CN108619016B - Whitening and wrinkle-improving application of aquilaria sinensis - Google Patents
Whitening and wrinkle-improving application of aquilaria sinensis Download PDFInfo
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- CN108619016B CN108619016B CN201810214265.XA CN201810214265A CN108619016B CN 108619016 B CN108619016 B CN 108619016B CN 201810214265 A CN201810214265 A CN 201810214265A CN 108619016 B CN108619016 B CN 108619016B
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
The invention relates to whitening and wrinkle-improving application of pistacia chinensis bunge. The present invention also relates to a cosmetic composition comprising an extract of pistachio or a isolate thereof as an active ingredient, which exhibits effects of inhibiting tyrosinase activity involved in melanin synthesis, inhibiting melanin production in melanocytes, protecting ultraviolet cells, inhibiting the expression of MMP-1 and MMP-3, which are wrinkle-related genes, and promoting the expression of elastin gene, and thus has excellent skin whitening and wrinkle improvement effects.
Description
Technical Field
The present invention relates to a cosmetic composition containing an extract or an isolate of Pistacia lentiscus (Pistacia weinmannifolia) as an active ingredient, and more particularly, to a cosmetic composition for skin whitening or wrinkle improvement, a skin external preparation composition, and a health functional food containing an extract or an isolate of Pistacia lentinus as an active ingredient.
Background
With the development of science, the life of people is prolonged, and the attention on functional cosmetics is also promoted. Human skin is the largest immune organ that is the 1 st defense tissue of the body from various harmful environments, and development of functional cosmetics having whitening, ultraviolet protection, wrinkle improvement, and other effects is required to protect it.
Human skin color is genetically determined by the concentration and distribution of melanin (melanin) in the skin, but is also affected by environmental or physiological conditions such as solar ultraviolet rays, fatigue, stress, and the like. The chemical effect on melanin production, which determines skin color, has been reported many times so far by various papers and the like as follows: melanocytes (melanosomes) as melanin-producing cells synthesize melanin pigments having a function of protecting cells from ultraviolet rays in small intracellular organs called melanosomes (melanosomes), and then the melanin pigments migrate from the melanocytes to keratinocytes (keratinocytes), and thus the melanin pigments migrating to the keratinocytes determine skin color.
It is known that a precursor required in the above melanin biosynthesis process is an amino acid called tyrosine (tyrosine), which is converted into Dihydroxyphenylalanine (DOPA), and then finally converted into melanin through a substance called dopaquinone (dopaquinone), or the like. Since melanin synthesized in this way darkens skin color, and is required for health or beauty, studies on a skin lightening method for lightening skin color by suppressing the synthesis of melanin are actively conducted.
Examples of generally known whitening ingredients include substances that inhibit tyrosinase activity, such as Kojic acid (Kojic acid) and Arbutin (Arbutin), hydroquinone (Hydroquinone), vitamin C (L-ascoic acid), and derivatives thereof, and various plant extracts. They can not only whiten the skin by blocking the synthesis of melanin to lighten the skin, but also improve skin hyperpigmentation such as black nevi, freckles, etc. caused by ultraviolet rays, hormones, or heredity. However, when applied to the skin, there are problems as follows: because of safety problems such as irritation and redness, the amount of use is limited, or because the effect is small, a substantial effect cannot be expected.
The mechanism for confirming the wrinkle-alleviating effect can be divided into two types. One is to confirm the ability of collagen and elastin to synthesize, and the other is to confirm the inhibition of MMP (proteolytic enzyme). Collagen is the major component of connective tissue, with Type i, iii playing an important role in skin cells and cell membrane formation. Elastin acts as a natural polymer in a fibrous state present in the connective tissue of the skin, and acts like a spring to support collagen and maintain the elasticity of the skin. MMP is a abbreviation of matrixmetalloprotenase, MMP-1 is a collagenase, and MMP-3 is an enzyme for denaturing collagen, fibronectin (fibronectin), gelatin, and laminin (laminin).
