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WO2022250313A1 - Composition d'hydratation de la peau, favorisant la régénération de la peau, et le traitement des plaies, comprenant de l'extrait de fleur d'aloe vera, ou de l'extrait de fleur d'aloe vera et des polysaccharides d'aloe vera, comme ingrédient(s) actif(s) - Google Patents

Composition d'hydratation de la peau, favorisant la régénération de la peau, et le traitement des plaies, comprenant de l'extrait de fleur d'aloe vera, ou de l'extrait de fleur d'aloe vera et des polysaccharides d'aloe vera, comme ingrédient(s) actif(s) Download PDF

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Publication number
WO2022250313A1
WO2022250313A1 PCT/KR2022/006170 KR2022006170W WO2022250313A1 WO 2022250313 A1 WO2022250313 A1 WO 2022250313A1 KR 2022006170 W KR2022006170 W KR 2022006170W WO 2022250313 A1 WO2022250313 A1 WO 2022250313A1
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WO
WIPO (PCT)
Prior art keywords
aloe vera
skin
flower extract
moisturizing
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2022/006170
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English (en)
Korean (ko)
Inventor
심규석
조은애
김선여
술타나라지아
강민철
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Univera Co Ltd
Industry Academic Cooperation Foundation of Gachon University
Original Assignee
Univera Co Ltd
Industry Academic Cooperation Foundation of Gachon University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020210067992A external-priority patent/KR102543123B1/ko
Priority claimed from KR1020220052745A external-priority patent/KR102563734B1/ko
Priority claimed from KR1020220052744A external-priority patent/KR102563733B1/ko
Application filed by Univera Co Ltd, Industry Academic Cooperation Foundation of Gachon University filed Critical Univera Co Ltd
Publication of WO2022250313A1 publication Critical patent/WO2022250313A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9794—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • 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.
  • 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.
  • 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.
  • Hyaluronan which is regulated by hyaluronan synthase (HASes) and degrading enzymes (hyaluronidase (HYALs)
  • HASes hyaluronan synthase
  • HYALs degrading enzymes
  • 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.
  • 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.
  • the most well-known material is Aloe vera
  • 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).
  • Korean Patent Publication No. 10-2020-0140520 describes the respiratory disease improvement effect of aloe vera gel
  • Korean Patent Publication No. 10-2016-0086809 discloses the ratio of aloe vera extract
  • the treatment effect of alcoholic chronic fatty liver is disclosed
  • 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.
  • 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.
  • 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.
  • aloe vera flower extract AVF
  • aloe vera polysaccharide PAG
  • MFAP4 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 ⁇ and ⁇ -SMA expression, also synergistically wound compared to AVF or PAG, respectively. It was confirmed that the treatment effect was shown.
  • an aloe vera flower extract and a compound derived therefrom can be usefully used as an active ingredient in a skin moisturizing composition
  • 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
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the aloe vera flower extract of the present invention significantly increases the migration of dermal fibroblasts, which is an important feature in wound healing
  • 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 ⁇ and ⁇ -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
  • FIGS. 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
  • 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
  • FIG. 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
  • 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.
  • FIG. 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
  • FIG. 3a shows involucrin through Western blot analysis (involucrin) and filaggrin expression confirmation results are shown
  • FIGS. 3b to 3c show the results of involucrin (FIG. 3b) and filaggrin (FIG. 3c) expression confirmation through densitometry analysis
  • FIG. 3d is qRT- PCR analysis shows the result of confirming the relative mRNA expression of involucrin
  • 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.
  • FIG. 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
  • Figure 4a shows the cytotoxicity confirmation result of AFWE under UVB conditions through MTT analysis
  • 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).
  • FIG. 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.
  • AFWE Aloe vera flower extract
  • FIGS. 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
  • 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
  • FIGS. 6d to 6h show involucrin, HAS1, and HYAL1 through densitometric analysis. , filaggrin, AQP3 expression confirmation results are shown.
  • FIG. 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.
  • IO isoorientin
  • V vitexin
  • isovitexin isovitexin
  • FIG. 7a shows the results of confirming the cytotoxicity of IO, V, and IV through MTT analysis
  • FIG. 7b shows the result of confirming the hyaluronan content through EL
  • FIG. 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.
  • FIG. 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
  • 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.
  • FIG. 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).
  • FIG. 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
  • 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).
  • FIG. 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)
  • 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) .
  • FIG. 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
  • 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).
  • FIG. 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.
  • FIG. 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- ⁇ , VEGF) through Western blot upon treatment with the AVF and PAG mixture of the present invention.
  • TGF- ⁇ , VEGF angiogenesis proteins
  • 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- ⁇ 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 ⁇ -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- ⁇ and IFN- ⁇ (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.
  • AVF is 25 and 100 ⁇ g / mL of aloe vera flower ethanol extract
  • PAG is 50 ⁇ g / mL of aloe vera polysaccharide.
  • AVF + PAG 25 represents a mixture of PAG (50 ⁇ g/ml) and AVF (25 ⁇ g/mL)
  • AVF + PAG 100 represents a mixture of PAG (50 ⁇ g/ml) and AVF (100 ⁇ g/mL).
  • 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.
  • 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.
  • 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.
  • 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.
  • cultiva plants may be used without limitation.
  • 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.
