WO2022250313A1

WO2022250313A1 - Composition for moisturizing skin, promoting skin regeneration, and treating wounds, comprising aloe vera flower extract, or aloe vera flower extract and aloe vera polysaccharides, as active ingredient(s)

Info

Publication number: WO2022250313A1
Application number: PCT/KR2022/006170
Authority: WO
Inventors: 심규석; 조은애; 김선여; 술타나라지아; 강민철
Original assignee: 주식회사 유니베라; 가천대학교 산학협력단
Priority date: 2021-05-27
Filing date: 2022-04-29
Publication date: 2022-12-01

Abstract

The present invention relates to a composition for moisturizing the skin, promoting skin regeneration, and treating wounds, comprising an Aloe vera flower extract as an active ingredient, or an Aloe vera flower extract and Aloe vera polysaccharides as active ingredients, and more particularly, to a composition for moisturizing the skin, regenerating the skin, or treating skin wounds, comprising an Aloe vera flower extract or a compound isolated therefrom as an active ingredient, or an Aloe vera flower extract and Aloe vera polysaccharides as active ingredients.

Description

Composition for moisturizing skin, promoting regeneration and treating wounds containing an aloe vera flower extract or an aloe vera flower extract and an aloe vera polysaccharide as active ingredients

The present invention relates to a composition for moisturizing skin, promoting regeneration, and treating wounds, containing an Aloe vera flower extract or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients, and more particularly, an Aloe vera flower extract or It relates to a composition for skin moisturizing, skin regeneration or skin wound treatment containing a compound isolated therefrom, or an aloe vera flower extract and an aloe vera polysaccharide as active ingredients.

The skin is a tissue that covers the outermost part of the human body and serves as a primary defense function to protect the body from external chemical and physical shocks by protecting the human body and functioning as a barrier. The skin is composed of three layers: the epidermis, the dermis, and the subcutaneous layer. When the skin is aged by ultraviolet rays, stress, etc., or damaged by wounds, its function is weakened, and at this time, the skin regeneration process begins. The skin regeneration process begins with an acute inflammatory response, then undergoes contraction of connective tissue through proliferation, migration, and proliferation of epidermal cells, generation of new blood vessels, and synthesis of collagen. At this time, migration and proliferation of keratinocytes and dermal fibroblasts, which occupy more than 90% of the epidermis, not only provide wound healing and skin regeneration, but also play an important role in promoting skin elasticity and forming a basement membrane by inducing collagen synthesis.

In addition, intracellular lipids and natural moisturizing factors in the epidermal layer help to maintain skin moisture by preventing epidermal water loss. Damage to the stratum corneum due to lack of moisture and poor environmental conditions results in dry skin, which makes it prone to various skin diseases such as dermatitis, itching, psoriasis and aging.

Based on molecular signaling pathways related to skin hydration, several types of epidermal proteins have been reported to be involved in regulating skin barrier function. Hyaluronan (HA, Hyaluronan), which is regulated by hyaluronan synthase (HASes) and degrading enzymes (hyaluronidase (HYALs)), is a skin moisturizing regulator. Profilaggrin, aquaporins (AQP), and involucrin contribute to the strong physical and permeation barrier function of the skin. All of these play an important role in maintaining skin hydration and skin integrity.

Natural sourced moisturizers have traditionally been used to treat a number of skin conditions. Aloe vera, centella asiatica, seaweed, and camellia are plants known for their moisturizing properties. Aloe vera (synonymous with Aloe barbadensis Mill.), belonging to the Asphodelaceae family, is a perennial shrub with leaves surrounding the stem in a rose-like pattern, with a central flower growing. Aloe vera has been reported for its moisturizing, anti-inflammatory, antibacterial, and wound healing properties and has been used in various cosmetics.

Aloe is a plant belonging to the genus Aloineae of the family Liliaceae, which lives in the tropics, and is used worldwide for food and medicine. Among them, the most well-known material is Aloe vera, and juice or gel processed from its leaves is one of the representative health functional foods, and is also one of the natural materials widely used as pharmaceutical or cosmetic materials. Aloe vera gel is about 99-99.5% water and about 0.5-1.0% solids, of which polysaccharides are 55%, sugars 17%, minerals 16%, proteins 7%, fats 4%, and phenols 1%. has been The polysaccharide that accounts for a large portion of the solid content of the aloe vera gel is the main physiologically active component of the aloe vera gel, and typically includes acetylated mannan (or acemannan).

Most studies, including human application tests, have been conducted on aloe vera leaf extract, gel, and specific ingredients. On the other hand, there is little literature on the flower of aloe vera, and research has mainly focused on identifying chemical components with antioxidant and antibacterial properties.

In addition, as a prior art using aloe vera, Korean Patent Publication No. 10-2020-0140520 describes the respiratory disease improvement effect of aloe vera gel, and Korean Patent Publication No. 10-2016-0086809 discloses the ratio of aloe vera extract The treatment effect of alcoholic chronic fatty liver is disclosed, and Korean Patent Registration No. 10-1520643 only discloses the treatment effect of aloe vera gel on diabetes, but nothing is known about its activity against aloe vera flowers.

Accordingly, the present inventors have tried to develop a natural product-derived moisturizer and skin wound treatment, and as a result, confirmed the skin moisturizing function of an aloe vera flower extract and a compound derived therefrom. In addition, it was confirmed that the aloe vera flower extract significantly increased the migration of dermal fibroblasts, which is an important feature in wound healing, and did not show cytotoxicity even at high concentrations. In addition, the mixture of aloe vera flower extract (AVF) and aloe vera polysaccharide (PAG) increases the expression of MFAP4, thereby increasing the proliferation, migration, and especially ECM (Extracellular) of NHDF (normal human dermal fibroblast) and HaCaT (human epidermal keratinocytes) cells. matrix), as well as increasing other extracellular components related to MFAP4, such as fibrillin, collagen, elastin, TGF β and α-SMA expression, also synergistically wound compared to AVF or PAG, respectively. It was confirmed that the treatment effect was shown.

Therefore, an aloe vera flower extract and a compound derived therefrom can be usefully used as an active ingredient in a skin moisturizing composition, and an aloe vera flower extract or an aloe vera flower extract and an aloe vera polysaccharide can be used as a composition for treating skin wounds or regenerating skin By revealing that it can be usefully used as an active ingredient of, the present invention was completed.

An object of the present invention is to provide a composition for moisturizing skin, promoting regeneration and treating wounds, containing an Aloe vera flower extract or a compound derived therefrom, or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients. .

In order to achieve the above object, the present invention provides a cosmetic composition for skin moisturizing or skin regeneration containing an Aloe vera flower extract or a compound isolated therefrom, or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients to provide.

In addition, the present invention provides a quasi-drug for skin moisturizing or skin regeneration containing an Aloe vera flower extract or a compound isolated therefrom, or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In addition, the present invention provides an external skin preparation for skin moisturizing or skin regeneration, containing an Aloe vera flower extract or a compound isolated therefrom, or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In addition, the present invention provides a food composition for moisturizing or regenerating skin, containing an Aloe vera flower extract or a compound isolated therefrom, or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In addition, the present invention provides a health functional food composition for skin moisturizing or skin regeneration, containing an Aloe vera flower extract or a compound isolated therefrom, or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In addition, the present invention provides a skin moisturizing or skin regeneration method comprising administering an effective amount of an aloe vera flower extract or a compound isolated therefrom, or a composition containing an aloe vera flower extract and an aloe vera polysaccharide as active ingredients. do.

In addition, the present invention provides an aloe vera flower extract or a compound isolated therefrom, or a composition containing an aloe vera flower extract and an aloe vera polysaccharide as active ingredients for use in skin moisturizing or skin regeneration.

In addition, the present invention provides a pharmaceutical composition for wound healing or skin regeneration containing an Aloe vera flower extract or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In addition, the present invention provides a wound treatment method comprising administering an effective amount of an Aloe vera flower extract or a composition containing an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In addition, the present invention provides an aloe vera flower extract for use in wound treatment, or a composition containing an aloe vera flower extract and an aloe vera polysaccharide as active ingredients.

The aloe vera flower extract and the compounds isolated therefrom of the present invention activate the PKC and MAPK signaling pathways to increase the expression of involucrin, which contributes to the skin barrier function, and the key skin moisturizing factors, AQP3 and filaggrin. , Since it increases the expression of HA and exhibits an effect of improving skin moisturizing reduced by skin damage caused by UVB, the aloe vera flower extract or a compound isolated therefrom is used in cosmetics, quasi-drugs, skin external agents, It can be usefully used as an active ingredient in food compositions and the like.

In addition, the aloe vera flower extract of the present invention significantly increases the migration of dermal fibroblasts, which is an important feature in wound healing, and the mixture of the aloe vera flower extract and aloe vera polysaccharide (PAG) increases the expression of MFAP4, thereby reducing NHDF (normal human dermal fibroblast), HaCaT (human epidermal keratinocytes) cell proliferation, migration, and especially ECM (Extracellular matrix) formation synergistically, as well as fibrillin, collagen, elastin, TGF β and α-SMA expression and It also increases other extracellular components related to the same MFAP4, and also shows a synergistic wound healing effect compared to each aloe vera flower extract and aloe vera polysaccharide (PAG), so the aloe vera flower extract, or the aloe vera flower extract and aloe A mixture of vera polysaccharide (PAG) can be usefully used as an active ingredient of a composition for skin wound treatment or skin regeneration targeting MFAP4 and related signal transduction pathways.

1A to 1D are diagrams confirming the active ingredient of an Aloe vera flower extract according to an embodiment of the present invention, FIG. 1A shows dried Aloe vera flowers, and FIG. 1B shows an example of the present invention. Aloe vera flower number (AFWE), 100% ethanol (AE), 50% ethanol (EE) extract and TLC patterns of functional components thereof are shown, Figure 1c shows the HPLC chromatogram of AFWE, Figure 1d is a standard active ingredient It shows the HPLC chromatogram of

2a to 2f confirm the effect of Aloe vera flower extract (AFWE) on skin hydration-related protein expression regulation in HaCaT cells according to an embodiment of the present invention. Figure 2b shows the results of confirming the cytotoxicity, and Figure 2b shows the results of confirming the hyaluronan content through ELISA analysis. Shows the results of confirming the expression of HAS1 (FIG. 2d), HYAL1 (FIG. 2e), and AQP3 (FIG. 2f) through.

3a to 3h show the effect of enhancing the expression of proteins related to skin barrier function in HaCaT cells of the aloe vera flower extract (AFWE) according to an embodiment of the present invention, and FIG. 3a shows involucrin through Western blot analysis (involucrin) and filaggrin expression confirmation results are shown, and FIGS. 3b to 3c show the results of involucrin (FIG. 3b) and filaggrin (FIG. 3c) expression confirmation through densitometry analysis, and FIG. 3d is qRT- PCR analysis shows the result of confirming the relative mRNA expression of involucrin, and FIG. 3e shows the expression of PCK and MAPK (pP38/p38, ERK1/2) expression of signaling pathway proteins involved in involucrin regulation through Western blot analysis. 3f to 3h show PCK and MAPK (pP38/p38, ERK1/2) confirmation results through densitometry analysis.