Collagen exists in extracellular matrix as a main matrix protein produced in fibroblasts (fibroplasts) of the skin, and mechanical firmness of the skin, resistance of connective tissue and binding force of tissue, support of cell adhesion, induction of cell division and differentiation (during growth of an organism or wound healing), and the like are known as important functions. Such collagen decreases due to aging and photoaging caused by ultraviolet irradiation, and the decrease of collagen is promoted by collagenase activity that decomposes collagen. This is known to be closely related to wrinkle formation of the skin.
In addition, elastin (Elastin) fibers form cross-links with collagen, which is an important skin constituent in the formation of wrinkles associated with skin elasticity. It has been found that lack and aggregation of elastin fibers, a significant increase in the activity of Elastase (Elastase), an Elastase decomposing enzyme, are one of the causes of skin wrinkle formation. Elastase is known to be the only enzyme capable of decomposing elastin, and its inhibition is capable of fundamentally alleviating skin wrinkles and improving them.
It is also known that collagen and elastin fibers function as matrix proteins that play an important role in retaining moisture in the dermis layer, and such matrix proteins absorb moisture and increase the moisture retention force within the structure formed by them, thereby maintaining the skin in a state containing appropriate moisture, and thus participating in maintaining the elasticity of the skin.
At present, as skin elasticity-improving and wrinkle-improving cosmetics, retinol, adenosine, proteins derived from animal placenta, chlorella extract, and the like are known. It is known that retinol, which is known to be a substance that promotes collagen synthesis and inhibits elastase, is unstable and has safety problems such as irritation and redness when applied to the skin, and therefore the amount of retinol used is limited, and the effects of chlorella extract and the like are minimal, and thus it is substantially difficult to expect skin elasticity-improving and wrinkle-improving effects.
Meanwhile, pistacia weinmannifolia j. Poiss, ex Franch) is a widely wild plant in the vicinity of Yunnan province in china, and has been used for headaches such as dysentery, enteritis, and influenza since ancient times. In previous research reports, two compounds isolated from this plant were reported to have the ability to scavenge reactive oxygen species (Zhao x. Et al., biochim biophysis Acta,1725,103-110, 2005). However, in addition to such reports, nothing is known about the skin whitening and wrinkle improvement activities of the pistacia weinmannifolia extract, its isolate or a compound isolated therefrom.
Disclosure of Invention
Problems to be solved by the invention
The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that a pistacia weinmannifolia extract or a isolate thereof shows no toxicity and has an excellent cell protective effect against ultraviolet rays, MMP (Matrix metalloproteinase) expression inhibition, cytokine IL-8 gene expression inhibition, and elastin gene expression increasing effect, thereby completing the present invention.
Means for solving the problems
An object of the present invention is to provide a cosmetic composition comprising an extract of Pistacia weinmannifolia (Pistacia weinmannifolia) or an isolate thereof as an active ingredient.
Another object of the present invention is to provide a skin external composition comprising an extract of Pistacia weinmannifolia (Pistacia weinmannifolia) or an isolate thereof as an effective ingredient.
Still another object of the present invention is to provide a health functional food comprising an extract of Pistacia weinmannifolia (Pistacia weinmannifolia) or an isolate thereof as an effective ingredient.
Still another object of the present invention is to provide a use of a composition comprising an extract of Pistacia weinmannifolia (Pistacia weinmannifolia) or an isolate thereof as an active ingredient for manufacturing a cosmetic for skin whitening or wrinkle improvement or a skin external preparation.
Still another object of the present invention is to provide a use of a composition comprising an extract of Pistacia weinmannifolia (Pistacia weinmannifolia) or an isolate thereof as an active ingredient for manufacturing a health functional food for skin whitening or wrinkle improvement.
Effects of the invention
The composition of the present invention comprising the extract or the isolate of the same as an active ingredient exhibits effects of inhibiting tyrosinase activity involved in melanin synthesis, inhibiting melanin production in melanocytes, protecting ultraviolet cells, inhibiting the expression of MMP-1 and MMP-3, which are wrinkle-related genes, and promoting the expression of elastin gene, and thus has excellent skin whitening and wrinkle-improving effects.
