

When the human-derived HaCaT cell culture solution is treated at a concentration of 100 μg/ml, it is characterized in that it has a higher regeneration rate compared to the group without the addition of the complex extract when cells are damaged by peroxide,

When the culture solution of human-derived HaCaT cells was treated at a concentration of 100 μg/ml, the collagen regeneration rate from collagen damage caused by UV-B was characterized in that it had a higher regeneration rate compared to the group without the addition of the complex extract,

When the human-derived HaCaT cell culture solution is treated at a concentration of 100 μg/ml, the collagen regeneration rate from UV-B-induced collagen damage in human-derived HaCaT cells is increased.

When the human-derived HaCaT cell culture solution is treated at a concentration of 100 μg/ml, the production amount of elastin and collagen protein increases from UV-B-induced collagen damage in human HaCaT cells,

It is characterized in that it has the effect of reducing epidermis from skin damage caused by UV-B, inhibiting infiltration of mast cells, and protecting skin collagen.

In particular, the composition according to the present invention is characterized in that when vitamin C is added, the collagen synthesis rate is increased.

According to the composition for skin cell regeneration comprising phyllophthora as an active ingredient according to the present invention, it is possible to provide a composition for skin cell regeneration based on an extract containing the active ingredients of phyllophyllum basilica, sagebrush and red ginseng.

1 shows the results of a cell culture experiment according to Experimental Example 1.
2 and 3 show the results of the cell culture experiment according to Experimental Example 2, FIG. 2 shows the confirmation of the regeneration rate from cell damage caused by peroxide, and FIG. 3 shows the collagen regeneration rate from the collagen damage caused by UV-B ELISA Kit The result of confirming the amount of collagen released into the culture medium was confirmed.
4 shows the experimental results according to Experimental Example 3.
5 shows the results according to Experimental Example 4.
6 shows the results according to Experimental Example 5.
7 is a histological analysis performed to confirm the results according to Experimental Example 5.
8 shows the results according to Experimental Example 6.
9 shows the results according to Experimental Example 7.

The terms or words used in the present specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventor may properly define the concepts of the terms to best describe his invention. Based on the principle, it should be interpreted as meaning and concept consistent with the technical idea of the present invention.

Therefore, the embodiments described in this specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention and do not represent all the technical spirit of the present invention, so various equivalents that can be substituted for them at the time of the present application It should be understood that there may be variations and examples.

Hereinafter, prior to the description with reference to the drawings, it is not shown or specifically described for the known configurations that are not necessary to reveal the gist of the present invention, that is, those skilled in the art that can be added obviously by those skilled in the art. reveal the sound

The present invention relates to a composition for regenerating skin cells, which includes P.

The composition for skin cell regeneration according to the present invention includes, as the main composition, sagebrush, sagebrush, sagebrush and red ginseng, and preferably, the ratio of sagebrush: sagebrush: sagebrush: red ginseng is 2 : 1 : 1 : 2, ,

Quantify at a weight ratio of 100 g in the above ratio, add 2 L of 50% (v/v) ethanol, and extract at room temperature for 5 days to obtain a mixed extract.

At this time, the mixed extract was applied to Advantec filter paper No. 2 (Toyo Roshi Kaisha, Tokyo, Japan), concentrated in a vacuum concentrator (EYELA, Tokyo, Japan), dried in a freeze dryer (EYELA FDU-2100, EYELA), recovered and stored at -20℃ A complex extract was obtained.

However, the conditions in the process of obtaining the above complex extract are not limited to the present invention. That is, if only the conditions for obtaining the mixed extract and the conditions for filtering the obtained mixed extract and then storing it at a temperature of -20°C through drying and recovery can be met, other conditions can be applied without limitation.

The composition according to an embodiment of the present invention includes skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, eye cream, moisture cream, hand cream, essence, It may have any one formulation selected from the group consisting of nutritional essence, pack, cleansing foam, cleansing water, cleansing lotion, cleansing cream, body lotion, body cleanser, soap and powder, but is not limited thereto. The composition consisting of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.

The composition according to another embodiment of the present invention may be processed into a nutraceutical for oral administration.

At this time, there is no particular limitation on the functional food. Examples of foods to which the above-described composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea drinks, There are alcoholic beverages, vitamin complexes, and the like, and includes all health foods in the ordinary sense.