4a to 4g show the effect of modulating the expression of skin moisturizing factors, specifically proteins related to skin hydration and proteins related to skin barrier function, when HaCaT cells according to an embodiment of the present invention are treated with aloe vera flower extract (AFWE) after UVB irradiation As confirmed, Figure 4a shows the cytotoxicity confirmation result of AFWE under UVB conditions through MTT analysis, and Figure 4b shows involucrin, HAS1, HYAL1, profilagrin, Fig. 4c to Fig. 4g show the results of confirming the expression of filaggrin and AQP3. Involucrin (Fig. 4c), HAS1 (Fig. 4d), HYAL1 (Fig. 4c), HAS1 (Fig. 4d), HYAL1 (Fig. 4e), shows the result of confirming the expression of filaggrin (FIG. 4f) and AQP3 (FIG. 4g).

5 is a diagram confirming morphological changes when HaCaT cells according to an embodiment of the present invention are treated with Aloe vera flower extract (AFWE) after UVB irradiation.

6a to 6h show skin moisturizing factors, specifically skin moisturizing factors, in HaCaT cells treated with aloe vera flower extract (AFWE), 100% ethanol (AE), and 50% ethanol (EE) extracts according to an embodiment of the present invention. Fig. 6a shows the results of confirming the cytotoxicity of AFWE, AE, and EE through MTT analysis, and Fig. 6b shows the result of confirming the hyaluronan content through ELISA analysis. 6c shows the results of confirming the expression of involucrin, HAS1, HYAL1, profilagrin, filaggrin, and AQP3 through Western blot analysis, and FIGS. 6d to 6h show involucrin, HAS1, and HYAL1 through densitometric analysis. , filaggrin, AQP3 expression confirmation results are shown.

7a to 7h show the active ingredients contained in the aloe vera flower extract according to an embodiment of the present invention, specifically isoorientin (IO), vitexin (V), isovitexin (isovitexin, As a diagram confirming the expression effect of skin moisturizing factors in HaCaT cells of IV), FIG. 7a shows the results of confirming the cytotoxicity of IO, V, and IV through MTT analysis, and FIG. 7b shows the result of confirming the hyaluronan content through ELISA analysis. 7c shows the results of confirming the expression of involucrin, HAS1, HYAL1, proflagrin, filaggrin, and AQP3 through Western blot analysis, and FIGS. 7d to 7h show involucrin through densitometry analysis (FIG. 7d) , HAS1 (FIG. 7e), HYAL1 (FIG. 7f), filaggrin (FIG. 7g), and AQP3 (FIG. 7h) expression confirmation results are shown.

8a to 8g are diagrams showing molecular bond analysis between IO and skin moisturizing factors according to an embodiment of the present invention, FIG. 8a shows a bonding structure between IO and involucrin, and FIG. 8b shows a bond between IO and PKC 8c shows the bonding structure of IO and P38. Figure 8d shows the bonding structure of IO and ERK1, Figure 8e shows the bonding structure of IO and filaggrin, Figure 8f shows the bonding structure of IO and AQP3, Figure 8g shows the formation of the conjugate shown in Figures 8a to 8f represents the combined score.

9 is a diagram confirming the molecular bond analysis between IO and involucrin according to an embodiment of the present invention, showing the binding pocket between IO and involucrin (Fig. 9, left) and the bond formed between IO and involucrin. Indicates the type (Fig. 9, right).

10a and 10b are diagrams confirming the molecular binding analysis of IO and PKC and IO and P38 according to an embodiment of the present invention, and FIG. 10a is a binding pocket between IO and PKC (Fig. 10a, left) and between IO and PKC. Shows the type of bond formed in (Fig. 10a, right), Figure 10b shows the binding pocket of IO and P38 (Fig. 10b, left) and the type of bond formed between IO and P38 (Fig. 10b, right).

11a and 11b are diagrams confirming the molecular binding analysis of IO and ERK and IO and filaggrin according to an embodiment of the present invention, Figure 11a is a binding pocket of IO and ERK (Fig. 11a, left) and IO and ERK Shows the type of bond formed between (FIG. 11a, right), and FIG. 11b shows the binding pocket of IO and filaggrin (FIG. 11b, left) and the type of bond formed between IO and filaggrin (FIG. 11b, right) .

12a and 12b are diagrams confirming the molecular binding analysis of IO and AQP3 and IO and HYAL1 according to an embodiment of the present invention, and FIG. 12a is a binding pocket between IO and AQP3 (Fig. 12a, left) and between IO and AQP3 Shows the type of bond formed in (Fig. 12a, right), Figure 12b shows the binding pocket of IO and HYAL1 (Fig. 12b, left) and the type of bond formed between IO and HYAL1 (Fig. 12b, right).

13 is a diagram schematically showing a mechanism in which an aloe vera flower extract according to an embodiment of the present invention and an active ingredient contained therein improve skin moisturizing through a signal transduction pathway.

14 is a diagram confirming cell viability in NHDF cells treated with water, 50% ethanol (50% EtOH), and 100% ethanol extract (EtOH) of Aloe vera flowers through MTT assay.

15 is a diagram showing migration of NHDF cells for 72 hours to confirm the wound healing activity of water, 50% ethanol (50% Ethanol), and 100% ethanol extract (Absolute Ethanol) of Aloe vera flowers.

Figure 16a is a diagram confirming the survival rates of AVF and PAG, or mixtures thereof, through MTT analysis.

16b and 16c are diagrams confirming migration of NHDF cells for 72 hours in order to confirm wound healing activity.

17a is a diagram showing the results of cell proliferation analysis using a BrdU cell proliferation assay kit.

Figure 17b is a diagram confirming the effects of phosphorylation activation of AKT and ERK pathways, and increased expression of angiogenesis proteins (TGF-β, VEGF) through Western blot upon treatment with the AVF and PAG mixture of the present invention.

17c is a diagram quantifying the relative activation profiles of the AKT and ERK pathways upon treatment with the AVF and PAG mixture of the present invention.

Figure 17d is a diagram quantifying the expression levels of TGF-β and VEGF upon treatment with the AVF and PAG mixture of the present invention.

18a is a diagram confirming the relative mRNA expression levels of MFAP4, COL1A, and α-SMA genes by qRT-PCR upon treatment with the AVF and PAG mixture of the present invention.

18B is a diagram confirming the expression levels of MFAP4, COL1A, fibrillin, and elastin, which are ECM synthesis-related proteins, through Western blot analysis when the AVF and PAG mixture of the present invention is treated.

18c is a diagram quantifying the expression profiles of MFAP4, COL1A, fibrillin, and elastin.

Figure 19a is a diagram confirming and quantifying siRNA-mediated knockdown of MFAP4 through Western blot analysis.

Figure 19b is a diagram showing cell migration analysis under MFAP4 siRNA-mediated knockdown conditions.

19C is a graph showing wound confluence in migration assays in MFAP4 siRNA-mediated knockdown conditions.

19D is a diagram confirming cell cycle progression in MFAP4 siRNA knockdown conditions and AVF + PAG treatment groups through flow cytometry.

19E is a diagram showing the cell cycle distribution of each experimental group through flow cytometry.

20a is a diagram showing cell viability after AVF and PAG treatment or non-treatment of HaCaT cells.

20b is a diagram showing BradU cell proliferation assay after treatment or non-treatment with AVF and PAG mixture.

20c is a diagram illustrating cell migration analysis after treatment of HaCaT cells with a mixture of AVF and PAG.

Figure 20d is a graph showing the migration rate in terms of wound confluence in the control group and the AVF and PAG mixture treatment group for 12 hours.

20E is a diagram confirming the relative mRNA expression levels of MFAP4 and IVN genes by qRT-PCR.

21a is a diagram confirming cell viability of HaCaT cells induced by TNF-α and IFN-γ (10 ng/ml) by MTT assay.

Figure 21b is a diagram confirming cell viability in normal and MFAP4 siRNA knockdown conditions, as well as AVF and PAG mixture treatment groups.

21c is a diagram confirming siRNA-mediated knockdown of MFAP4 through qPCR.

Figure 22 is a diagram showing the mechanism showing the synergistic effect of the AVF and PAG mixture treatment of the present invention on wound healing.

16a to 21c, C is a control group, PC is a positive control (medakasoside) 5 μM treatment group, AVF is 25 and 100 μg / mL of aloe vera flower ethanol extract, and PAG is 50 μg / mL of aloe vera polysaccharide. indicate In addition, AVF + PAG 25 represents a mixture of PAG (50 μg/ml) and AVF (25 μg/mL), and AVF + PAG 100 represents a mixture of PAG (50 μg/ml) and AVF (100 μg/mL). indicate

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Accordingly, the embodiments of the present invention can be modified in many different forms, and the scope of the present invention is not limited to the embodiments described below.

Throughout the specification of the present invention, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.

The present invention relates to a composition for skin moisturizing, skin regeneration, or wound healing, containing an Aloe vera flower extract or a compound isolated therefrom, or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In addition, the present invention provides an effective amount of an aloe vera flower extract or a compound isolated therefrom, or a composition containing an aloe vera flower extract and an aloe vera polysaccharide as active ingredients, for skin moisturizing, skin regeneration or wound healing. It is about treatment methods.

Further, the present invention relates to an Aloe vera flower extract or a compound isolated therefrom, or a composition containing an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients for use in skin moisturizing, skin regeneration or wound healing.

As used herein, the term "extract" refers to an extract obtained by the extraction treatment of the aloe vera flower, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, or mixtures thereof, and extracts of all formulations that can be formed using the extract itself and the extract. The extract may be extracted from natural, hybrid, or mutant plants of Aloe vera flowers, and may also be extracted from plant tissue culture.

In the present invention, cultivated or commercially available aloe vera flowers may be used without limitation.

In the present invention, the extract may use water, an organic solvent, or a mixed solvent thereof, and the organic solvent is a C ₁ to C ₅ lower alcohol, a polar solvent such as ethyl acetate and acetone, and a non-polar solvent such as hexane and dichloromethane. A solvent or a mixed solvent thereof may be used, and preferably may be an extract extracted with water, C ₁ to C ₄ lower alcohol, or a mixed solvent thereof. The alcohol may be methanol, ethanol, propanol, butanol and isopropanol, preferably ethanol. As the extraction method, ultrasonic extraction, shaking extraction, Soxhelt extraction or reflux extraction method may be used, but is not limited thereto. It is preferable to perform extraction by adding 1 to 20 times the amount of washed and well-dried aloe vera flowers with the extraction solvent, and more preferably to perform extraction by adding 5 to 20 times, but is not limited thereto. In addition, the extraction time is preferably 1 to 72 hours, more preferably 1 to 48 hours, but is not limited thereto. In addition, the number of times of extraction is preferably 1 to 5 times, but is not limited thereto.

