KR20190020530A - Cosmetic Composition which comprises Diplectria barbata Extract as an active ingredient - Google Patents

Abstract

The present invention relates to cosmetic, quasi-drug, food, and pharmaceutical compositions containing a Diplectria barbata extract as an active ingredient and a cosmetic composition preparing method. According to the present invention, the Diplectria barbata extract activates cell growth in wounded areas by promoting the phosphorylation of proteins related to cell differentiation and growth such as PERK and pPKB as well as reducing the expression of MMP-3 accelerating skin aging while increasing synthesis of the extracellular matrix such as collagen or elastin, thereby having wound healing and regeneration capabilities. Furthermore, the Diplectria barbata extract can be used in cosmetic, quasi-drug, food, and pharmaceutical products for antioxidant, anti-aging, wound healing purposes by increasing the expression of antioxidant proteins and antioxidant enzyme activities.

Description

[0001] The present invention relates to a cosmetic composition comprising Diplectriptan barbata extract as an active ingredient,

The present invention relates to a cosmetic composition, a quasi-drug composition, a food composition, a pharmaceutical composition, and a method for producing the cosmetic composition containing Diplectria barbata extract as an active ingredient.

In response to the well-being trend of eating well and living well, cosmetics are becoming an indispensable element, not an option, but a high value-added industry. As a result, various materials, including natural products, microorganisms, protein cultures and stem cell- . In recent years, there has been a problem caused by genetic modification of cosmetic ingredients and environmental hormones, and there is a growing demand for functional cosmetics for natural substances that can be safely used without being harmful to human body in the long term.

Plants have long been a source of a variety of natural materials that help human welfare. Most of these substances are secondary metabolites and are used for medicines, food additives, raw materials for cosmetics, fragrance, pigment and so on. Despite the synthesis of numerous new compounds due to advances in organic synthesis, plants are still recognized as the source of new materials. Plants are important raw materials considering that there are thousands of new substances with new chemical structure found in plants every year, and many of them have physiological activity.

 Diplectria barbata (Wall. Ex CB Clarke) Franken & Roos. (Hereinafter Diplectria barbata) is native to Vietnam and its scientific name to date is Melastomataceae family It is a plant. Recently, research on the efficacy of natural products has been actively conducted worldwide, but studies on the components, efficacy and the like of Diprecetria barbata have not been conducted yet.

The present inventors have searched for natural products having antioxidative, anti-aging, or wound regenerating effects and less side effects, and found that the extract of Diplectria barbata has antioxidant, anti-aging and wound regeneration effects, Completed.

Accordingly, an object of the present invention is to provide a cosmetic composition containing an extract of Diprecetria barbata as an active ingredient, a quasi-drug composition for improving skin condition, a food composition for antioxidant or anti-aging, a pharmaceutical composition for wound regeneration or skin regeneration, Extracting barbata; And a method for producing a cosmetic composition comprising the same.

In order to achieve the above object, the present invention provides a cosmetic composition comprising Diplectria barbata extract as an active ingredient.

In addition, the present invention provides a quasi-drug composition for improving skin condition, which contains Diplectria barbata extract as an active ingredient.

In addition, the present invention provides a food composition for antioxidant or anti-aging comprising an extract of Diplectria barbata as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for wound regeneration or skin regeneration, which comprises an extract of Diplectria barbata as an active ingredient.

The present invention also relates to a method for producing a microorganism, comprising the steps of: extracting Diplectria barbata; And a method for producing the cosmetic composition.

The extract of Diplectria barbata according to the present invention promotes phosphorylation of pERK and pPKB proteins, which are cell differentiation and growth related proteins, to activate cell proliferation at the wound site, and to inhibit the extracellular matrix such as collagen or elastin Increases synthesis and reduces the expression of MMP-3, which promotes skin aging, and has wound healing and regenerating efficacy. Furthermore, the antioxidant enzyme activity and the expression of antioxidant protein can be increased and used for cosmetics, quasi-drugs, foods, medicines and the like for the purpose of antioxidation, anti-aging or wound regeneration.