Brief description of the drawings
FIG. 1 is a table showing the total flavonoid content and LO inhibitory activity of 14 plant extracts, the concentration of cytotoxicity, and the effect on gene expression of IL-6, IL-8, and N-kB 1.
FIG. 2 is a table showing the total polyphenol content, DPPH radical scavenging activity, tyrosinase inhibitory activity, and melanin synthesis inhibitory activity of 14 plant extracts.
FIG. 3 is a graph showing DPPH free radical scavenging activity of 14 plant extracts.
FIG. 4 is a table showing the cell protective effect of 14 plant extracts against ultraviolet rays, the expression levels of MMP-1 and MMP-3, and the effect on the gene expression level of Elastin (Elastin).
FIG. 5 is a graph showing the cytoprotective effects of 14 plant extracts against ultraviolet rays.
FIG. 6 is a diagram showing each solvent isolate obtained from an aerial part extract of Pimenta dioica.
FIG. 7 is a graph showing the MPLC results associated with each active isolate (Fr.1 to 6) obtained from an aerial part extract of P.aquifolium.
FIG. 8 is a graph showing the results of an analysis of a Sasa Veitchii Rehd extract and each column fraction by UPLC (ultra Performance liquid chromatography) at a wavelength of 254 nm.
Fig. 9 is a diagram showing a result of UPLC (ultra performance liquid chromatography) analysis using CAD (charged aerosol detection) on a pistacia stratiotes extract and each column isolate.
FIG. 10 is a graph showing the results of UPLC (ultra Performance liquid chromatography) analysis of a Sasa Veitchii Rehd extract and each column isolate by MS (Mass Spectrometry).
FIG. 11 is a graph showing cell viability in the case of treatment with the extract of pistacia weinmannifolia and each solvent isolate.
FIG. 12 is a graph showing NO inhibitory activity in the case of treatment with the extract of Aquilaria sinensis and each solvent isolate.
FIG. 13 is a graph showing the NO inhibitory activity and the degree of cytotoxicity in the case of treatment with the extract of Aquilaria sinensis and each solvent isolate.
FIG. 14 is a graph showing the degree of cytotoxicity of extracts and column isolates of Cestrum album.
FIG. 15 is a graph showing TNF-alpha blocking activity of extracts and column isolates of P.aquilaria sinensis.
FIG. 16 is a graph showing NO inhibitory activity of extracts and column isolates of pistacia weinmannifolia.
FIG. 17 is a graph showing IL-6 inhibitory activity of extracts and column isolates of pistacia weinmannifolia.
FIG. 18 is a table showing DPPH free radical scavenging activity, ABTS free radical scavenging activity, and superoxide scavenging activity of extracts and column isolates of Sasa Veitchii.
FIG. 19 is a table showing DPPH free radical scavenging activity of extracts and column isolates of pistacia weinmannifolia.
FIG. 20 is a graph showing ABTS free radical scavenging activity of extracts and column isolates of Sasa veitchii.
Fig. 21 is a graph showing cell viability in the case of treatment with the extract of pistacia weinmannifolia at different concentrations.
FIG. 22 is an electrophoretogram showing inhibitory effects on gene expression of MMP-1, MMP-3 and MMP-12 and gene expression of cytokine IL-8 in the case of BJ cells treated with the extracts of Aquilaria sinensis at different concentrations.
Detailed Description
This will be described in detail below. On the other hand, the various descriptions and embodiments disclosed in the present invention can be applied to various other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present invention are within the scope of the present invention. The scope of the present invention is not limited to the specific description below.
As one mode for achieving the above object, the present invention provides a cosmetic composition comprising an extract of pistacia weinmannifolia (pistacia weinmannifolia) or an isolate thereof as an active ingredient.
As another embodiment for achieving the above objects, the present invention provides a use of a composition comprising an extract of Pistacia weinmannifolia (Pistacia weinmannifolia) or an isolate thereof as an active ingredient for manufacturing a cosmetic for skin whitening or wrinkle improvement.