As used herein, the term "polysaccharide" is preferably an aloe polysaccharide obtained by enzymatically hydrolyzing aloe vera gel, removing pigments and anthraquinones, and then drying it, but may not be limited thereto. . By the enzymatic hydrolysis process, a specific polysaccharide among the “polysaccharides” contained in “aloe” vera gel (e.g., a low molecular weight “aloe” polysaccharide having a molecular weight of 5 to 400 kDa, preferably a low molecular weight having a molecular weight of 50 to 200 kDa) The ratio of aloe to polysaccharide) may be increased. In addition, the enzymatic hydrolysis process preferably uses cellulase, but is not limited thereto.

Accordingly, the present invention provides a cosmetic composition for skin moisturizing or skin regeneration containing an Aloe vera flower extract or a compound isolated therefrom, or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

More specifically, the present invention provides a cosmetic composition for moisturizing the skin, containing an aloe vera flower extract or a compound isolated therefrom as an active ingredient.

In the present invention, the aloe vera flower extract is preferably extracted with water.

In the present invention, the compound may be a C-glycoside flavonoid glycoside, and the C-glycoside flavonoid glycoside may be issoorientin, vitexin, or isovitexin. And, specifically, it may be isoorientin.

In the present invention, the aloe vera flower extract or a compound isolated therefrom can control skin moisturizing factors, thereby exhibiting a skin moisturizing effect, and improving skin moisturizing reduced due to skin damage caused by UVB.

More specifically, the aloe vera flower extract or a compound isolated therefrom is a skin moisturizing factor that modulates skin hydration-related proteins or skin barrier function-related proteins, thereby exhibiting a skin moisturizing effect and reducing skin damage caused by UVB can improve skin hydration.

More specifically, the skin hydration-related proteins are HAS1, HYAL1, hyaluronan or AQP3, and the aloe vera flower extract or a compound isolated therefrom increases HAS1, hyaluronan or AQP3 expression, and decreases HYAL1 expression. , It shows a skin moisturizing effect, and can improve skin moisturizing that has been reduced due to skin damage caused by UVB.

In addition, the skin barrier function-related protein is involuclin or filaggrin, and the aloe vera flower extract or a compound isolated therefrom increases the expression of involucrin or filaggrin, thereby increasing the skin moisturizing effect It can improve skin moisturization that has been reduced due to skin damage caused by UVB.

More specifically, the present invention provides a cosmetic composition for skin regeneration containing an Aloe vera flower extract or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In the present invention, the aloe vera flower extract is preferably extracted with ethanol or a mixed solvent of ethanol and water.

In the present invention, the aloe vera flower extract and the aloe vera polysaccharide are preferably mixed at a concentration ratio of 0.5:1 to 1:2, and when mixed at various concentrations, compared to the aloe vera flower extract or the aloe vera polysaccharide, respectively It exhibits synergistic wound healing and skin regeneration effects.

In the present invention, the aloe vera flower extract or the aloe vera flower extract and the aloe vera polysaccharide exhibit wound healing and skin regeneration effects by increasing the migration of dermal fibroblasts.

As used herein, the term "cosmetic composition" is a composition that is topically applied to human skin to improve the appearance and health of the skin without affecting the structure or function of the body.

In the present invention, the cosmetic composition is a skin-adhesive cosmetic formulation, for example, basic product cosmetics (toilet, cream, essence, cleansing foam, cleansing water, pack, soap), body product cosmetics (body lotion, body oil, Body gel, soap), color product cosmetics (foundation, lipstick, mascara, makeup base), hair product cosmetics (shampoo, conditioner, hair conditioner, hair gel), etc. In addition, transdermal dosage forms such as ointments, liquids, dressings, patches, or sprays may be prepared, but are not limited thereto.

The cosmetic composition may include a carrier acceptable in cosmetic formulations, and the carrier is a compound or composition that is already known and used, or a compound or composition to be developed in the future that can be included in a cosmetic formulation, and the human body adapts when in contact with the skin. It means that there is no more toxicity, instability or irritation than possible. The carrier may be included in about 1% to about 99.99% by weight based on the total weight of the cosmetic composition, preferably about 90% to about 99.99% by weight of the weight of the cosmetic composition. However, since the above ratio varies according to the above-described formulation in which the composition for external application for skin of the present invention is prepared, and also according to its specific application site (face, neck, etc.) or its preferred application amount, the above ratio is It should not be construed as limiting the scope of the invention.

Meanwhile, as the carrier, alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscosity modifier, emulsion, stabilizer, sunscreen, coloring agent, fragrance, and the like may be exemplified. Compounds/compositions that can be used as alcohols, oils, surfactants, fatty acids, silicone oils, humectants, humectants, viscosity modifiers, emulsions, stabilizers, sunscreens, coloring agents, fragrances, etc. are already known in the art, so those skilled in the art An appropriate corresponding material/composition can be selected and used.

More specifically, the present invention provides a quasi-drug for skin moisturizing, containing an aloe vera flower extract or a compound isolated therefrom as an active ingredient. In addition, the present invention provides a quasi-drug for skin regeneration containing an Aloe vera flower extract or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In the present invention, since the contents of the extract, extraction method, compound, and polysaccharide are the same as those described above, specific descriptions will be made using the above contents, and only the unique configuration of the quasi-drug will be described below.

As used herein, the term "quasi-drugs" refers to items with a milder effect than pharmaceuticals among items used for the purpose of diagnosing, treating, improving, mitigating, treating or preventing diseases of humans or animals, for example, according to the Pharmaceutical Affairs Act. According to the Act, quasi-drugs are products excluding items used for pharmaceutical purposes, and may be products used to treat or prevent diseases in humans or animals, or products with minor or no direct action on the human body.

The quasi-drug composition is a body cleanser, disinfectant cleanser, detergent, kitchen detergent, cleaning detergent, toothpaste, gargle, wet tissue, detergent, soap, hand wash, hair wash, hair softener, humidifier filler, mask, ointment and filter filler in the group consisting of It can be prepared in the formulation of your choice.

More specifically, the present invention provides an external skin preparation for skin moisturizing, containing an aloe vera flower extract or a compound isolated therefrom as an active ingredient. In addition, the present invention provides an external skin preparation for skin regeneration containing an Aloe vera flower extract or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In the present invention, since the contents of the extract, the extraction method, the compound, and the polysaccharide are the same as those described above, specific descriptions will be made using the above contents, and only the specific composition of the skin external preparation will be described below.

As used herein, the term "external use agent" is a preparation provided for external use.

In the present invention, the external skin preparation may be formulated into a dosage form selected from the group consisting of powders for external use, tablets for external use, liquids for external use, ointments, creams, gels, warning agents, dressings, patches, sprays, and suppositories.

In addition, the external skin preparation may be a parenteral preparation formulated in a solid, semi-solid or liquid form by adding a commercially available inorganic or organic carrier, excipient, and diluent. The preparation for parenteral administration may be a transdermal dosage form selected from the group consisting of drops, ointments, lotions, gels, creams, patches, sprays, suspensions and emulsions, but is not limited thereto. Carriers, excipients and diluents that may be included in the external preparation include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

More specifically, the present invention provides a food composition for moisturizing the skin, containing an aloe vera flower extract or a compound isolated therefrom as an active ingredient. In addition, the present invention provides a food composition for skin regeneration containing an Aloe vera flower extract or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In the present invention, since the contents of the extract, the extraction method, the compound, and the polysaccharide are the same as those described above, specific descriptions will be made using the above contents, and only the specific configuration of the food composition will be described below.

As used herein, the term "food composition" refers to any kind of composition that can be eaten and/or drunk without causing toxicity symptoms when eating or drinking each composition.

The term used in the present invention, "functional food" is the same term as food for special health use (FOSHU), medicine processed to efficiently display bioregulatory functions in addition to nutritional supply, It is a food with high medical effect.

In the present invention, the food composition is a beverage, meat, sausage, bread, biscuits, rice cake, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, soup, beverages, alcoholic beverages and vitamin complexes, dairy products and milk-processed products, processed foods in a conventional sense, and health functional foods, but are not limited thereto. The beverage composition is not particularly limited in other ingredients, and as in conventional beverages, various flavors (natural flavors such as thaumatin, stevia extract, rebaudioside A, glycyrrhizin, etc.; and synthetic flavors, such as For example, saccharin, aspartame, etc.) or natural carbohydrates (monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, Conventional sugars such as cyclodextrin and sugar alcohols such as xylitol, sorbitol, and erythritol) may be contained as additional components. The proportion of the natural carbohydrate is generally about 1 g to 20 g, preferably about 5 g to 10 g per 100 g of the composition of the present invention.

In addition, in addition to the above, the food of the present invention contains nutrients, vitamins, minerals (electrolytes), synthetic flavors, natural flavors, colorants, enhancers (cheese, chocolate, etc.), pectic acid, pectic acid salts, alginic acid, alginates, organic acids. , protective colloidal thickener, pH adjusting agent, stabilizer, preservative, glycerin, alcohol, carbonating agent and fruit flesh, etc. These components may be used independently or in combination, and the ratio of these additives may also be determined appropriately by those skilled in the art. can be chosen The proportion of the additives may be generally about 0.1 g to 20 g, preferably about 1 g to 20 g per 100 g of the composition of the present invention.

In addition, the health functional food may be added to food as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be suitably determined depending on the purpose of its use (for prevention or improvement). In general, when preparing food or beverage, the health functional food of the present invention may be added in an amount of preferably 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range.

More specifically, the present invention provides a skin moisturizing method comprising the step of administering a composition containing an effective amount of an Aloe vera flower extract or a compound isolated therefrom as an active ingredient. In addition, the present invention provides a skin regeneration method comprising administering an effective amount of an Aloe vera flower extract or a composition containing an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In the present invention, the contents of the extract, extraction method, compound, polysaccharide, and composition are the same as those described above.

More specifically, the present invention provides a composition containing, as an active ingredient, an aloe vera flower extract or a compound isolated therefrom for use in moisturizing the skin. In addition, the present invention provides an Aloe vera flower extract for use in skin regeneration, or a composition containing an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.

In the present invention, the aloe vera flower extract and the aloe vera polysaccharide exhibit a wound healing effect through the microfibril-associated glycoprotein 4 (MFAP4) signaling pathway and activate the AKT and ERK signaling pathways.

In the present invention, the pharmaceutical composition is a group consisting of solutions, suspensions, granules, tablets, capsules, pills, extracts, emulsions, syrups, gels, pastes, ointment frosts, powders, emulsions and aerosols It is preferably any one agent selected from, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors confirmed the effects of water, 50% ethanol and 100% ethanol extract of aloe vera flowers on NHDF cell migration, and as a result, 50% ethanol extract and 100% ethanol extract of aloe vera flowers were significant. It was confirmed that the wound closure rate was increased. In addition, it was confirmed that there was no cytotoxicity at high concentrations (see FIGS. 14 and 15).