Figure 1 is the leaf and tree shape of Diplectria barbata.
FIG. 2 is a graph showing the results of MTT analysis of Diplectria barbata extract treated with human fibroblast cell line at different concentrations. FIG.
FIG. 3 is a view showing a result of microscopic observation of wound regeneration progression after treatment of Diplectria barbata extract with concentration in human fibroblast cell line. FIG.
FIG. 4 is a graph showing the results of confirming the changes in the amounts of pERK and pPKB-S473 protein, which are cell differentiation and growth-related proteins, after treatment of Diplectria barbata extract with concentration in human fibroblast cells.
FIG. 5 is a graph showing the results of the treatment of Diplectria barbata extract with concentrations of human fibroblast cell line extract, collagen, elastin and MMP, an extracellular matrix active protein, -3 < / RTI > protein.
FIGS. 6 to 9 are graphs showing the antioxidant activity of extracts obtained by measuring the expression levels of antioxidant enzymes after treatment of Diplectria barbata extract with concentrations in human fibroblast cells. FIG. 6 is a graph showing the antioxidant activity of the extract-treated group as compared with the comparative group BHT (butylated hydroxyl toluene). FIG. 7 shows the results of Western blot analysis of the expression levels of the antioxidative enzymes HO-1 and NRF2 (GAPDH is used as a control group), and FIG. 8 is a graph showing the quantification thereof. FIG. 9 is a graph showing the result of confirming the total antioxidant capacity according to the concentration of the extract. FIG.
FIGS. 10 to 12 show results obtained by treating Diplectria barbata extract by concentration at wound sites of wound model mice. FIG. 10 is a diagram showing the result of visually checking the extent of wound regeneration in a mouse. FIG. FIG. 11 is a graph showing the results of visually checking the wound regeneration degree of the mouse according to the concentration of the extract, and FIG. 12 is a graph showing quantification of the extent of wound area.
FIG. 13 is a view showing a result of microscopic observation of the recovery of the skin layer caused by treatment of Diplectria barbata extract at the wound site by concentration. FIG.
FIG. 14 is a graph showing the results of confirming the amount of expression of collagen, elastin, and MMP-3 as a result of treating Diplectria barbata extract by concentration at the wound site. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides a cosmetic composition comprising an extract of Diplectria barbata as an active ingredient.

In the present invention, Diplectria barbata has the full name Diplectria barbata (Wall. Ex C. B. Clarke) Franken & Roos. And the scientific name is not yet known. The specific ingredients and efficacy of Diprecetria barbata have not been known yet. In the present invention, the antioxidative, anti-aging and wound regeneration effects of Diprecetria barbata were first confirmed.

In the present invention, the cosmetic composition may be an antioxidant or anti-aging composition.

In the present invention, " antioxidant " means an oxidation-inhibiting action. Although the human body has a balance of prooxidant and antioxidant, when the balance is imbalanced due to various factors and it is inclined toward the promotion of oxidation, oxidative stress is induced Resulting in potential cellular damage and pathological disease. Reactive oxygen species (ROS), which is a direct cause of oxidative stress, is unstable and highly reactive. It reacts easily with various bio-materials, attacks irreversible damage to cells and tissues, Cytotoxicity and carcinogenesis. Active nitrogen species (RNS) such as NO, HNO ₂ , and ONOO ^- are produced by the immune response of macrophage neutrophils and other immune cells during inflammatory reaction, and ROS is also produced. Such active oxygen oxidizes and destroys cells in the body, thereby exposing them to various diseases.

In the present invention, the term " anti-aging " is intended to mean skin protection and skin condition improvement, skin whitening, prevention or improvement of skin aging and wrinkles, pore contraction and reduction, skin protection and alleviation of skin inflammation, Skin barrier function improvement, skin irritation mitigation, skin cell proliferation and regeneration ability, antioxidant ability, and collagen synthesis promoting ability.

In the present invention, " containing as an active ingredient " means a cosmetic composition containing an effective amount of such a degree as to exhibit skin-improving effect upon antioxidation or anti-aging. Therefore, the Difflexible Barbara extract of the present invention can contribute to health promotion by achieving antioxidative and anti-aging effects.

In the present invention, the Diplectria barbata extract may be one or more extracts selected from the group consisting of leaves, stems, roots and outbreaks of plants (whole plants), preferably leaves or stems But are not limited thereto.