Hereinafter, an embodiment of the present invention will be described in detail. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

experimental material

Dulbecoos modified Eagle's medium (DMEM), fetal bovine serum (FBS) and RIPA buffer were purchased from Invitrogen (Waltham, MA, USA). EZ-Cytox was purchased from Dogenbio (Seoul, Korea).

Type1 collagen and elastin antibodies and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-actin antibody was purchased from BD Biosciences (CA, USA).

Preparation Example 1. Composition for skin cell regeneration

The halophytes used in the present invention were collected from the tidal flats of Saemangeum and Gochang-gun, and then used in the experiment. After quantifying 100 g of each plant in the ratio of sphagnum 2: fern 1: mugwort 1: red ginseng 2, 2L of 50% ethanol was added, and extracted for 5 days at room temperature to obtain a mixed extract.

The mixed extract was prepared using Advantec filter paper No. 2 (Toyo Roshi Kaisha, Tokyo, Japan), concentrated in a vacuum concentrator (EYELA, Tokyo, Japan), dried in a freeze dryer (EYELA FDU-2100, EYELA), recovered and stored at -20℃ A complex extract was obtained.

Experimental Example 1. Cell culture experiment

The HaCaT cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and used.

B16/F10 cells and HaCaT cells were cultured using DMEM medium containing 10% FBS and 1% antibiotics (penicillin & streptomycin) in a 5% CO2 incubator maintained at 37°C with sufficient humidity.

The results according to the experiment may be referred to FIG. 1 of the accompanying drawings.

1 shows the results of a cell culture experiment according to Experimental Example 1.

According to FIG. 1 of the accompanying drawings, as a result of confirming cytotoxicity in human-derived HaCat cells up to a concentration of 25 to 400 μg/ml of the complex extract according to Preparation Example 1, the cell viability was not affected at 100 μg/ml or less. sound could be confirmed.

Experimental Example 2. Cytotoxicity evaluation

HaCaT cells were dispensed into 96 well plates at ^{a concentration of 2×10 5} cells/ml and cultured, and each complex extract quantified by concentration was injected and cultured for 20 hours. Absorbance was then measured at 450 nm. The survival rate was calculated as a percentage compared to the non-additive group of the complex extract.

Results according to the experiment may be referred to FIGS. 2 and 3 of the accompanying drawings.

2 and 3 show the results of the cell culture experiment according to Experimental Example 2, FIG. 2 shows the confirmation of the regeneration rate from cell damage caused by peroxide, and FIG. 3 shows the collagen regeneration rate from the collagen damage caused by UV-B ELISA Kit. The result of confirming the amount of collagen released into the culture medium was confirmed.

According to FIG. 2 of the accompanying drawings, as a result of confirming the regeneration rate from cell damage by peroxide in human-derived HaCat cells, when the complex extract according to Preparation Example 1 was treated at a concentration of 100 μg/ml in the cell culture medium, compared to the positive control group It was confirmed that there was a cell regeneration effect by showing a regeneration rate of about 25% or more.

In addition, according to FIG. 3 of the accompanying drawings, the collagen regeneration rate from UV-B-induced collagen damage in human-derived HaCat cells was confirmed by ELISA-Kit to determine the amount of collagen released into the culture medium. When treated at a concentration of ㎖, it was confirmed that there was an effect of increasing collagen production by showing a production amount of about 28% or more compared to the positive control group.

Experimental Example 3. Immunofluorescence staining

HaCaT cells were dispensed on a slide glass at ^{a concentration of 2×10 5} cells/ml and cultured for 24 hours. Each extract was injected at a concentration of 100 μg/ml, followed by UV-B (20 mJ/cm ² ) irradiation 1 hour later. After incubation for 24 hours, the cells were fixed with methanol, blocked with PBS containing 1% BSA, injected with collagenI antibody, and reacted at 4°C for 16 hours.

After washing 3 times for 10 minutes with PBST, the secondary antibody was reacted with Goat anti-Rabbit IgG Alexa Fluor 488, followed by mounting with a mounting solution containing DAPI. After drying the slide for 24 hours, images were obtained using a fluorescence microscope.

This will refer to FIG. 4 of the accompanying drawings.

4 shows the experimental results according to Experimental Example 3.