  • the extraction solvent 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.
  • the extraction time is preferably 1 to 72 hours, more preferably 1 to 48 hours, but is not limited thereto.
  • the number of times of extraction is preferably 1 to 5 times, but is not limited thereto.
  • 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. .
  • 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.
  • the enzymatic hydrolysis process preferably uses cellulase, but is not limited thereto.
  • 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.
  • 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.
  • the aloe vera flower extract is preferably extracted with water.
  • the compound may be a C-glycoside flavonoid glycoside
  • the C-glycoside flavonoid glycoside may be issoorientin, vitexin, or isovitexin. And, specifically, it may be isoorientin.
  • 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.
  • 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.
  • 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.
  • the skin barrier function-related protein is involuclin or filaggrin
  • 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.
  • 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.
  • the aloe vera flower extract is preferably extracted with ethanol or a mixed solvent of ethanol and water.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • external use agent is a preparation provided for external use.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the amount may be less than the above range.
  • 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.
  • 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.
  • 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.
  • the contents of the extract, extraction method, compound, polysaccharide, and composition are the same as those described above.
  • 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.
  • 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.
  • 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.
  • the contents of the extract, extraction method, compound, polysaccharide, and composition are the same as those described above.
  • 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.
  • the aloe vera flower extract is preferably extracted with ethanol or a mixed solvent of ethanol and water.
  • 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.
  • 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.
  • 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.
  • MFAP4 microfibril-associated glycoprotein 4
  • 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.
  • 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).
  • 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.
  • 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).
  • 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).
  • the present inventors confirmed that the AVF + PAG mixture dose-dependently increased the expression levels of TGF- ⁇ 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).
  • the present inventors confirmed the synergistic effect of AVF and PAG in skin wound healing through the MFAP4 signaling pathway.
  • 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).
  • MFAP4 siRNA knockdown reduced the cell migration rate compared to groups transfected with scramble siRNA and untreated groups.
  • 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).
  • the mixture of the present invention increases HaCaT cell migration and releases inflammatory cytokines (IL-6 and IL-1 ⁇ ). It was confirmed that it was suppressed (see FIGS. 20a to 20f).
  • 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.
  • MFAP4 aloe vera flower extract
  • MFAP4 aloe vera polysaccharide
  • 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.
  • 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.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, manni
  • 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.
  • oral, rectal or intravenous, intramuscular, or subcutaneous injection have.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the contents of the extract, extraction method, compound, polysaccharide, and composition are the same as those described above.
  • Dried Aloe vera flowers (Fig. 1a) were provided by Univera Co., Ltd. (Seoul, Korea).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin and streptomycin 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 TM 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • each extract of Example 1 was separated by 10 mg/ml.
  • 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.
  • 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.
  • 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 ⁇ g/mL extract sample was prepared using the stock for administration to cells, and 5 ⁇ m IO, V, and IV samples diluted in serum-free medium were prepared.
  • AFWE samples at concentrations of 1, 2.5, and 5 ⁇ g/mL were additionally prepared using AFWE stock.
  • centella asiatica ethanol extract known to improve skin moisturizing (Postepy Dermatologii Alergologii 2013: XXX, 1: 46-49) 5 ⁇ g/mL sample was prepared.
  • the cell group not treated with the extract was used as a negative control.
  • MTT assay was performed to confirm the cytotoxicity of AFWE.
  • 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 ⁇ l DMSO, the absorbance was measured at 570 nm using a microplate reader (Molecular Device, CA, USA).
  • 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.
  • ELISA Enzyme Linked-Immuo sorbent assay
  • hyaluronan secretion increased in a concentration-dependent manner according to the concentration of AFWE (1, 2.5, or 5 ⁇ g/ml).
  • 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).
  • HAS1 expression increased in a concentration-dependent manner according to the concentration of AFWE, while HYAL1 decreased compared to the positive control.
  • Example 1-5-2 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).
  • Example 1-5-2 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).
  • Quantitative real time-reverse transcription-polymerase chain reaction was performed to examine the effect of involucrin regulation on the mRNA level by AFWE.
  • 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).
  • Example 1-5-2 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).
  • 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.
  • 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.
  • a UVB irradiation machine Bio-Link BLX-312; Vilber Lourmat GmbH, Marne -la-Vallee, France
  • MTT assay was performed to confirm the cytotoxicity of AFWE under UVB conditions.
  • Example 1-7-1 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.
  • Example 1-7-1 Western blot and densitometric analysis were performed in the same manner as described in Example 1-5.
  • involucrin expression was increased in a concentration-dependent manner in the AFWE-treated group, similar to that observed in normal conditions.
  • HAS1 expression increased in a concentration-dependent manner
  • HYAL1 expression decreased in a concentration-dependent manner.
  • filaggrin and AQP3 expression were increased.
  • 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.