In the present invention, " Diplectria barbata extract " means an extract obtained by extracting Diplectria barbata. For the purpose of the present invention, the Dipolectria barbata extract is a Dipectria barbata extract, The rumen may be extracted with water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, a hydrocarbon solvent or a mixed solvent thereof, preferably methanol or ethanol, more preferably methanol It may be extracted. In addition, in the present invention, the extract may contain at least one of an extract obtained by the extraction treatment, a diluted solution or concentrate of the extract, a dried product obtained by drying the extract, or any of these adjusted products or purified products.

In the present invention, the above extract may be used by concentration or dilution, and a distillate of the extract may be used.

In addition, the present invention provides a quasi-drug composition for improving skin condition, which contains Diplectria barbata extract as an active ingredient.

In the present invention, the term " quasi-drug " refers to a substance, a machine or an apparatus, which is used for diagnosing, treating, alleviating, treating or preventing a disease of a human or an animal and a pharmacological effect Means goods, machinery, or apparatus other than those not used for the purpose of reducing the value of goods.

The quasi-drug composition may be a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment or a filter filler. In addition, the quasi-drug composition may include external preparations for skin and personal hygiene products. The external preparation for skin may be prepared and used preferably in the form of an ointment, a lotion, a spray, a patch, a cream, a powder, a suspension, a gel or a gel, But are not limited to, soap, cosmetics, wet tissues, tissue paper, shampoo, skin creams, face creams, toothpaste, lipstick, perfume, make-up, foundation, ball touch, mascara, eye shadow, sunscreen lotion, , Air freshner gel, cleaning gel, and the like.

In addition, the present invention provides a food composition for antioxidant or anti-aging comprising an extract of Diplectria barbata as an active ingredient.

In the present invention, the " food composition " may include all conventional foods, and may be used in combination with terms known in the art such as functional foods, health functional foods, and the like.

When the Difflexible Barbara extract of the present invention is used as a food additive, the Difflexible Barbara extract can be used as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment), and may further include a food-acceptable food-aid additive. Since the composition of the present invention contains an extract derived from a natural substance as an active ingredient, there is no problem in terms of stability, so there is no great limitation on the amount of the mixture.

In the present invention, the term " functional food " means a food prepared and processed by using a raw material or a component having functionality useful to the human body according to Law No. 6727 on health functional foods. Structure and function of the nutrient to control or physiological effects, such as to obtain a beneficial effect for health is intended to eat.

In the present invention, the term " health functional food " refers to a food prepared by processing a specific ingredient as a raw material for the purpose of health assisting or by extracting, concentrating, refining, mixing, or the like a specific ingredient contained in the raw material .

There is no limitation on the kind of food in which the extract of the present invention can be used. In addition, the food composition containing the diplexible barbata extract of the present invention as an active ingredient can be prepared by blending known additives with other appropriate auxiliary ingredients that may be contained in the food according to the selection of a person skilled in the art. Examples of foods that can be added include dairy products, such as meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component.

Examples of foods that can be used in the present invention include special nutritional foods such as crude oil, milk, baby food, meat products, fish meat products, tofu, mushrooms, noodles such as ramen noodles, (Such as soy sauce, doenjang, kochujang, mixed potatoes), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickles ), Beverages (such as fruit, vegetable beverages, beverages, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a drink, it may contain various sweeteners, flavors, or natural carbohydrates as an additional ingredient such as ordinary beverages. In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

In addition, the present invention provides a pharmaceutical composition for wound regeneration or skin regeneration, which comprises an extract of Diplectria barbata as an active ingredient.

The Diprectactia barbata extract may be active in wound regeneration or skin regeneration, but is not limited thereto.

The dipeptry barbata extract of the present invention has an effect of increasing the reconstitution and synthesis of extracellular matrix such as collagen and elastin and lowering the expression of MMP-3 which accelerates skin aging by decomposing the substrate protein. Therefore, It can be used for skin regeneration.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In addition, the pharmaceutical compositions may further comprise suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions. Preferably, the composition is in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze- The composition may have any one of formulations selected from the group consisting of ointments, creams, patches, cataplasms, pastes, sprays, skin emulsions, skin suspensions, transdermal patches, drug-containing dressings and suppositories, It may, but is not limited to, various forms of parenteral administration.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In the present invention, the preferable dosage of the pharmaceutical composition varies depending on the condition and body weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for more effective prevention or treatment, it is preferable to administer the diplex trita barbata extract of the present invention at a dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day, It may be administered once or divided into several doses.