According to FIG. 4 of the accompanying drawings, as a result of confirming the collagen regeneration rate from UV-B-induced collagen damage in human-derived HaCaT cells through immunofluorescence staining, the complex extract according to Preparation Example 1 was added to the cell culture medium at a concentration of 100 μg/ml. When treated with , it was confirmed that the collagen synthesis rate increased.

Experimental Example 4. Western blot analysis

HaCaT cells were dispensed on 6 well plates at ^{a concentration of 2×10 5} cells/ml and cultured for 24 hours. After each extract was injected at a concentration of 100 μg/ml, 1 hour after UVB (20 mJ/cm ² ) irradiation, 24 After culturing for time, the cells were centrifuged twice with PBS, injected with RIPA buffer, and centrifuged at 12,000 RPM to extract proteins.

The extracted protein was quantified by measuring absorbance at 595 nm using Bradford protein assay reagent. The quantified protein was separated through SDS-PAGE and transferred to PVDF (polyvinylidene difluoride) membrane (Bio-Rad) at 100V for 1 hour. Membrane was blocked in Tris buffered saline with 1% Tween20 (TBST) containing 5% BSA at room temperature and washed 3 times with TBST for 10 minutes. and reacted at 4°C for 16 hours.

Then, after washing 3 times with TBST for 10 minutes, secondary antibodies (goat anti-rabbit IgG HRP, rabbit anti-mouse IgG HRP) were reacted in 5% BSA at room temperature for 2 hours, and then washed 3 times with TBST for 10 minutes.

This is to confirm the result by attaching FIG. 5 of the accompanying drawings.

5 shows the results according to Experimental Example 4.

Figure 5 is the result confirmed by Western blot using an antibody that specifically acts on these molecules to examine the intracellular elastin and collagen protein expression from UV-B-induced collagen damage in human-derived HaCaT cells, both proteins extract When the cell culture medium was treated at a concentration of 100 μg/ml, it was confirmed that the production amount increased.

Experimental Example 5. Animal Experiment

7-week-old male C57/BL and ICR mice bred in a sterile environment were purchased from Orient Bio (Iksan, Korea), and were acclimatized for 1 week while sufficiently supplied with feed and water, and then used in the experiment.

The breeding environment had a day and night cycle of 12 hours each, and the temperature (20~22℃) and humidity (50~60%) were kept constant. ^{After that, UVB (300mJ/cm 2} ) was irradiated every 3 days while orally administering the complex extract according to Preparation Example 1 at a concentration of 200 mg/kg of the extract every day. After conducting the experiment for 2 weeks, the results were analyzed according to each analysis method.

On the 14th day, the last day of the experiment, the mice were sacrificed, the skin tissue was cut out, fixed with 10% neutral formalin (pH 7.4) for 24 hours, dehydrated using 50-100% ethanol, washed with xylene, etc. samples were made

The prepared paraffin memol specimens of each experimental group were sectioned to a thickness of 5 μm, attached to silane-coated micro slides, stained with hematoxylin & eosin (H&E), toluidine blue, and trichrome, and then examined under an optical microscope at 100X and 400X field of view.

Referring to FIG. 6 of the accompanying drawings, the protective effect of the complex extract from skin damage caused by UV-B in male mice was confirmed.

6 shows the results according to Experimental Example 5.

As a result according to FIG. 6 of the accompanying drawings, when the complex extract was orally administered at a concentration of 200 mg/kg for 2 weeks, the protective effect as shown in FIG. 6 was confirmed, and the effect of reducing the epidermal thickness was also confirmed.

Histological analysis was performed to confirm the protective effect of FIG. 6 of the accompanying drawings, which is shown in FIG. 7 of the accompanying drawings.

7 is a histological analysis performed to confirm the results according to Experimental Example 5.

As shown in FIG. 7 of the accompanying drawings, the epidermal reduction effect of the complex extract according to Preparation Example 1 was confirmed through H&E staining (column 1 of FIG. 7), and inhibition of mast cell infiltration through toluidine blue staining was also confirmed. It was confirmed (column 2 in FIG. 7).

In addition, the skin collagen protective effect was confirmed through trichrome staining (column 3 in FIG. 7 ).