  • 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.
  • Example 1-9-1 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.
  • Example 1-9-2 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.
  • 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.
  • Dried Aloe vera (A. vera ) flowers (UV-AVF1001) were provided by Univera Co., Ltd. (Seoul, Korea).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin and streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA).
  • NHDF Normal human dermal fibroblasts
  • Korean Cell Line Bank 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 ⁇ .
  • NHDF cells used in the present invention were used between passage numbers 5 and 10.
  • 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.
  • Example 2-1 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 ⁇ g/mL and 100 ⁇ g/mL in serum-free media and used.
  • 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.
  • 10 ⁇ g/mL madecassoside was used as a positive control (PC).
  • 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 ⁇ g/mL and 100 ⁇ g/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 ⁇ L 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).
  • An in vitro wound healing assay was performed to examine the wound healing activity using the dose confirmed by the MTT assay.
  • 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.
  • the cells were cultured for 72 hours in a serum-free medium containing 25 ⁇ g/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).
  • 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
  • FBS fetal bovine serum
  • penicillin and streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA).
  • ELISA kits used for the detection of IL-6 and IL-1 ⁇ were purchased from R&D Systems Inc. (Minneapolis, MN, USA).
  • GAPDH GAPDH, MFAP4, type I collagen (COL1A), fibrillin-1, elastin, TGF- ⁇ , VEGF-C, MMP-9, AKT, pAKT, ERK1/2, and pERK1/2 used for Western blot analysis
  • HRP horseradish peroxidase
  • 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).
  • BrdU cell proliferation assay kit was purchased from Cell Signaling (Danvers, USA).
  • NHDF normal human dermal fibroblasts
  • HaCaT cells immortal human epidermal keratinocytes
  • 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
  • NHDF cells used in the present invention were used between passage numbers 5 and 10.
  • 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.
  • a 100% ethanol extract of Aloe vera flowers was prepared in the same manner as in Example 1-1.
  • PAG processed aloe gel
  • na ⁇ ve aloe vera gel was incubated with cellulase and heated to obtain a high concentration of medium-sized (50-200 kDa) polysaccharide.
  • 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.
  • 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 ⁇ g/mL, 50 ⁇ g/mL and 100 ⁇ g/mL in serum-free medium.
  • 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 ⁇ g/mL, 50 ⁇ g/mL and 100 ⁇ g/mL) and PAG (25 ⁇ g/mL, 50 ⁇ g/mL and 100 ⁇ g /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.
  • NHDF cells 3 ⁇ 10 3 cells/well
  • AVF + PAG 25 and AVF + PAG 100 in serum-free medium with BrdU.
  • further incubation was performed for 48 hours.
  • integration of BrdU-positive cells was confirmed by ELISA method according to the manufacturer's method.
  • serum-free medium containing AVF + PAG 25 or AVF + PAG 100 respectively blood-free medium containing individual concentrations of AVF (25 ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL) or PAG (25 ⁇ g/mL) ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL) cells were cultured in serum-free media for 24 hours and 72 hours, respectively.
  • the wound healing effect was confirmed by capturing images of cells every 2 hours using IncuCyte ZOOM (Essen Bioscience, MI, USA).
  • NHDF 1.2 ⁇ 10 5 cells/well
  • HaCaT 4 ⁇ 10 5 cells/well
  • NHDF 1.2 ⁇ 10 5 cells/well
  • HaCaT 4 ⁇ 10 5 cells/well
  • a confluent cell monolayer was formed, and several wounds (5 horizontal and vertical lines) were applied using a p200 pipette tip.
  • 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.
  • primer sequences used for quantitative real-time polymerase chain reaction are shown in Table 1 below.
  • the cells were collected and washed with cold PBS (1X). Then, it was dissolved in PRO-PREPTM containing protease, and the protein was quantified through Bradford's assay. In addition, 6-12% SDS-PAGE gel electrophoresis was performed, and then 40 ⁇ g 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- ⁇ , VEGF-C, AKT, p-AKT, ERK, and p-ERK.
  • each protein band was visualized using a ChemiDoc XRS + imaging system (Bio-Rad, Hercules, CA, USA).
  • 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.
  • 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- ⁇ and IFN- ⁇ , 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-1 ⁇ in serum was measured by ELISA according to the manufacturer's protocol.
  • 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.
  • cell migration assay was performed in the knockdown condition of NHDF cells.
  • Example 3-10 Scratch wound healing assay and cell cycle analysis by flow cytometry in microfibril-associated glycoprotein 4 (MFAP4) specific siRNA knockdown state
  • 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.
  • an in vitro wound healing assay was performed to examine the wound healing activity using the optimized dose confirmed through the MTT assay.
  • 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.
  • AVF and PAG 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).
  • the AVF + PAG treatment group improved NHDF cell proliferation compared to the control and PC groups.
  • the AVF + PAG 100 treatment group showed DNA It was confirmed that the synthesis was increased by more than 0.5 times (FIG. 17a).
  • the AVF + PAG mixture of the present invention promotes cell proliferation and migration by activating the AKT and ERK signaling pathways.
  • TGF- ⁇ and VEGF which are angiogenic proteins related to wound healing
  • FIGS. 17B and 17D it was confirmed that the AVF + PAG mixture dose-dependently increased the expression levels of TGF- ⁇ and VEGF.
  • the expression of TGF- ⁇ was increased by about 1.5 times or more in the AVF + PAG mixture treatment group (FIGS. 17b and 17d).
  • MFAP4 can initiate cell migration and proliferation and assist in the synthesis of ECM (Extracellular Matrix).
  • MFAP4 and related pathways in NHDF cells were confirmed through qRT-PCR and Western blot analysis.
  • 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 ⁇ -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).
  • 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.
  • MFAP4 knockdown was performed to evaluate the role of MFAP4 in wound healing.
  • 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).
  • 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.
  • 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.
  • 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).
  • HaCaT cells were exposed to TNF- ⁇ and IFN- ⁇ (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-1 ⁇ 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.