The present invention also relates to a method for producing a microorganism, comprising the steps of: extracting Diplectria barbata; And a method for producing the cosmetic composition.

In the present invention, the composition may be an antioxidant or anti-aging cosmetic composition.

In the present invention, the extraction may be one or more extracts selected from the group consisting of leaves, stems, roots and outposts of Diprecetia barbata (whole plant), preferably an extract of leaves or stems , But is not limited thereto.

It is also within the scope of the present invention that the above-described contents of the present invention are applied mutatis mutandis to each other, and those skilled in the art will make appropriate changes. Hereinafter, the present invention will be described in detail with reference to Experimental Examples and Examples, but the scope of the present invention is not limited to the following Experimental Examples and Examples.

Statistical analysis was performed using ± SD, and p <0.05 ( ^* ), p <0.01 ( ^** ) was recorded.

Example  1. Experimental preparation

1-1. Diplexia Barbara ( Diplectria barbata ) Preparation of extract

The leaves and stems of Diplectria barbata (Wall. Ex CB Clarke) Franken & Roos. (Hereinafter referred to as Diplectria barbata), a Vietnamese origin belonging to the Melastomataceae family, Was supplied and used by the Center for Biotechnology Research. Specifically, Dipreparia barbata was immersed in methanol (Methyl alcohol 99.9%, HPLC grade) at 45 ° C, sonicated for 15 minutes, and allowed to stand at room temperature for 2 hours for 10 times a day for a total of 3 days To obtain an extract solution. Thereafter, the extract was filtered, concentrated and dried, and stored in a low-temperature room (-4 ° C). The leaves and stems of Diprecetria barbata are shown in Fig. In the following experiments, leaf and stem extracts of Diprecetria barbata (hereinafter referred to as 'extract') were used.

1-2. Human skin Fiber  Cell culture

Human dermal fibroblasts, NHDF, were cultured in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum and 10% antibiotics at 37 ° C in a 5% CO ₂ incubator.

Example  2. Diplexia Barbara  Measurement of cell survival rate by extract treatment

Human fibroblast cells were treated with extracts at different concentrations and cell viability was measured by MTT assay.

NHDF, a human fibroblast cell line, was divided into three groups (1 × 10 ³ cells / well) in 96-well plates divided into non-treated group and treated group. After 24 hours of incubation, 100 .mu.l of new media and 0, 10, 25, and 50 .mu.g / ml of fresh media were further treated with dipeptia barbata treatment group (concentration group) for 24 hours. Twenty-four hours later, 10 μl of Ez-Cyto (MTT) reagent was added to 90 μl of fresh media per well, followed by reaction for 30 minutes. After incubation, the absorbance was measured at 450 nm (reference 650 nm) using an enzyme-linked immunosorbent assay plate reader (ELISA) and plotted. The results are shown in Fig.

As shown in Fig. 2, the cell survival rate was maintained even when the extract was treated to 50 占 퐂 / ml, confirming that the extract of the present invention was not cytotoxic.

Example  3. Diplexia Barbara  Measurement of wound regeneration effect by extract treatment

Human fibroblast cell lines were treated with extracts at different concentrations, and cell wound regeneration experiments were performed in general anti - aging experiments.

NHDF cell lines cultured according to the method described in Example 1-2 were placed in 4 wells at 1 × 10 ⁶ cells on a 6-well plate. The next day, when the cells grew by about 70%, the cells were scratched by scraping the cell surface at equal intervals with a 10 [mu] l tip. Thereafter, 2 ml of fresh media was added to the control wells, and 2 ml of the medium was added to each of the remaining 3 wells at a concentration of 1 μg / ml, 25 μg / ml and 50 μg / ml, , And observed with a microscope. FIG. 3A shows the results of photographing on a microscope, and 5 straight lines were drawn between the dotted lines A in FIG. 3 (the wound area), and the average value of the five numerical values was digitized and quantified. 3B.

As shown in Fig. 3 (A), it was confirmed that wound closure was excellent as the concentration of the extract treated at the wound site increased. In addition, as shown in Fig. 3B, it was confirmed that when the extract was treated, the recovery rate of wound area was significantly higher than that of the control group at all concentrations.