Experimental Example 6. Expression of elastin and collagen protein in the skin from collagen damage caused by UV-B

In addition, in order to examine the expression of elastin and collagen protein in the skin from collagen damage caused by UV-B in animal experiments, using an antibody known to specifically act on elastin and collagen molecules, confirming by Western blot Experiments were performed.

This is illustrated in FIG. 8 of the accompanying drawings.

8 shows the results according to Experimental Example 6.

According to FIG. 8 of the accompanying drawings, both collagen and elastin, when the complex extract according to Preparation Example 1 was orally administered to mice at a concentration of 200 mg/kg, it was confirmed that the amount of collagen and elastin production increased.

Experimental Example 7. Experiment with artificial skin

The artificial skin used in the experiment was purchased from Tego Science Co., Ltd., and cultured according to the manufacturer's manual, and each extract was pretreated into the cells.

After completion of the experiment, a paraffin block was prepared and sectioned to a thickness of 5 μm, stained with trichrome, and then examined under a 100-fold field of view under an optical microscope.

Experimental results according to Experimental Example 7 are shown in FIG. 9 of the accompanying drawings.

9 shows the results according to Experimental Example 7.

According to the evaluation result of the collagen synthesis rate of the complex extract of Preparation Example 1 through the artificial cell culture experiment according to FIG. 9 of the accompanying drawings, the effect of increasing the collagen synthesis rate in the experimental group to which the mixed extract and vitamin C was added have.

What has been described above using the drawings is to describe only the main points of the present invention, and it is obvious that the present invention is not limited to the configuration of the drawings as much as various designs are possible within the technical scope.

Claims (12)

A composition for skin cell regeneration containing Psyllium as an active ingredient.
The method according to claim 1,
The composition is, characterized in that the extract using the syphilis, sagebrush, mugwort and red ginseng, a composition for skin cell regeneration containing the syphilis as an active ingredient.
3. The method according to claim 2,
The composition is a composition for skin cell regeneration containing a plantain plant as an active ingredient, characterized in that the ratio of spearmint: sagebrush: wormwood: red ginseng is in a ratio of 2: 1: 1: 2.
4. The method according to claim 3,
The composition is
The ratio is quantified as 100 g, and the mixed extract is obtained by adding 2 L of 50% (v/v) ethanol and extracting at room temperature for 5 days.
5. The method according to claim 4,
The composition is
After filtering the mixed extract, it is concentrated through a vacuum concentrator, dried through a freeze dryer and recovered, and then stored at -20 ° C. A composition for cell regeneration.
6. The method of claim 5,
The composition is 100㎍ / ㎖ or less concentration, characterized in that it does not affect the viability of human-derived HaCat cells, a composition for skin cell regeneration containing phyllophthora as an active ingredient.
6. The method of claim 5,
When the composition is treated at a concentration of 100 μg/ml in a culture solution of human-derived HaCaT cells, it has a higher regeneration rate compared to the group without the addition of the complex extract when cells are damaged by peroxide. A composition for skin cell regeneration.
6. The method of claim 5,
When the composition is treated at a concentration of 100 μg/ml in a culture solution of human-derived HaCaT cells, the collagen regeneration rate from UV-B-induced collagen damage is higher than that of the group without the addition of the complex extract. A composition for skin cell regeneration containing as an active ingredient.
6. The method of claim 5,
The composition, when treated at a concentration of 100㎍ / ㎖ in the culture medium of human-derived HaCaT cells, characterized in that the collagen regeneration rate from UV-B-induced collagen damage in human-derived HaCaT cells is increased. A composition for skin cell regeneration containing
6. The method of claim 5,
The composition is characterized in that when the human-derived HaCaT cell culture solution is treated at a concentration of 100 μg/ml, the production of elastin and collagen protein increases from UV-B-induced collagen damage in human-derived HaCaT cells. A composition for skin cell regeneration containing plantain as an active ingredient.
6. The method of claim 5,
The composition, characterized in that it has the effect of reducing epidermal dermatoma from skin damage caused by UV-B, inhibiting the infiltration of mast cells and protecting skin collagen, a composition for skin cell regeneration containing Psyllium as an active ingredient.
6. The method of claim 5,
The composition is characterized in that when vitamin C is added, the collagen synthesis rate is increased, the composition for skin cell regeneration containing Psyllium as an active ingredient.

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