"Pistacia weinmannifolia j. Poiss, ex Franch" is a widely wild plant in Yunnan province of china and has been used for headaches such as dysentery, enteritis, and influenza since ancient times. In previous research reports, two compounds isolated from this plant were reported to have the ability to scavenge reactive oxygen species (Zhao x. Et al., biochim biophysis Acta,1725,103-110, 2005). However, in addition to such reports, nothing is known about the skin whitening and wrinkle improvement activities of the pistacia weinmannifolia extract, its isolate or a compound isolated therefrom.
In the present invention, the term "pistachio extract" may include any one or more of an extract obtained by extracting a stem of pistachio with an appropriate solvent, a diluted solution or a concentrated solution of the extract, a dried product obtained by drying the extract, and a crude purified product or a purified product thereof.
The extract of the present invention can be prepared by a conventional extraction method in the art, such as an ultrasonic extraction method, a filtration method, a reflux extraction method, etc., and commercially available products or natural or cultivated pistachio can be used as the pistachio.
The pistachio extract of the present invention can be isolated according to a conventional method known in the art for producing an extract from a natural product, that is, under a conventional condition of temperature and pressure using a conventional solvent.
In the production of the pistacia chinensis bunge extract of the present invention, water and C can be used 1 ~C 4 The anhydrous or hydrous lower alcohol of (2) or a mixed solvent thereof is not particularly limited. Preferably, the balsamiferous wood extract is produced using ethanol.
In the present invention, the term "isolate" refers to a product obtained by a separation method for separating a specific component or a specific group (group) from the aforementioned produced pistachio extract.
Specifically, the isolate may be a solvent isolate of a pistacia weinmannifolia extract, and more specifically, the solvent isolate may be a solvent isolate separated using hexane, chloroform, ethyl acetate or butanol. More specifically, a conventional separation solvent known in the art, for example, a polar solvent such as water, an anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms such as ethanol or methanol, a nonpolar solvent such as hexane, ethyl acetate, chloroform or dichloromethane, or a mixed solvent thereof can be used, and is not particularly limited.
The aromatic wood isolate of the present invention may include an isolate obtained by further applying a purification process. For example, a separation product obtained by passing the aquilaria sinensis extract of the present invention through an ultrafiltration membrane having a certain molecular weight threshold value, or a column separation product obtained by a variety of purification methods further performed by separation using a variety of chromatographs (chromatographs prepared for separation according to size, charge, hydrophobicity, or affinity) such as MPLC (medium pressure liquid chromatography) are also included in the aquilaria sinensis extract of the present invention.
Further, the above-mentioned extract or isolate of the present invention may be extracted or isolated from the aerial parts of pistacia weinmannifolia, more specifically, may be extracted or isolated from leaves of pistacia weinmannifolia, and is not particularly limited.
In one embodiment of the present invention, leaves of pistacia weinmannifolia are collected, naturally dried, and then pulverized, and methanol is added to the pulverized powder sample to produce an overground part extract of pistacia weinmannifolia. In addition, hexane, chloroform, ethyl acetate, and butanol isolates (fig. 6) were obtained using the above extracts, and active isolates (fr.1 to 6) were obtained using MPLC (medium pressure chromatography) (fig. 7 and table 1).
The cosmetic composition of the present invention can be used for skin whitening or wrinkle improvement, and more specifically, can be used for skin whitening and wrinkle improvement.
The present inventors have found that the lignum aquilariae resinatum of the present invention has an effect on headaches such as diarrhea, enteritis, and influenza and has an ability to remove active oxygen, but it is unknown about the skin-beautifying and wrinkle-improving effects of the lignum aquilariae resinatum for the first time.
In one embodiment of the invention, it is confirmed that: among 14 different plant species, the pistachio extract shows excellent tyrosinase inhibitory effect and melanin production inhibitory effect and thus has excellent skin whitening effect (fig. 2), cell protective effect against ultraviolet rays (fig. 4 and 5), wrinkle-related gene expression inhibition effect, elastin gene expression promotion effect (fig. 4), and wrinkle improvement effect.