Therefore, the aloe vera flower extract of the present invention significantly increases the migration of dermal fibroblasts, which is an important feature in wound healing, and does not exhibit cytotoxicity even at high concentrations, so it is an active ingredient of a pharmaceutical composition for skin wound treatment or skin regeneration. can be usefully used.

In addition, the present inventors confirmed the synergistic effect of aloe vera flower ethanol extract (AVF) and aloe vera polysaccharide (PAG) on NHDF cell migration, and as a result, AVF and PAG, and mixtures thereof (AVF + PAG 25 and AVF + PAG 100) was confirmed to significantly increase the wound closure rate. In particular, it was confirmed that the mixture of AVF and PAG increased the cell migration rate 24 hours faster than the other experimental groups (see FIGS. 16b and 16c).

In addition, the present inventors confirmed the synergistic effect of the AVF and PAG mixture on cell proliferation through activation of the AKT and ERK signaling pathways, and as a result, the AVF + PAG mixture of the present invention activates the AKT and ERK signaling pathways to promote cell proliferation and migration It was confirmed that it promotes (see Figs. 17a to 17d).

In addition, as a result of confirming the effect of the AVF and PAG mixture to induce expression of angiogenesis-related proteins, the present inventors confirmed that the AVF + PAG mixture dose-dependently increased the expression levels of TGF-β and VEGF. + It was confirmed that the expression was increased by about 1.5 times or more in the PAG mixture treatment group (see FIGS. 17b and 17d).

In addition, the present inventors confirmed the synergistic effect of AVF and PAG in skin wound healing through the MFAP4 signaling pathway. As a result, the AVF + PAG treatment group upregulated the expression of MFAP4 compared to the control and positive control groups, and ECM (Extracellular matrix ) It was confirmed that the expression of COL1A, fibrillin and elastin proteins, which are synthesis-related factors, was increased in a dose-dependent manner (see FIGS. 18a to 18c).

In addition, as a result of confirming cell migration and cell cycle under MFAP4 siRNA knockdown conditions, the present inventors confirmed that MFAP4 siRNA knockdown reduced the cell migration rate compared to groups transfected with scramble siRNA and untreated groups. In addition, it was confirmed that the AVF + PAG mixture treatment could modify cell cycle progression by enhancing DNA replication through the S phase of the cell cycle, whereas the MFAP4 siRNA knockdown condition indicated the initiation of the apoptotic process (FIGS. 19a to 19e Reference).

In addition, as a result of confirming the effects of AVF and PAG mixtures on inducing HaCaT cell migration and inhibiting the release of inflammatory cytokines, the mixture of the present invention increases HaCaT cell migration and releases inflammatory cytokines (IL-6 and IL-1β). It was confirmed that it was suppressed (see FIGS. 20a to 20f).

Thus, the mixture of aloe vera flower extract (AVF) and aloe vera polysaccharide (PAG) of the present invention exhibits a synergistic wound healing effect compared to either AVF or PAG. In addition, by increasing the expression of MFAP4, it synergistically induces NHDF, HaCaT cell proliferation, migration, and especially ECM formation, and other extracellular activities associated with MFAP4, such as fibrillin, collagen, elastin, TGF β and α-SMA expression. Since the components are also increased, the mixture of the aloe vera flower extract (AVF) and the aloe vera polysaccharide (PAG) of the present invention is useful as an active ingredient of a pharmaceutical composition for skin wound treatment or skin regeneration targeting MFAP4 and related signal transduction pathways can be used

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one commonly used in formulation, and carbohydrate compounds (eg, lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc.) ), gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solutions, alcohol, gum arabic, vegetable oils (e.g. corn oil, cotton) seed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, and the like, but are not limited thereto.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered by various routes such as oral or parenteral, and all modes of administration can be expected, for example, it can be administered by oral, rectal or intravenous, intramuscular, or subcutaneous injection. have. At this time, as a parenteral route, transdermal administration is preferred, and among them, topical application by application is most preferred.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity. It can be. The oral dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg/kg (body weight) for adults. In addition, in the case of an external preparation, it can be applied 1 to 5 times a day in an amount of 1.0 to 3.0 ml based on adults and continued for 1 month or longer. However, the dosage is not limited to the scope of the present invention.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

In addition, the present invention provides an aloe vera flower extract for use in wound treatment, or a pharmaceutical composition containing an aloe vera flower extract and an aloe vera polysaccharide as active ingredients.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to aid understanding of the present invention and are not intended to limit the scope of the present invention thereto.

<실시예 1> 알로에 베라 꽃 추출물 또는 이로부터 분리된 화합물의 피부 보습 효과 확인<Example 1> Confirmation of skin moisturizing effect of aloe vera flower extract or a compound isolated therefrom

화학물 및 시약chemicals and reagents

Dried Aloe vera flowers (Fig. 1a) were provided by Univera Co., Ltd. (Seoul, Korea). DMEM (Dulbecco's modified Eagle's medium) medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA). An ELISA kit for hyaluronan detection was purchased from R&D System Inc. (Minneapolis, Minn., USA). PRO-PREP ^™ protein extraction solution and ECL (Enhanced chemiluminescence) detection kit were purchased from Intron (Seongnam, Korea). Antibodies of GAPDH, involucrin, HAS1, HYAL1, AQP3, filaggrin, PKC, P38, ERK 1/2, and HRP (horseradish peroxidase)-conjugated secondary antibodies were from Santa Cruz (Dallas, TX) USA), Cell Science (Canton, MA, USA), and Cell Signaling Technology (Beverly, MA, USA). All other chemicals were purchased from Sigma-Aldrich (Steinheim, Germany).

세포 배양cell culture

HaCaT cells, human keratinocytes, were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in high-glucose DMEM medium containing 10% heat-inactivated fetal bovine serum (heat-inactivated FBS) and 1% penicillin-streptomycin at 37°C and 5% CO ₂ conditions.

통계적 분석statistical analysis

To compare the experimental groups, a one-dimensional analysis of variance (ANOVA) was performed using GraphPad Prism5 (La Jolla, CA, USA), and a p -value <0.05 was considered statistically significant. Mean±standard error is expressed for three independent experiments.

<실시예 1-1> 알로에 베라 꽃 추출물 제조<Example 1-1> Preparation of Aloe Vera Flower Extract

Three kinds of aloe vera flower extracts were prepared by using different extraction solvents.

Specifically, 1 g of each dried and powdered aloe vera flower was put into 10 mL of water (AFWE), 100% ethanol (AE), and 50% ethanol (EE), extracted by ultrasonic waves at 40 ° C. for 60 minutes, and then centrifuged. and filtered. Thereafter, each extract was concentrated under reduced pressure using a rotary vacuum concentrator and then lyophilized. Each extract prepared was stored at -4°C until use.

<실시예 1-2> 알로에 베라 꽃 추출물에 포함된 활성성분 분석<Example 1-2> Analysis of Active Components in Aloe Vera Flower Extract

In order to analyze the components contained in the aloe vera flower extract, TLC and HPLC were performed using the three types of aloe vera flower extracts prepared in Example 1-1. TLC was performed according to a conventional method (The Scientific World Journal Volume 2014, Article ID 724267, page 6). HPLC analysis was performed according to a conventional method using a separation module (e2695) and a water system (Water Corp., Milford, MA, USA) equipped with a photodiode array detector (Phytochemical Analysis Volume 16, Issue 5, pages 295- 301). For the analysis of the contents, each extract of Example 1 was separated by 10 mg/ml. As active ingredients, C-glycoside flavonoids glycosides, orientin (O), isoorientin (IO), vitexin (V), and isovitexin (IV) are standard ingredients. and was used at a concentration of 1 mg/mL.

As a result, as shown in FIGS. 1B to 1D, isoorientin (IO), B. It was confirmed that vitexin (V) and isovitexin (IV) were contained, and in particular, the content of these active ingredients was higher in AFWE than in other extracts. In addition, the concentration of IO was higher among these active ingredients. The content of IO in AFWE was 22.24%, and it was confirmed that it was contained in the order of IO>V>IV.

<실시예 1-3> 샘플 제조 및 세포 처리<Example 1-3> Sample preparation and cell treatment

Each of the extracts of Example 1-1 was dissolved in PBS, and filtered to make a stock at a concentration of 10 mg/mL. Thereafter, a 25 μg/mL extract sample was prepared using the stock for administration to cells, and 5 μm IO, V, and IV samples diluted in serum-free medium were prepared. In order to evaluate the concentration-dependent effect of AFWE among the extracts of Example 1-1, AFWE samples at concentrations of 1, 2.5, and 5 μg/mL were additionally prepared using AFWE stock. In addition, to use as a positive control, a centella asiatica ethanol extract known to improve skin moisturizing (Postepy Dermatologii Alergologii 2013: XXX, 1: 46-49) 5 μg/mL sample was prepared. In addition, the cell group not treated with the extract was used as a negative control.

<실시예 1-4> AFWE의 세포 독성 분석<Example 1-4> Cytotoxicity analysis of AFWE

MTT assay was performed to confirm the cytotoxicity of AFWE.

Specifically, HaCaT cells (4×10 ⁴ cells/well) were seeded in a 96-well plate containing a culture medium containing 10% FBS, and after culturing for 24 hours, the AFWE samples of Examples 1-3 were applied to the cells, respectively. administered. After culturing for 24 hours, the culture medium was removed, and 0.5 mg/mL of MTT solution was added, followed by incubation at 37°C and 5% CO ₂ for 1 hour. Then, after removing the MTT solution from each well and adding 100 μl DMSO, the absorbance was measured at 570 nm using a microplate reader (Molecular Device, CA, USA).

As a result, as shown in FIG. 2a, it was confirmed that the AFWE-treated group did not exhibit cytotoxicity compared to the negative control group (NCtr) or the positive control group (PCtr) under normal conditions.

<실시예 1-5> AFWE의 피부 보습 인자 조절 효과<Example 1-5> Skin moisturizing factor control effect of AFWE

In order to investigate the effect of AFWE on skin moisturizing, HaCaT cells were treated with AFWE, and then hyaluronan (HA), HSA1, HYAL1, and AQP3, proteins related to skin hydration, and skin barrier function-related proteins, pillars, were used as skin moisturizing factors. Secretion of filaggrin was confirmed.

<실시예 1-5-1> AFWE의 히알루로난 분비 조절 효과<Example 1-5-1> Hyaluronan secretion regulating effect of AFWE

ELISA (Enzyme Linked-Immuo sorbent assay) was performed to examine the effect of AFWE on the regulation of hyaluronan (HA) secretion.

Specifically, after treating and culturing HaCaT cells with AFWE in the same manner as described in Examples 1-4, the supernatant was collected and ELISA was performed according to the manufacturer's instructions.

As a result, as shown in FIG. 2B, hyaluronan secretion increased in a concentration-dependent manner according to the concentration of AFWE (1, 2.5, or 5 μg/ml).

<실시예 1-5-2> AFWE의 HAS1, HYAL1 조절 효과<Example 1-5-2> HAS1, HYAL1 regulatory effect of AFWE

To investigate the regulatory effect of AFWE on HAS1 and HYAL1 that regulate hyaluronan secretion, Western blot and densitometric analysis were performed.