Example  4. Diplexia Barbara  Measurement of cell differentiation and growth-related protein expression by extract treatment

Anti-pKK, anti-PKB, anti-pPKB (S473), and anti-pPKB (anti-pKK) were used to treat the human fibroblast cell line by concentration and to measure the expression of the cell- Western blotting was performed using GAPDH antibody.

The cultured NHDF cell lines were plated in a total of 4 wells on a ⁶ -well plate at 1 × 10 ⁶ cells / well. On the next day, 2 ml of new medium was added to the control wells in the next day, and the extracts were added to 2 ml of the media in the remaining 3 wells at a concentration of 1 / / ml, 25 / / ml and 50 / / After removing the media and washing with 1X PBS, the cells were washed with 20 mM Tris-HCl (pH 7.5), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% NP40, 10 mM beta-glycerophosphate, Cells were lysed with lysis buffer made up of sodium vanadate, 10 mM PNPP, 5 mM PMSF and 10 μg / ml leupeptin. The concentration of the protein dissolved in the Bradford protein assay (Bio-Rad, Hercules, Calif.) Was measured and then 10 μg was quantitated, mixed with Laemmli sample buffer and concentration, and loaded on SDS-PAGE. Then, the cells were transferred, blocked with milk, washed with TBS-T, and incubated overnight with the primary antibody. The next day, after washing with TBS-T, the Rabbit secondary antibody was incubated for 1 hour, washed three times with TBS-T for 20 minutes, and developed with a film. The primary antibodies were collagen (ab138492) -AbCam, Elastin (sc-374638), MMP-3 (sc-21732), pERK (sc-7383), ERK GAPDH (# 5174s), pPKB (# 3787s), PKB (# 9272s) and HO-1 (# 5853) -Cell Signaling Tech were used and mouse and rabbit-Komabiotech were used as secondary antibodies. The results are shown in Fig.

As shown in Fig. 4A, the extracellular signal-regulated kinases (ERKs) and AKT / PKB (protein kinases), which are sub-proteins of MAPK, which are well known for their differentiation and invasion functions in dermal fibroblasts, B) protein phosphorylation, and the amount of phosphorylated protein was increased as the concentration of the extract increased. The quantification result is shown in Fig. 4B. Thus, it was confirmed that Diprecetria barbata extract can actively induce cell proliferation by ERK and PKB signal transduction and thus can be effectively used for wound regeneration.

Example  5. Diplexia Barbara  Collagen, elastin and MMP -3 expression level measurement

The human fibroblast cell line was treated with the extracts at different concentrations and the expression levels of collagen, elastin and matrix metalloproteinase-3 (MMP-3), an extracellular matrix active protein, Blot.

Crushing was a human fibroblast cell lines cultured in each 3 X 10 ⁵ on a 6-well plate. On the next day, the extracts were treated with the extracts at different concentrations (0, 1, 10, 25, 50 μg / ml) and cultured for 24 hours. After washing with 1X PBS, the protein was extracted and the protein expression level was measured by Western blotting. The results are shown in Fig.

As shown in Fig. 5A, it was confirmed that the expression levels of collagen and elastin, which are extracellular matrix active genes, were increased depending on the concentration of the extract treated. On the other hand, the expression level of MMP-3, an intercellular protease, decreased with concentration. The quantification result thereof is shown in Fig. 5B. As a result, the extract of the present invention increased the reconstitution and synthesis of the extracellular matrix to facilitate wound regeneration and greatly inhibited the expression of MMP-3, an intercellular zygotic enzyme.

Example  6. Diplexia Barbara  Antioxidant efficacy by extract treatment

6.1 DPPH  Antioxidant experiments using

The compound DPPH (1,1-diphenyl-2-picryl hydrazyl) generates free radicals in ethanol. The amount of free radicals generated by mixing the extract of the present invention and DPPH at a certain ratio is reduced to some extent by a spectrophotometer , And the extracts were tested for antioxidant activity by indirectly measuring the antioxidative activity of the samples.