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Abstract

La présente invention concerne une composition d'hydratation de la peau, favorisant la régénération de la peau, et traitant les plaies, comprenant un extrait de fleur d'Aloe vera comme ingrédient actif, ou un extrait de fleur d'Aloe vera et des polysaccharides d'Aloe vera comme ingrédients actifs, et plus particulièrement, une composition d'hydratation de la peau, de régénération de la peau, ou de traitement des plaies cutanées, comprenant un extrait de fleur d'Aloe vera ou un composé isolé de celui-ci comme ingrédient actif, ou un extrait de fleur d'Aloe vera et des polysaccharides d'Aloe vera comme ingrédients actifs.
PCT/KR2022/006170 2021-05-27 2022-04-29 Composition d'hydratation de la peau, favorisant la régénération de la peau, et le traitement des plaies, comprenant de l'extrait de fleur d'aloe vera, ou de l'extrait de fleur d'aloe vera et des polysaccharides d'aloe vera, comme ingrédient(s) actif(s) Ceased WO2022250313A1 (fr)

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KR1020210067992A KR102543123B1 (ko) 2021-05-27 2021-05-27 알로에 꽃 추출물을 유효성분으로 함유하는 피부 보습용 조성물
KR10-2021-0067992 2021-05-27
KR10-2022-0052744 2022-04-28
KR1020220052745A KR102563734B1 (ko) 2022-04-28 2022-04-28 알로에 베라 꽃 추출물 및 알로에 베라 다당체를 유효성분으로 함유하는 상처 치료 또는 피부 재생용 조성물
KR10-2022-0052745 2022-04-28
KR1020220052744A KR102563733B1 (ko) 2022-04-28 2022-04-28 알로에 베라 꽃 추출물을 유효성분으로 함유하는 상처 치료 또는 피부 재생용 조성물

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CN116270383A (zh) * 2023-03-01 2023-06-23 上海瑞帝安生物科技有限公司 一种防uv损伤的芦荟花提取物及其制备工艺与应用
CN118978567A (zh) * 2024-09-05 2024-11-19 完美（广东）日用品有限公司 一种水解芦荟花蛋白及其制备方法与在制备抗糖基化产品中的应用
CN119185093A (zh) * 2024-09-05 2024-12-27 完美（广东）日用品有限公司 一种水解芦荟花蛋白在皮肤修复产品中的应用
CN119185093B (zh) * 2024-09-05 2025-06-20 完美（广东）日用品有限公司 一种水解芦荟花蛋白在皮肤修复产品中的应用

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