Specifically, HaCaT cells were recovered after treating and culturing HaCaT cells with AFWE in the same manner as described in Examples 1-4 above. The recovered HaCaT cells were washed with a cold PBS solution, and proteins were extracted by dissolving in PRO-PREP ^TM containing protease and phosphatase inhibitors. Western blot and densitometry analysis of the extracted proteins was performed according to a previously established method (Phytomedicine 28, 19-26) (Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam, Korea).

As a result, as shown in Figures 2c to 2e, compared to the negative control (NCtr) and positive control (PCtr), HAS1 expression increased in a concentration-dependent manner according to the concentration of AFWE, while HYAL1 decreased compared to the positive control.

<실시예 1-5-3> AFWE의 AQP3 및 필라그린(filaggrin) 조절 효과<Example 1-5-3> Modulating effect of AFWE on AQP3 and filaggrin

Western blot and densitometric analysis were performed to investigate the regulatory effect of AFWE on AQP3 and filaggrin involved in the regulation of skin barrier function.

Specifically, the protein extracted in Example 1-5-2 was subjected to Western blot and densitometric analysis (Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam , Korea).

As a result, as shown in Figures 2c and 2f, the expression of AQP3 increased in a concentration-dependent manner according to the concentration of AFWE.

In addition, as shown in Figures 3a and 3c, the expression of filaggrin was also increased in a concentration-dependent manner compared to the positive control according to the concentration of AFWE.

<실시예 1-6> AFWE의 PKC와 MAPK 신호전달경로를 통한 인볼루크린(involucrin) 발현 조절<Example 1-6> Regulation of involucrin expression through the PKC and MAPK signaling pathway of AFWE

The following analysis was conducted to investigate the regulation mechanism of involucrin, a protein related to the skin barrier function of AFWE.

<실시예 1-6-1> AFWE의 인볼루크린 발현 조절<Example 1-6-1> Control of Involucrin Expression of AFWE

Western blot and densitometric analysis were performed to examine the effect of involucrine regulation by AFWE.

As a result, as shown in FIGS. 3A and 3B , it was confirmed that the expression of involucrin was increased in a concentration-dependent manner by AFWE.

<실시예 1-6-2> AFWE의 mRNA 수준에서의 인볼루크린 조절<Example 1-6-2> Involucrin regulation at the mRNA level of AFWE

Quantitative real time-reverse transcription-polymerase chain reaction (qRT-PCR) was performed to examine the effect of involucrin regulation on the mRNA level by AFWE.

Specifically, mRNA was extracted from the HaCaT cells recovered in Example 1-5-2 using an RNA extraction kit (KeyGEN BioTECH, Nanjing, China). cDNA was synthesized from the extracted mRNA, and qRT-PCR was performed using the PrimeScript RT agent kit (Takara, Beijing, China) according to the manufacturer's instructions. The primer sequences used for qRT-PCR are as follows: IVL, forward: 5'-GTGGGGGAGAGAGGGAATTA-3' (SEQ ID NO: 7); reverse, 5′-CTCACCTGAGGTTGGGATTG-3′ (SEQ ID NO: 8); GAPDH, forward: 5'-TCCACTGGCGTCTTCACC-3' (SEQ ID NO: 9), reverse: 5'-GGCAGAGATGATGACCCTTTT-3' (SEQ ID NO: 10).

As a result, as shown in FIG. 3D , it was confirmed that the involucrin mRNA expression levels of the AFWE-treated group and the positive control group were similar.

<실시예 1-6-3> PKC와 MAPK 신호전달경로를 통한 인볼루크린 발현 조절<Example 1-6-3> Regulation of involucrin expression through PKC and MAPK signaling pathway

In regulating the expression of involucrin, the activation of PKC, P38, and the ERK1/2 signaling pathway of MAPK play an important role. Therefore, Western blot and densitometric analysis were performed to investigate the effect of AFWE on the PKC and MAPK signaling pathways.

Specifically, Western blot and densitometric analysis were performed on the protein extracted in Example 1-5-2 in a previously established manner (Phytomedicine 28, 19-26) (Enhanced chemiluminescence from ECL, detection kit from Intron., Sungnam , Korea).

As a result, as shown in Figures 3e and 3f, PKC expression increased in the AFWE-treated group compared to the positive control group.

In addition, as shown in Figures 3e, 3g and 3h, it was confirmed that P38 phosphorylation and ERK1/2 dephosphorylation increased in the AFWE-treated group compared to the negative control group.

<실시예 1-7> UVB 조건에서 AFWE의 피부 보습 향상 효과<Example 1-7> Skin moisturizing improvement effect of AFWE under UVB conditions

UVB exposure causes skin damage, which reduces skin moisturization. Accordingly, the following analysis was conducted to confirm whether AFWE could induce skin moisturizing factor expression under UVB conditions to improve skin moisturizing caused by UVB-induced skin damage.

<실시예 1-7-1> UVB 조사<Example 1-7-1> UVB irradiation

HaCaT cells (4 × 10 ⁴ cells/well) were dispensed into a 96-well plate containing a culture medium containing 10% FBS, and after culturing for 24 hours, a UVB irradiation machine (Bio-Link BLX-312; Vilber Lourmat GmbH, Marne -la-Vallee, France) was irradiated with UVB (200 mj/cm ² ) in a conventional manner (Pharmaceutical Biology, 55:1, 1032-1040). After that, the AFWE extract of Example 1-1 was immediately treated and cultured for 24 hours. Cells not irradiated with UVB were used as a negative control.

<실시예 1-7-2> UVB 조건에서 AFWE의 세포독성 분석<Example 1-7-2> Cytotoxicity analysis of AFWE under UVB conditions

MTT assay was performed to confirm the cytotoxicity of AFWE under UVB conditions.

Specifically, after completion of the treatment and culture of the AFWE extract in Example 1-7-1, the MTT assay was performed in the same manner as described in Example 1-4.

As a result, as shown in FIG. 4a, it was confirmed that AFWE did not exhibit cytotoxicity under UVB conditions.

In addition, as shown in Figure 5, the AFWE-treated group under UVB conditions showed no change in cell morphology.

<실시예 1-7-3> UVB 조건에서 AFWE의 피부 보습 인자 조절 효과 확인<Example 1-7-3> Confirmation of skin moisturizing factor control effect of AFWE under UVB conditions

Western blot and densitometric analysis were performed to investigate the effect of AFWE on skin moisturizing factor regulation under UVB conditions.

Specifically, after completion of AFWE extract treatment and culture in Example 1-7-1, Western blot and densitometric analysis were performed in the same manner as described in Example 1-5.

As a result, as shown in FIGS. 4B to 4G , involucrin expression was increased in a concentration-dependent manner in the AFWE-treated group, similar to that observed in normal conditions. In addition, HAS1 expression increased in a concentration-dependent manner, and HYAL1 expression decreased in a concentration-dependent manner. In addition, filaggrin and AQP3 expression were increased.

<실시예 1-8> 추출용매에 따른 알로에 베라 꽃 추출물의 피부 보습 효과 비교<Example 1-8> Comparison of skin moisturizing effect of aloe vera flower extract according to extraction solvent

In order to compare the skin moisturizing effect of the aloe vera flower extract according to the extraction solvent, MTT assay, Western blot and densitometric analysis were performed.

Specifically, HaCaT cells (4×10 ⁴ cells/well) were dispensed into a 96-well plate containing a culture medium containing 10% FBS, and after culturing for 24 hours, the samples of Examples 1-3 were administered to the cells, respectively. did Thereafter, MTT assay was performed in the same manner as described in Examples 1-4, and Western blot and densitometric analysis were performed in the same manner as in Examples 1-5.

As a result, as shown in FIG. 6a, it was confirmed that the 100% ethanol extract (AE), the 50% ethanol extract (EE), and the water extract (AFWE) of Aloe vera flowers did not show any cytotoxicity.

In addition, as shown in Figures 6b to 6h, all three extracts showed similar aspects in skin moisturizing factor control, and among them, AFWE was found to have the best skin moisturizing factor control effect.

<실시예 1-9> 알로에 베라 꽃 추출물에 함유된 유효성분들의 피부 보습 효과 비교<Example 1-9> Comparison of skin moisturizing effect of active ingredients contained in aloe vera flower extract

The following analysis was conducted to compare the function of the active ingredients contained in the aloe vera flower extract and the skin moisturizing effect.

<실시예 1-9-1> 알로에 베라 꽃 추출물에 함유된 유효성분들의 세포 독성 분석<Example 1-9-1> Cytotoxicity analysis of active ingredients contained in aloe vera flower extract

In order to confirm the cytotoxicity of the active ingredients IO, V, and IV confirmed in Example 1-2, MTT assay was performed.

Specifically, HaCaT cells (4×10 ⁴ cells/well) were dispensed into a 96-well plate containing a culture medium containing 10% FBS, and after culturing for 24 hours, the cells were cultured with IO, V, IV samples were administered individually. Then, after culturing for 24 hours, MTT assay was performed in the same manner as described in Examples 1-4.

As a result, as shown in FIG. 7a, it was confirmed that the cell viability of the IO, V, and IV treatment groups was similar to that of the negative control group.

<실시예 1-9-2> 알로에 베라 꽃 추출물에 함유된 유효성분들의 피부 보습 인자 조절 효과 비교<Example 1-9-2> Comparison of skin moisturizing factor control effect of active ingredients contained in aloe vera flower extract

Western blot and densitometric analysis were performed to compare the skin moisturizing factor control effect by the active ingredients IO, V, and IV confirmed in Example 1-2.

Specifically, cells that had been treated and cultured for each sample in Example 1-9-1 were recovered. Then, using the recovered cells, Western blot and densitometric analysis were performed in the same manner as described in Examples 1-5 above.

As a result, as shown in FIGS. 7B to 7H , it was confirmed that the expression of involucrin increased in the IO-treated group and the IV-treated group compared to the negative control group. In addition, it was confirmed that hyaluronan secretion increased as the expression of HYAL1 decreased in the IO-treated group, the V-treated group, and the IV-treated group. In addition, it was confirmed that the expression of filaggrin significantly increased in the IO-treated group, the V-treated group, and the IV-treated group. In particular, it was confirmed that the control effect of the skin moisturizing factors was the most excellent in the IO treatment group.

<실시예 1-10> 알로에 베라 꽃 추출물에 함유된 유효성분 IO의 피부 보습 인자 결합친화도 분석<Example 1-10> Analysis of skin moisturizing factor binding affinity of active ingredient IO contained in aloe vera flower extract

In order to find out the binding affinity between IO and skin moisturizing factor having the best skin moisturizing factor control effect in Example 1-9-2, a molecular docking study was performed on IO and skin moisturizing factor.

Specifically, the molecular binding between IO and skin moisturizing factors was confirmed using AutoDock Vina according to the previous method (Phytomedicine 31, 1-9).

As a result, as shown in Figs. 8a to 12b, it was confirmed that IO exhibits strong binding affinity between the skin moisturizing factor.