In addition, in another example of the present invention, in order to confirm the wrinkle-inhibiting effect of extracts at different concentrations, the expression level of MMP (Matrix metalloproteinase) was examined, and as a result, it was confirmed that: the obtained extract of Sasa Veitchii Rehder has reduced gene expression of cytokine IL-8 (FIG. 22) and excellent wrinkle-improving effect, depending on the concentration of MMP-1, MMP-3 and MMP-12.
Thus, it was confirmed that: a composition comprising a pistacia stratiotes extract or an isolate thereof as an active ingredient can be effectively used as a cosmetic composition for skin whitening or wrinkle improvement.
The cosmetic composition of the present invention may be an anti-inflammatory or antioxidant cosmetic composition.
In one embodiment of the invention, it is confirmed that: among 14 different plant species, the extract of pistacia weinmannifolia contains a considerable amount of flavonoids, shows LO (lipoxygenase) inhibitory activity, and shows excellent anti-inflammatory effect by suppressing the expression of inflammation-related genes (fig. 1). Further, it was confirmed that: the obtained Pimpinella brachycarpa extract contains more polyphenols than green tea, shows DPPH (1, 1-diphenyl-2-picryl-hydrzyl) radical scavenging activity (fig. 2 and 3), and has excellent antioxidant effect.
In another embodiment of the invention, it is again confirmed that: the extract or isolate of Pimpinella saxicola shows a Nitric Oxide (NO) inhibitory activity (FIG. 12), a cytokine inhibitory activity (FIGS. 13 to 17) and has an excellent anti-inflammatory effect, and shows a DPPH radical scavenging activity (FIGS. 18 and 19), an ABST radical scavenging activity (FIGS. 18 and 20), and an SOD (Superoxide dismutase) activity (FIG. 18) and has an excellent antioxidant effect.
Thus, it was confirmed that: the composition comprising the extract of pistacia weinmannifolia or the isolate thereof as an active ingredient can be effectively used as an anti-inflammatory or antioxidant cosmetic composition.
As another means for achieving the above object, the present invention provides a composition for external application to skin comprising an extract of Pistacia weinmannifolia (Pistacia weinmannifolia) or an isolate thereof as an active ingredient.
As another embodiment for achieving the above object, the present invention provides a use of a composition comprising an extract of Pistacia weinmannifolia (Pistacia weinmannifolia) or an isolate thereof as an active ingredient for manufacturing a skin external preparation for skin whitening or wrinkle improvement.
In the skin external preparation composition, the descriptions of the pistacia weinmannifolia, the extract and the isolate are the same as above.
The term "external preparation for skin" of the present invention is used as a concept including all substances generally used in external preparations for skin, AND non-limiting examples of dosage forms of external preparations for skin include PLASTERS (PLASTERS), LOTIONS (lotiones), LINIMENTS (LINIMENTS), LIQUIDS (liquoids AND solids), AEROSOLS (AEROSOLS), EXTRACTS (extractants), OINTMENTS (ointements), FLUIDEXTRACTS (FLUIDEXTRACTS), EMULSIONS (EMULSIONS), SUSPENSIONS (SUSPENSIONS), CAPSULES (caps), CREAMS (CREAMS), soft, hard gelatin CAPSULES, patches, or sustained-release agents.
The external skin preparation composition of the present invention can be used for skin whitening or wrinkle improvement, and more specifically, can be used for skin whitening and wrinkle improvement.
In one embodiment of the invention, it is confirmed that: among 14 different plant species, the pistacia stratiotes extract showed excellent tyrosinase inhibitory effect and melanin production inhibitory effect and thus excellent skin whitening effect (fig. 2), cell protective effect against ultraviolet rays (fig. 4 and 5), expression inhibition of wrinkle-related genes, and expression promotion effect of elastin genes (fig. 4), and thus excellent wrinkle improvement effect.