As a comparative group, BHT (Butylated Hydroxyl Toluene), which is a standard substance used in the experiment using DPPH, was used and the antioxidant ability was measured by comparing the activity as an antioxidant with BHT. BHT was dissolved in ethanol and then added to the 96-well plate by concentration. 100 μl of 0.2 mM 1-diphenyl-2-picrylhydrazyl (DPPH) prepared in ethanol was added to each well, and 100 μl of the extract sample was added. After allowing to stand at room temperature for 30 minutes, absorbance was measured at 517 nm. After measuring the absorbance of the test group and the control group, the degree of antioxidant activity was expressed as a percentage based on the absorbance intensity of the control group using the following equation. The results are shown in Fig.

As shown in Fig. 6, it was confirmed that the extract had an excellent antioxidative effect comparable to that of the comparative BHT, as compared with the comparative group. In addition, it was confirmed that the antioxidant ability was increased as the concentration of the extract increased.

6.2 NRF2  And HO-1 expression level

To examine the antioxidant activity of extracts, we measured the expression levels of NRF2 and antioxidant enzyme HO-1, which are known to protect cells against inflammation and harmful stimuli by increasing the expression of antioxidant enzymes under oxidative or stress conditions Respectively. Crushing was a human fibroblast cell lines cultured in each 3 X 10 ⁵ on a 6-well plate. On the next day, extracts were treated with different concentrations (0, 10, 25, 50 μg / ml) on new media and cultured for 24 hours. After washing with 1X PBS, the protein was extracted and the protein expression level was measured by Western blotting. The results are shown in Fig. The results of quantification are shown in FIG.

As shown in FIGS. 7 and 8, it was confirmed that the higher the concentration of the extract, the greater the expression amount of NRF-2 and HO-1. Therefore, it was confirmed that the extract of the present invention can protect cells and induce antioxidative effect.

6.3 Total antioxidant capacity ( Total antioxidant ability ) analysis

The cultured NHDF cell line was plated on a 6-well plate at 1 × 10 ⁶ cells / well. On the next day, extracts were added to each 1 μg / ml, 25 μg / ml and 50 μg / ml concentrations, and cultured for 24 hours. After the trypsin treatment, cells were collected with 1 × PBS and centrifuged at 1,000-2,000 × g for 10 minutes at 4 ° C. After removal of PBS, 100 μl of cold 60 mM NaOH was added, the cells were pulverized using a needle, and centrifuged at 1,500 × g at 4 ° C. for 4 minutes. The supernatant was transferred to a tube and placed in ice, and the total antioxidant capacity was analyzed with supernatant. This was done according to the protocol of total antioxidant capacity assay (cell-biolabs Cat. STA-360). Specifically, 20 μl of the Uric acid standard and each sample were placed in 3-wells of a 96-well plate, and 180 μl of 1 × Reaction Buffer was added thereto. The mixture was carefully mixed. After measuring the absorbance at 490 nm, 50 Cop of ionic reagent (Copper Ion Reagent) was added to each well and shaken in a shaker at room temperature for 5 minutes. After adding 50 1 of 1X Stop Solution to each well, the plate was again measured at 490 nm and calculated according to the protocol formula. The results are shown in Fig.

As shown in FIG. 9, it was confirmed that the total antioxidant capacity was increased as the concentration of the extract increased.

The DPPH antioxidant activity test, the antioxidant protein expression assay, and the total antioxidant activity analysis showed that the extract of the present invention had an antioxidative effect.

Example  7. In the wound model mouse Diplexia Barbara  Identification of wound regeneration effect of extract

7.1 Mouse Wound healing assay

In order to confirm the cell regeneration efficiency of the extracts observed in the cells in vivo , the following experiment was conducted. B / J male The hair of the mouse was hairdressed, and immediately after 4 identical sized lesions were removed with biopsy punch, the control group was applied with water only and the remaining three lesions were treated with 10 / / ml, 25 / / Ml, and then taken daily for 6 days. The experimental method is shown in Fig. 10, the wound regeneration is visually observed in Fig. 11, and the results are shown in Fig.

As shown in FIG. 11 and FIG. 12, it was confirmed that the wound treated with the extract was more active than the control. In addition, it was confirmed that as the concentration of the extract increased, the wound regeneration became more active and the wound area decreased in speed.

7.2 Mouse skin immunostaining

The skin of the control group and extract group treated with concentration was stained with hematoxylin. In addition, the skin tissue of the injured area of the mouse was fixed with 10% neutralized formalin for 16 hours, and fixed on paraffin. The tissue was cut to a thickness of about 5 mm and then stained with H & E stain and then stained with antibody Anti-collagen (ab138492) -AbCam, Anti-MMP3 (sc-21732) and Anti-Elastin (sc-374638) Respectively. The results are shown in Fig. 13 and Fig.