Through the above results, as shown in the schematic diagram of FIG. 13, the aloe vera flower extract of the present invention and the compounds derived therefrom modulate skin moisturizing factors, specifically proteins related to skin barrier function and proteins related to skin hydration, resulting in skin moisturizing effect It can be seen that represents In addition, it can be seen that it exhibits an excellent effect in improving skin moisturization reduced by skin damage caused by UVB.

<실시예 2> 알로에 베라 꽃 추출물의 상처 치유 및 피부 재생 효과 확인<Example 2> Confirmation of wound healing and skin regeneration effects of aloe vera flower extract

화학물 및 시약chemicals and reagents

Dried Aloe vera (A. vera ) flowers (UV-AVF1001) were provided by Univera Co., Ltd. (Seoul, Korea). DMEM (Dulbecco's modified Eagle's medium) medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA).

세포 배양cell culture

Normal human dermal fibroblasts (NHDF) were purchased from the Korean Cell Line Bank (Seoul National University, Korea). All of the cells were cultured in high-glucose DMEM (Thermo Scientific, Waltham, MA) containing 10% FBS and 1% penicillin-streptomycin (PS), and the cultured cells were humidified at 37°C and 5% CO _{2 .} kept in \. In addition, NHDF cells used in the present invention were used between

passage numbers

5 and 10.

통계적 분석statistical analysis

Results are expressed as mean ± standard error from three independent experiments, and statistical analysis was performed using GraphPad Prism 5 (GrahPad Software Inc., La Jolla, CA, USA). In addition, One-way analysis of variance was followed by Newman-Keuls multiple comparison test, and p < 0.05 was considered statistically significant.

<실시예 2-1> 알로에 베라 꽃 추출물 제조<Example 2-1> Preparation of Aloe Vera Flower Extract

Three types of aloe vera flower extracts were prepared by using different extraction solvents in the same manner as in Example 1-1.

Specifically, 1 g of dried and powdered aloe vera flowers were put into 10 mL of 100% ethanol, 50% ethanol, and water, respectively, extracted with ultrasonic waves at 40 ° C. for 60 minutes, and then centrifuged and filtered. Thereafter, each extract was concentrated under reduced pressure using a rotary vacuum concentrator and then lyophilized. Each extract prepared was stored at -4°C until use.

<실시예 2-2> 샘플 제조 및 세포 처리<Example 2-2> Sample preparation and cell treatment

Each extract prepared in Example 2-1 was dissolved in PBS and filtered to make a stock solution at a concentration of 10 mg/mL. In addition, stock solutions were diluted to concentrations of 25 μg/mL and 100 μg/mL in serum-free media and used.

On the other hand, in the cell treatment, NHDF cells were washed twice with PBS at 80% confluency, treated at the desired concentration, and then cultured for 24 hours and 48 hours, respectively. In addition, 10 μg/mL madecassoside was used as a positive control (PC).

<실시예 2-3> 세포 생존율 및 세포 증식 효과 확인<Example 2-3> Confirmation of cell viability and cell proliferation effect

MTT assay was performed to confirm cell viability. Specifically, NHDF (3 × 10 ³ cells/well) cells were seeded in a 96-well plate. Then, 25 μg/mL and 100 μg/mL of the samples prepared in Example 2-2 were treated, and incubated at 37° C. in 5% CO ₂ for 24 hours. After removing the culture medium and culturing for 24 hours and 48 hours, 0.5 mg/mL of MTT solution was added. After 1 hour, after removing the MTT solution, 100 μL of DMSO was added to each well. Then, absorbance was measured at a wavelength of 570 nm using a microplate reader (Molecular Devices E09090; San Francisco, CA, USA).

Through cell viability confirmation, all NHDF cells treated with aloe vera flower 100% ethanol extract (EtOH), aloe vera flower 50% ethanol extract (50% EtOH), and aloe vera flower extract (water), as shown in FIG. It was confirmed that there was no toxicity (FIG. 14).

<실시예 2-4> 스크래치 상처-치료 분석을 통한 NHDF 세포 이동에 대한 알로에 베라 꽃 추출물의 효과 확인<Example 2-4> Confirmation of effect of aloe vera flower extract on NHDF cell migration through scratch-healing assay

An in vitro wound healing assay was performed to examine the wound healing activity using the dose confirmed by the MTT assay.

On the other hand, cell migration is an important feature of skin wound healing, and the NHDF cell migration rate was determined based on the percentage of wound closure relative to the wound gap induced through scratch analysis.

Specifically, a scratch wound was applied to a monolayer of cultured NHDF cells with WoundMaker ^™ (Essen Bioscience, Michigan, USA).

Then, the cells were cultured for 72 hours in a serum-free medium containing 25 μg/mL of the sample prepared in Example 2-2.

The wound healing effect was confirmed by capturing images of cells every 2 hours using IncuCyte ZOOM (Essen Bioscience, MI, USA).

As shown in FIG. 15, Aloe vera flower 100% ethanol extract (Absolute Ethanol) and Aloe vera flower 50% ethanol extract (50% Ethanol) significantly increased the wound closure rate compared to the untreated control (Control). It was confirmed (FIG. 15).

<실시예 3> 알로에 베라 꽃 추출물 및 알로에 베라 다당체의 상처 치유 및 피부 재생 효과 확인<Example 3> Confirmation of Wound Healing and Skin Regeneration Effects of Aloe Vera Flower Extract and Aloe Vera Polysaccharide

화학물 및 시약chemicals and reagents

Dried Aloe vera (A. vera ) flowers (UV-AVF1001) and aloe gel were provided by Univera Co., Ltd. (Seoul, Korea). DMEM (Dulbecco's modified Eagle's medium) medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA). ELISA kits used for the detection of IL-6 and IL-1β were purchased from R&D Systems Inc. (Minneapolis, MN, USA). In addition, GAPDH, MFAP4, type I collagen (COL1A), fibrillin-1, elastin, TGF-β, VEGF-C, MMP-9, AKT, pAKT, ERK1/2, and pERK1/2 used for Western blot analysis Primary antibodies against HRP (horseradish peroxidase) and secondary antibodies conjugated were Santa Cruz (Dallas, TX, USA), Cell Sciences (Canton, MA, USA), Abcam (UK), and Cell Signaling Technology (Beverly, MA), respectively. , USA). Fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit Ⅰ containing propidium iodide was purchased from BD Biosciences Pharmingen (San Diego, CA, USA). In addition, BrdU cell proliferation assay kit was purchased from Cell Signaling (Danvers, USA).

세포 배양cell culture

Normal human dermal fibroblasts (NHDF) and immortal human epidermal keratinocytes (HaCaT cells) were purchased from the Korean Cell Line Bank (Seoul National University, Korea). All of the cells were cultured in high-glucose DMEM (Thermo Scientific, Waltham, MA) containing 10% FBS and 1% penicillin-streptomycin (PS), and the cultured cells were humidified at 37°C and 5% CO _{2 .} maintained in In addition, NHDF cells used in the present invention were used between

passage numbers

5 and 10.

통계적 분석statistical analysis

<실시예 3-1> 알로에 베라 꽃 추출물 제조<Example 3-1> Preparation of Aloe Vera Flower Extract

A 100% ethanol extract of Aloe vera flowers was prepared in the same manner as in Example 1-1.

Specifically, 1 g of dried and powdered aloe vera flowers were put in 10 mL of 100% ethanol, extracted with ultrasonic waves at 40° C. for 60 minutes, and then centrifuged and filtered. Afterwards, the extract was concentrated under reduced pressure using a rotary vacuum concentrator and then lyophilized. The prepared extract (AVF) was stored at -4°C until use.

<실시예 3-2> 알로에 베라 다당체의 제조<Example 3-2> Preparation of Aloe Vera Polysaccharide

To prepare processed aloe gel (PAG), which is an aloe vera polysaccharide, naïve aloe vera gel was incubated with cellulase and heated to obtain a high concentration of medium-sized (50-200 kDa) polysaccharide. . Then, the cellulase-treated gel was filtered through a charcoal column to remove anthraquinone and other coloring substances. After concentrating under reduced pressure using a rotary vacuum concentrator, it was lyophilized and stored at -4 ° C until use.

<실시예 3-3> 샘플 제조 및 세포 처리<Example 3-3> Sample preparation and cell treatment

The AVF and PAG prepared in Examples 3-1 and 3-2 were dissolved in PBS, and filtered to prepare a stock solution having a concentration of 10 mg/mL. Stock solutions were also diluted to concentrations of 25 μg/mL, 50 μg/mL and 100 μg/mL in serum-free medium.

In addition, in order to confirm the synergistic effect of PAG and AVF, 50 μg/mL PAG was mixed with 25 μg/mL AVF and 100 μg/mL AVF in PBS, respectively, and marked as [AVF + PAG 25] and [AVF + PAG 100]. did In addition, the mixture was stored at 4°C for treatment to cells.

Meanwhile, cell treatment was performed by washing HaCaT and NHDF cells twice with PBS at 80% confluency, treating them at the desired concentration, and incubating them for 24 hours and 48 hours, respectively. In addition, 5 μM madecassoside was used as a positive control (PC).

<실시예 3-4> 세포 생존율 및 세포 증식 효과 확인<Example 3-4> Confirmation of cell viability and cell proliferation effect

MTT assay was performed to confirm cell viability. Specifically, HaCaT (3 × 10 ⁴ cells/well) and NHDF (3 × 10 ³ cells/well) cells were seeded in a 96-well plate. Both cell groups received AVF + PAG 25 and AVF + PAG 100 as well as individual concentrations of AVF (25 μg/mL, 50 μg/mL and 100 μg/mL) and PAG (25 μg/mL, 50 μg/mL and 100 μg /mL), and then cultured at 37°C for 24 hours in 5% CO ₂ . After removing the culture medium and culturing for 24 hours and 48 hours, 0.5 mg/mL of MTT solution was added. After 1 hour, after removing the MTT solution, 100 μL of DMSO was added to each well. Then, absorbance was measured at a wavelength of 570 nm using a microplate reader (Molecular Devices E09090; San Francisco, CA, USA).

In addition, in order to perform cell proliferation analysis, analysis was performed using the BrdU cell proliferation assay kit (Cell signaling, Danvers, USA). Specifically, NHDF cells (3 × 10 ³ cells/well) were seeded in a 96-well plate, incubated for 24 hours, and then treated with various concentrations of AVF + PAG 25 and AVF + PAG 100 in serum-free medium with BrdU. , further incubation was performed for 48 hours. Then, integration of BrdU-positive cells was confirmed by ELISA method according to the manufacturer's method.

<실시예 3-5> 스크래치 상처-치료 분석<Example 3-5> Scratch wound-healing assay

A scratch wound was applied to the cultured HaCaT and NHDF cell monolayers with WoundMaker ^™ (Essen Bioscience, Michigan, USA).