In addition, in another example of the present invention, in order to confirm the wrinkle-inhibiting effect of extracts at different concentrations, the expression level of MMP (Matrix metalloproteinase) was examined, and as a result, it was confirmed that: the obtained extract of Satureja verniciflua has reduced gene expression of cytokine IL-8 (FIG. 22) and excellent wrinkle improving effect, depending on the concentration of the extract when it inhibits gene expression of MMP-1, MMP-3 and MMP-12.
Thus, it was confirmed that: a composition comprising a pistacia stratiotes extract or an isolate thereof as an active ingredient can be effectively used as a cosmetic composition for skin whitening or wrinkle improvement.
The skin external composition of the present invention may be an anti-inflammatory or antioxidant skin external composition.
In one embodiment of the invention, it is confirmed that: among 14 different plant species, the extract of pistacia weinmannifolia contains a considerable amount of flavonoids, shows LO (lipoxygenase) inhibitory activity, and shows excellent anti-inflammatory effect by suppressing the expression of inflammation-related genes (fig. 1). Further, it was confirmed that: the obtained Pimpinella brachycarpa extract contains more polyphenols than green tea, shows DPPH (1, 1-diphenyl-2-picryl-hydrzyl) radical scavenging activity (FIG. 2 and FIG. 3), and has excellent antioxidant effect.
In another embodiment of the invention, it is again confirmed that: the extract or isolate of the same shows a Nitric Oxide (NO) inhibitory activity (fig. 12), a cytokine inhibitory activity (fig. 13 to 17) and an excellent anti-inflammatory effect, and shows a DPPH radical scavenging activity (fig. 18 and 19), an ABST radical scavenging activity (fig. 18 and 20), and an SOD (Superoxide dismutase) activity (fig. 18) and an excellent antioxidant effect.
Thus, it was confirmed that: the composition comprising the extract of pistacia weinmannifolia or the isolate thereof as an active ingredient can be effectively used as an anti-inflammatory or antioxidant composition for external skin application.
As still another means for achieving the above objects, the present invention provides a health functional food comprising an extract of Pistacia weinmannifolia (Pistacia weinmannifolia) or an isolate thereof as an effective ingredient.
In another aspect of the present invention for achieving the above objects, there is provided a use of a composition comprising an extract of Pistacia weinmannifolia (Pistacia weinmannifolia) or an isolate thereof as an active ingredient for manufacturing a functional health food for skin whitening or wrinkle improvement.
In the above health functional food, the descriptions of the pistacia weinmannifolia, the extract and the isolate are the same as above.
The term "health functional food" as used herein refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients having functionality useful for the human body. Here, the term "functionality" refers to an effect useful for health care purposes such as regulation of nutrients and physiological actions with respect to the structure and function of the human body. The functional health food of the present invention can be produced by a method conventionally used in the art, and can be produced by adding raw materials and ingredients conventionally added in the art. Further, since food is used as a raw material unlike ordinary medicines, there is no advantage in that side effects and the like that can be produced when medicines are taken for a long time are not present, and portability is excellent.
The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (treatment for prevention, health or treatment). In general, in the production of food, the pistachio extract or the isolate thereof of the present invention is added to the raw material composition in an amount of 1 to 10% by weight, preferably 5 to 10% by weight. However, in the case of long-term ingestion for the purpose of health and hygiene or for the purpose of health regulation, the above amount may be below the above range.
The health functional food of the present invention can be used for skin whitening or wrinkle improvement, and more specifically, can be used for skin whitening and wrinkle improvement.
In one embodiment of the invention, it is confirmed that: among 14 different plant species, the pistacia stratiotes extract showed excellent tyrosinase inhibitory effect and melanin production inhibitory effect and thus excellent skin whitening effect (fig. 2), cell protective effect against ultraviolet rays (fig. 4 and 5), expression inhibition of wrinkle-related genes, and expression promotion effect of elastin genes (fig. 4), and thus excellent wrinkle improvement effect.
In addition, in another example of the present invention, in order to confirm the wrinkle-inhibiting effect of extracts at different concentrations, the expression level of MMP (Matrix metalloproteinase) was examined, and as a result, it was confirmed that: the obtained extract of Sasa Veitchii Rehder has reduced gene expression of cytokine IL-8 (FIG. 22) and excellent wrinkle-improving effect, depending on the concentration of MMP-1, MMP-3 and MMP-12.