As shown in Fig. 13, it was confirmed that the skin layer treated with the extract in concentration was thicker than the skin without extract. This study confirmed the recovery effect of skin layer by skin regeneration by extract.

In addition, as shown in Fig. 14, the expression level of MMP-3, an intercellular protease, was decreased in skin tissues in which extracts were applied to extracts of collagen and elastin in mouse skin. Respectively. It is confirmed that this is a result corresponding to the cell test result. The results showed that the extract increased the expression of collagen and elastin, which are extracellular matrix active genes, not only in vitro but also in vivo , and decreased the expression of MMP-3 which promotes skin aging by degrading such substrate proteins , Wound healing and wound regeneration. Therefore, it has been confirmed that the diplex trita barbata extract of the present invention can be effectively used as antioxidants, anti-aging cosmetic compositions and wound regeneration compositions.

As described above, the Diprepectia barbata extract of the present invention is effective for increasing cell proliferation, reconstruction and synthesis of extracellular matrix, increasing antioxidant enzyme activity and antioxidant protein expression, and degrading matrix proteins to promote skin aging. -3 in the wound healing, wound regeneration, antioxidant and anti-aging effects.

Hereinafter, the antioxidant or antioxidant food composition containing the diplexetar barbata extract of the present invention as an active ingredient and the pharmaceutical composition for wound regeneration or skin regeneration will be described, but the present invention is not limited to specific examples .

Formulation example  1: Manufacture of food preparation

1-1: Manufacture of health food

Diprecetria barbata extract 100 mg

Vitamin mixture quantity

70 g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 g of vitamin B12

Vitamin C 10 mg

Biotin 10 g

Nicotinic acid amide 1.7 mg

Folate 50 g

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

1-2: Manufacture of health drinks

Diprecetria barbata extract 100 mg

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3 g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered to obtain a sterilized container, which was sealed and sterilized, Used in the manufacture of health beverage compositions.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Formulation example  2: Preparation of pharmaceutical preparations

2-1: Sanje  Produce

Diprecetria barbata extract 200 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

2-2: Preparation of tablets

Diprecetria barbata extract 100 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

2-3: Preparation of capsules

Diprecetria barbata extract 100 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

2-4: Preparation of injection

Diprecetria barbata extract 100 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ .2H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

2-5: Liquid  Produce

Diprecetria barbata extract 200 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the conventional liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto to adjust the whole volume to 100 ml. The solution was filled in a brown bottle and sterilized, .

Claims (13)

A cosmetic composition comprising an extract of Diplectria barbata as an active ingredient.
2. The cosmetic composition according to claim 1, wherein the Difflexa barbata is Diplectria barbata (Wall. Ex CB Clarke) Franken & Roos.
The cosmetic composition according to claim 1, wherein the composition is an antioxidant or anti-aging cosmetic composition.
The cosmetic composition according to claim 1, wherein the extract is extracted from leaves or stems of Diprecetia barbata.
The cosmetic composition according to claim 1, wherein the extract is extracted with water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, a hydrocarbon solvent or a mixed solvent thereof.
A quasi-drug composition for improving skin condition containing an extract of Diplectria barbata as an active ingredient.
The quasi-drug composition for improving skin condition according to claim 6, wherein the skin condition improvement is skin regeneration, skin elasticity improvement, skin wrinkle prevention or improvement, skin inflammation improvement or wound regeneration.
An antioxidant or anti-aging food composition comprising an extract of Diplectria barbata as an active ingredient.
A pharmaceutical composition for wound regeneration or skin regeneration, comprising an extract of Diplectria barbata as an active ingredient.
10. The pharmaceutical composition according to claim 9, wherein the extract increases the expression of collagen or elastin and reduces the expression of MMP-3.
Extracting Diplectria barbata; &Lt; / RTI &gt;
12. The method for producing a cosmetic composition according to claim 11, wherein the composition is a cosmetic composition for antioxidation or anti-aging.
12. The method of claim 11, wherein the extraction is performed from a leaf or stalk of Diprecetia barbata.

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