Then, serum-free medium containing AVF + PAG 25 or AVF + PAG 100 respectively, blood-free medium containing individual concentrations of AVF (25 μg/mL, 50 μg/mL, 100 μg/mL) or PAG (25 μg/mL) μg/mL, 50 μg/mL, 100 μg/mL) cells were cultured in serum-free media for 24 hours and 72 hours, respectively.

<실시예 3-6> 정량적 실시간 PCR(qRT-PCR)<Example 3-6> Quantitative real-time PCR (qRT-PCR)

NHDF (1.2 × 10 ⁵ cells/well) and HaCaT (4 × 10 ⁵ cells/well) cells were seeded in 60-mm dishes and 6-well plates in complete medium, respectively. After culturing for 24 hours, a confluent cell monolayer was formed, and several wounds (5 horizontal and vertical lines) were applied using a p200 pipette tip. Then, both the HaCaT and NHDF cells were treated with AVF + PAG 25 and AVF + PAG 100, respectively, and cultured for 24 hours. After washing again twice with PBS, RNA purification was performed. Total RNA was extracted from NHDF and HaCaT cells using TRIzol ^® Reagent (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions, and then approximately 0.5 μg of RNA was extracted using the PrimeScript RT Reagent Kit (Takara, Beijing, China). cDNA was synthesized from purified total RNA. In addition, the primer sequences used for quantitative real-time polymerase chain reaction (qRT-PCR) are shown in Table 1 below. In addition, the expression of MFAP4, COL1A, IVL and α-SMA mRNA was normalized to GAPDH.

Gene nameGene name	Forward primer sequences Forward primer sequences	Reverse primer sequencesReverse primer sequences

Human MFAP4Human MFAP4	5′-GGCTCAGTAAGTTTCTTCCGCG-3′ (서열번호 1)5′-GGCTCAGTAAGTTTCTTCCGCG-3′ (SEQ ID NO: 1)	5′-CCAAGTCCACTCGCAGCTCATA-3′ (서열번호 2)5′-CCAAGTCCACTCGCAGCTCATA-3′ (SEQ ID NO: 2)
Human COL Human COL	5′-GATTCCCTGGACCTAAAGGTGC-3′ (서열번호 3)5′-GATTCCCTGGACCTAAAGGTGC-3′ (SEQ ID NO: 3)	5′AGCCTCTCCATCTTTGCCAGCA-3′ (서열번호 4)5′AGCCTCTCCATCTTTGCCAGCA-3′ (SEQ ID NO: 4)
Human α-SMAHuman α-SMA	5′-CTATGCCTCTGGACGCACAACT-3′ (서열번호 5)5′-CTATGCCTCTGGACGCACAACT-3′ (SEQ ID NO: 5)	5′-CAGATCCAGACGCATGATGGCA-3′ (서열번호 6)5′-CAGATCCAGACGCATGATGGCA-3′ (SEQ ID NO: 6)
Human IVL Human IVL	5′-GTGGGGGAGAGAGGGAATTA-3′ (서열번호 7)5′-GTGGGGGAGAGAGGGAATTA-3′ (SEQ ID NO: 7)	5′-CTCACCTGAGGTTGGGATTG-3′ (서열번호 8)5′-CTCACCTGAGGTTGGGATTG-3′ (SEQ ID NO: 8)
Human GAPDH Human GAPDH	5′-TCCACTGGCGTCTTCACC-3′ (서열번호 9)5′-TCCACTGGCGTCTTCACC-3′ (SEQ ID NO: 9)	5′-GGCAGAGATGATGACCCTTTT-3′ (서열번호 10)5′-GGCAGAGATGATGACCCTTTT-3′ (SEQ ID NO: 10)

<실시예 3-7> 웨스턴 블랏 분석<Example 3-7> Western blot analysis

After 48 hours of treating the experimental group with NHDF cells, the cells were collected and washed with cold PBS (1X). Then, it was dissolved in PRO-PREP™ containing protease, and the protein was quantified through Bradford's assay. In addition, 6-12% SDS-PAGE gel electrophoresis was performed, and then 40 μg of protein from each group was transferred to a nitrocellulose membrane and blocked with 5% skim milk to block non-specific binding. Subsequently, the cells were incubated overnight at 4°C using primary antibodies against GAPDH, MFAP4, fibrillin 1, COL1A, elastin, TGF-β, VEGF-C, AKT, p-AKT, ERK, and p-ERK. The next day, the membrane was incubated with each of the secondary antibodies conjugated to horseradish peroxidase (HRP), and an enhanced chemiluminescence reagent. Then, each protein band was visualized using a ChemiDoc XRS + imaging system (Bio-Rad, Hercules, CA, USA). In addition, densitometric analysis was performed by Image Master TM 17 2D Elite software version 3.1 (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and protein bands were quantified.

<실시예 3-8> ELISA(Enzyme-linked immunosorbent assay) 분석<Example 3-8> ELISA (Enzyme-linked immunosorbent assay) analysis

HaCaT cells (4 × 10 ⁴ cells/well) were seeded in a 48-well plate for 24 hours. Then, inflammation was induced with 10 ng/ml of TNF-α and IFN-γ, each treated with a desired concentration of AVF+PAG mixture, and cultured for 24 hours. Then, the supernatant was collected, and the secretion of IL-6 and IL-1β in serum was measured by ELISA according to the manufacturer's protocol.

<실시예 3-9> siRNA(small interfering RNA)를 사용한 일시적 형질감염<Example 3-9> Transient transfection using siRNA (small interfering RNA)

NHDF cells were seeded in 6-well plates (~60% confluence) and transfected with 100 nM of MFAP4-specific siRNA (BIONEER) using Lipofectamine (Invitrogen) according to the manufacturer's instructions. After 6 hours of transfection, the Lipofectamine-containing medium was replaced with a serum-free medium containing or without a sample at the desired concentration, and the culture was maintained at 37° C. for 48 hours, and then the supernatant was removed. Cells were then harvested, lysed, and Western blot analysis was performed to confirm MFAP4-specific siRNA knockdown.

In addition, to perform qRT-PCR, RNA was extracted and the expression of the MFAP4 gene was confirmed in the same manner as in Examples 3-6.

In addition, cell migration assay was performed in the knockdown condition of NHDF cells.

<실시예 3-10> MFAP4(microfibril-associated glycoprotein 4) 특이적 siRNA 녹다운 상태에서 스크래치 상처 치유 분석 및 유세포 분석에 의한 세포 주기 분석<Example 3-10> Scratch wound healing assay and cell cycle analysis by flow cytometry in microfibril-associated glycoprotein 4 (MFAP4) specific siRNA knockdown state

After 6 hours of MFAP4-specific siRNA treatment, the medium was removed and cells were wounded for migration assays before being treated or not treated with AVF+PAG 25 and AVF+PAG 100 mixtures. Then, cell migration was analyzed by capturing images every 2 hours.

Cell cycle analysis was performed by flow cytometry in the presence of MFAP4-specific siRNA knockdown based on Khamchun and Thongboonkerd, 2018. After knockdown, cells were treated with AVF+PAG 25 and AVF+PAG 100 mixtures for 24 hours. Then, the cells were fixed with 70% ethanol at 4° C. for 1 hour and then centrifuged at 1000×g for 5 minutes. Afterwards, the cells were resuspended in PBS and the mixture was centrifuged. Then, the cells were resuspended in 500 μL PBS containing 10 μL each of RNase A and propidium iodide (PI, 1 mg/mL). In addition, flow cytometry (FACSCalibur™; Becton-Dickinson, San Jose, CA, USA) was performed within 1 hour.

<실험예 3-1> NHDF 세포 이동에 대한 AVF와 PAG의 상승적 효과 확인<Experimental Example 3-1> Confirm synergistic effect of AVF and PAG on NHDF cell migration

First, through cell viability confirmation, NHDF cells treated with individual concentrations of AVF and PAG extracts and mixtures of AVF and PAG up to 100 μg/mL were all in the control or positive control (PC) Medacassoside treatment groups, as shown in FIG. 16a. It was confirmed that there was no cytotoxicity compared to (FIG. 16a).

Therefore, an in vitro wound healing assay was performed to examine the wound healing activity using the optimized dose confirmed through the MTT assay.

As shown in FIGS. 16B and 16C , it was confirmed that AVF and PAG, and mixtures thereof (AVF + PAG 25 and AVF + PAG 100) significantly increased the wound closure rate. In particular, it was confirmed that the mixture of AVF and PAG increased the cell migration rate 24 hours faster than the other experimental groups (FIGS. 16b and 16c).

<실험예 3-2> AKT 및 ERK 신호 전달 경로 활성화를 통한 세포 증식에 있어서 AVF 및 PAG 혼합물의 시너지 효과 확인<Experimental Example 3-2> Confirmation of synergistic effect of AVF and PAG mixtures on cell proliferation through activation of AKT and ERK signaling pathways

As a result of cell proliferation analysis using the BrdU cell proliferation assay kit, as shown in FIG. 17a, the AVF + PAG treatment group improved NHDF cell proliferation compared to the control and PC groups. In particular, the AVF + PAG 100 treatment group showed DNA It was confirmed that the synthesis was increased by more than 0.5 times (FIG. 17a).

On the other hand, activation of the AKT and ERK signaling pathways is involved in the migration and proliferation of skin fibroblasts (Beserra et al., 2018).

Accordingly, as a result of confirming the effect of the AVF + PAG mixture treatment group of the present invention on the AKT and ERK signaling pathways through Western blotting, as shown in Figs. 17b and 17c, AVF + It was confirmed that the PAG mixture treatment group significantly increased phosphorylation of AKT and ERK1/2. In particular, it was confirmed that AKT was increased in a dose-dependent manner in the AVF + PAG mixture, and that ERK phosphorylation was significantly increased in the AVF + PAG 100 treated group (FIGS. 17b and 17c).

Therefore, it was confirmed that the AVF + PAG mixture of the present invention promotes cell proliferation and migration by activating the AKT and ERK signaling pathways.

<실험예 3-3> AVF와 PAG 혼합물의 혈관신생 관련 단백질 발현 유도 확인<Experimental Example 3-3> Confirmation of induction of angiogenesis-related protein expression of AVF and PAG mixture

As a result of confirming the expression levels of TGF-β and VEGF, which are angiogenic proteins related to wound healing, as shown in FIGS. 17B and 17D, it was confirmed that the AVF + PAG mixture dose-dependently increased the expression levels of TGF-β and VEGF. In particular, it was confirmed that the expression of TGF-β was increased by about 1.5 times or more in the AVF + PAG mixture treatment group (FIGS. 17b and 17d).

<실험예 3-4> MFAP4 신호 전달 경로를 통한 피부 상처 치유에 있어서 AVF와 PAG의 시너지 효과 확인<Experimental Example 3-4> Confirmation of the synergistic effect of AVF and PAG in skin wound healing through the MFAP4 signal transduction pathway

It is known that MFAP4 can initiate cell migration and proliferation and assist in the synthesis of ECM (Extracellular Matrix).

Therefore, in order to elucidate the mechanism of AVF + PAG in the wound healing process, MFAP4 and related pathways in NHDF cells were confirmed through qRT-PCR and Western blot analysis.

As shown in Figures 18a to 18c, it was confirmed that the AVF + PAG treatment group upregulated the expression of MFAP4 compared to the control group and the PC group. Specifically, it was confirmed that the protein expression of MFAP4 was increased 3-fold and the mRNA level was increased 2-fold. In addition, it was confirmed that the expression of COL1A and α-SMA genes were also up-regulated, and in particular, the expression of COL1A, fibrillin, and elastin protein was increased in a dose-dependent manner in the AVF + PAG 100 treatment group (FIG. 18a to FIG. 18c).

Therefore, it was confirmed that the mixture of AVF and PAG of the present invention exhibits a synergistic effect on skin wound healing through the MFAP4 signal transduction pathway in a dose-dependent manner.

<실험예 3-5> MFAP4 siRNA 녹다운 조건에서 세포 이동과 세포 주기 확인<Experimental Example 3-5> Confirmation of cell migration and cell cycle under MFAP4 siRNA knockdown conditions

MFAP4 knockdown was performed to evaluate the role of MFAP4 in wound healing.

Specifically, the protein and RNA levels of MFAP4 expression were significantly downregulated in MFAP4-transfected cells compared to scramble siRNA and untransfected controls, which was confirmed by Western blot analysis (FIG. 19A) and qPCR (FIG. 21C). ) were confirmed in all. In addition, it was confirmed that there was no cytotoxicity (FIG. 21b).

Then, cell migration and cell cycle analysis were performed to confirm the correlation with MFAP4 in the siRNA-mediated knockdown state.

As a result, as shown in FIGS. 19B and 19C , it was confirmed that MFAP4 siRNA knockdown reduced the cell migration rate compared to the scramble siRNA transfected group and the untreated group, and the cell migration remained similarly slow up to 72 hours.

However, it was confirmed that the interval for immediate wound closure was reduced in the group treated with the mixture of AVF and PAG due to increased movement speed (Figs. 19b and 19c).

In addition, flow cytometry was performed to investigate different stages of the cell cycle in MFAP4 siRNA knockdown conditions.

As a result, as shown in FIGS. 19D and 19E , it was confirmed that the AVF and PAG treated groups increased the S phase after 24 hours compared to the control group and the positive control group. Specifically, it was confirmed that the number of S-phase cell populations was 20% in the control group, but about 30% in the experimental group (FIGS. 19d and 19e).

However, both the control group and the AVF + PAG treatment group showed no difference in the G2/M phase, and in the case of cells transfected with MFAP4 siRNA and scramble siRNA, most of the cell population was distributed in the G2/M phase, while the lowest distribution of cells was shown in FIGS. 19D and 19D. As shown in FIG. 19e, it was confirmed that it was in the S phase.

Thus, it was confirmed that the AVF + PAG mixture treatment could modify the cell cycle progression by enhancing DNA replication through the S phase of the cell cycle, whereas the MFAP4 siRNA knockdown condition indicated the initiation of the apoptotic process.

<실험예 3-6> AVF 및 PAG 혼합물의 세포 이동 유도 효과 및 염증성 사이토카인의 방출 억제 효과 확인<Experimental Example 3-6> Confirmation of cell migration inducing effect and inhibitory effect of inflammatory cytokine release of AVF and PAG mixture

Cytotoxicity and cell migration were confirmed in HaCaT cells in the same manner as in Experimental Example 3-1.

As a result, as shown in FIGS. 20a to 20f, there was no cytotoxicity when the AVF + PAG mixture was treated (FIG. 20a), and it was confirmed that cell proliferation increased compared to the control group (FIG. 20b).

Also, similar to the case of NHDF cells, it was confirmed that the AVF + PAG mixture significantly induced the migration of HaCaT cells compared to the control group and the positive control group in a dose-dependent manner (FIG. 20c).

In addition, a monolayer of HaCaT cells also began migrating rapidly to the wound site after 6 hours, and maximum confluence was observed at 12 hours, and about 1.5-fold increase in IVL and MFAP4 gene expression was confirmed (FIG. 20E).

In addition, HaCaT cells were exposed to TNF-α and IFN-γ (10 ng/mL) and then treated with an AVF + PAG mixture to induce inflammation, followed by ELISA analysis. As a result, it was confirmed that the serum levels of IL-6 and IL-1β decreased (FIG. 20f). In addition, it was confirmed that there was no cytotoxicity even under this condition (FIG. 21a).

The present invention relates to an aloe vera flower extract or a composition for skin moisturizing, promoting regeneration and wound healing, containing an aloe vera flower extract and an aloe vera polysaccharide as active ingredients, the aloe vera flower extract of the present invention or a compound derived therefrom Alternatively, the aloe vera flower extract and the aloe vera polysaccharide may be usefully used as active ingredients of a skin moisturizing or skin regenerating composition.

Claims

Aloe vera ( Aloe vera ) Flower extract or a compound isolated therefrom, or Aloe vera flower extract and Aloe vera polysaccharide containing as active ingredients, a cosmetic composition for skin moisturizing or skin regeneration.
The cosmetic composition according to claim 1, wherein the aloe vera flower extract is extracted with water, C 1 to C 4 lower alcohol or a mixed solvent thereof.
The cosmetic composition for skin moisturizing or skin regeneration according to claim 1, wherein the aloe vera flower extract is extracted with water, ethanol or a mixed solvent thereof.
According to claim 1, wherein the skin moisturizing cosmetic composition contains an aloe vera flower extract or a compound isolated therefrom as an active ingredient, skin moisturizing or skin regeneration cosmetic composition.
The cosmetic composition for moisturizing or regenerating skin according to claim 4, wherein the aloe vera flower extract is extracted with water.
According to claim 4, wherein the compound is C- glycoside flavonoid (C-glycoside flavonoid) glycoside, skin moisturizing or skin regeneration cosmetic composition.
The method of claim 6, wherein the C-glycoside flavonoid glycoside is at least one selected from the group consisting of isoorientin, vitexin, and isovitexin, a cosmetic for moisturizing or regenerating skin. composition.
According to claim 4, wherein the aloe vera flower extract or a compound isolated therefrom is to adjust the skin moisturizing factor, skin moisturizing or skin regeneration cosmetic composition.
The cosmetic composition according to claim 8, wherein the skin moisturizing factor is a skin hydration related protein or a skin barrier function related protein.
The method of claim 9, wherein the skin hydration-related protein is HAS1, HYAL1, hyaluronan or AQP3, and the aloe vera flower extract or a compound isolated therefrom increases expression of HAS1, hyaluronan or AQP3, and reduces HYAL1 expression The skin barrier function-related protein is involucrin or filaggrin, and the aloe vera flower extract or a compound isolated therefrom increases the expression of involucrin or filaggrin. A cosmetic composition for moisturizing or regenerating skin.
According to claim 4, wherein the skin moisturizing cosmetic composition is to improve the skin moisturizing reduced by skin damage caused by UVB, skin moisturizing or skin regeneration cosmetic composition.
The cosmetic composition for skin moisturizing or skin regeneration according to claim 1, wherein the cosmetic composition for skin regeneration contains an Aloe vera flower extract or an Aloe vera flower extract and an Aloe vera polysaccharide as active ingredients.
[Claim 13] The cosmetic composition for moisturizing or regenerating skin according to claim 12, wherein the aloe vera flower extract is extracted with ethanol or a mixed solvent of ethanol and water.
[Claim 13] The cosmetic composition according to claim 12, wherein the aloe vera polysaccharide has a molecular weight of 5 to 400 kDa.
[Claim 13] The cosmetic composition for moisturizing or regenerating skin according to claim 12, wherein the aloe vera polysaccharide is obtained by enzymatically hydrolyzing aloe vera gel.
The cosmetic composition for moisturizing or regenerating skin according to claim 15, wherein the enzymatic hydrolysis uses cellulase.
[Claim 13] The cosmetic composition for moisturizing or regenerating skin according to claim 12, wherein the aloe vera flower extract and the aloe vera polysaccharide are mixed in a concentration ratio of 0.5:1 to 1:2.
According to claim 12, wherein the aloe vera flower extract, or the aloe vera flower extract and aloe vera polysaccharide is to increase the migration of dermal fibroblasts, skin moisturizing or skin regeneration cosmetic composition.
A quasi-drug for skin moisturizing or skin regeneration, containing an aloe vera flower extract or a compound isolated therefrom, or an aloe vera flower extract and an aloe vera polysaccharide as active ingredients.
An external skin preparation for skin moisturizing or skin regeneration, containing an aloe vera flower extract or a compound isolated therefrom, or an aloe vera flower extract and an aloe vera polysaccharide as active ingredients.
A food composition for moisturizing or regenerating skin, containing an aloe vera flower extract or a compound isolated therefrom, or an aloe vera flower extract and an aloe vera polysaccharide as active ingredients.
A pharmaceutical composition for wound healing or skin regeneration, containing an aloe vera flower extract or an aloe vera flower extract and an aloe vera polysaccharide as active ingredients.
The pharmaceutical composition for wound healing or skin regeneration according to claim 22, wherein the aloe vera flower extract is extracted with water, C 1 to C 4 lower alcohol or a mixed solvent thereof.
The pharmaceutical composition for wound treatment or skin regeneration according to claim 22, wherein the aloe vera flower extract is extracted with ethanol or a mixed solvent of ethanol and water.
23. The method of claim 22, wherein the aloe vera flower extract or the aloe vera flower extract and the aloe vera polysaccharide has a wound healing effect through the MFAP4 ((microfibril-associated glycoprotein 4) signal transduction pathway, for wound treatment or skin regeneration pharmaceutical composition.
The pharmaceutical composition for wound healing or skin regeneration according to claim 22, wherein the aloe vera flower extract and the aloe vera polysaccharide activate AKT and ERK signal transduction pathways.
23. The method of claim 22, wherein the pharmaceutical composition is selected from the group consisting of solutions, suspensions, granules, tablets, capsules, pills, extracts, emulsions, syrups, gels, pastes, ointment frosts, powders, emulsions and aerosols. Any one formulation, a pharmaceutical composition for wound healing or skin regeneration.
A skin moisturizing or skin regeneration method comprising administering an effective amount of an aloe vera flower extract or a compound isolated therefrom, or a composition containing an aloe vera flower extract and an aloe vera polysaccharide as active ingredients.
A wound treatment method comprising administering an effective amount of an aloe vera flower extract or a composition containing an aloe vera flower extract and an aloe vera polysaccharide as active ingredients.
A composition containing an Aloe vera flower extract or a compound isolated therefrom, or an Aloe vera flower extract and an Aloe vera polysaccharide for use in skin moisturizing or skin regeneration as active ingredients.
The composition according to claim 30, wherein the composition is a cosmetic composition, a quasi-drug, an external skin preparation, a food composition or a health functional food composition.
An aloe vera flower extract for use in wound treatment, or a composition containing an aloe vera flower extract and an aloe vera polysaccharide as active ingredients.