Thus, it was confirmed that: a composition comprising a pistacia weinmannifolia extract or an isolate thereof as an active ingredient can be effectively used as a health functional food for skin whitening or wrinkle improvement.
The health functional food of the present invention may be an anti-inflammatory or antioxidant health functional food.
In one embodiment of the invention, it is confirmed that: among 14 different plant species, the extract of pistacia weinmannifolia contains a considerable amount of flavonoids, shows LO (lipoxygenase) inhibitory activity, and shows excellent anti-inflammatory effect by suppressing the expression of inflammation-related genes (fig. 1). Further, it was confirmed that: the obtained Pimpinella brachycarpa extract contains more polyphenols than green tea, shows DPPH (1, 1-diphenyl-2-picryl-hydrzyl) radical scavenging activity (FIG. 2 and FIG. 3), and has excellent antioxidant effect.
In another embodiment of the invention, it is again confirmed that: the extract or isolate of Pimpinella saxicola shows a Nitric Oxide (NO) inhibitory activity (FIG. 12), a cytokine inhibitory activity (FIGS. 13 to 17) and has an excellent anti-inflammatory effect, and shows a DPPH radical scavenging activity (FIGS. 18 and 19), an ABST radical scavenging activity (FIGS. 18 and 20), and an SOD (Superoxide dismutase) activity (FIG. 18) and has an excellent antioxidant effect.
Thus, it was confirmed that: the composition comprising the extract of pistacia weinmannifolia or the isolate thereof as an active ingredient can be effectively used as a health functional food for anti-inflammation or anti-oxidation.
Examples
The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited to these examples.
Example 1:14 cosmetic raw material screening of plant and activity of aromatic wood
In order to select cosmetic raw materials, experiments for anti-inflammatory, antioxidant, whitening, skin-protecting, and wrinkle-improving effects of 14 plant extracts were performed.
1-1.14 anti-inflammatory effects of plant extracts
1-1-1. Measurement of flavonoid content
Flavonoids are known to have antibacterial, anticancer, antiviral and anti-inflammatory activities, and to exert antioxidant effects in the living body. In order to measure the amount of flavonoids, the absorbance of yellow flavan (flavan) or water-soluble flavonol (flavonol) glycoside produced by treating a sample with alkali was measured. The total flavonoid content in the 14 plant extract samples was calculated according to the standard curve.
As a result, the total flavonoid content in the plant extract of 14 plants is shown in FIG. 1. It can be predicted that the obtained extract of pistacia stratiotes (AKS-7) exhibits antibacterial, anticancer, antiviral and anti-inflammatory activities by confirming that it contains 0.41mg/g of flavonoids.
1-1-2 LO (lipoxygenase) blocking Activity
To determine the LO inhibitory activity of 14 plant extracts, lipoxygenase Inhibitor Screening Assay kits (Lipoxygenase inhibiting Screening Assay kit (Cayman, MI, USA)) were used and conducted according to the manufacturers protocol.
As a result, LO blocking activity of 14-plant extracts is shown in FIG. 1. In particular, indomethacin (indomethacin) in the positive control group showed an inhibitory effect of about 22%, whereas balsamiferous wood extract (AKS-7) showed an inhibitory effect of 53%, and was found to have an anti-inflammatory effect.
1-1-3 cell viability assay
In order to confirm that 14 plant extract samples were non-toxic, dark blue formazan (formazan) obtained by oxidizing yellow MTT (3- [4, 5-dimethylthiozol-2-yl ] -2,5-diphenyltetrazolium bromide) by the mitochondria of cells was dissolved in DMSO, and the absorbance was measured at 570nm using a microplate.
As a result, the 14 plant extracts showed no toxicity as shown in FIG. 1. In particular, since it was confirmed that the obtained extract of Satureja balsamifera (AKS-7) had no toxicity at 125ppm or less, the anti-inflammatory effect test described below was carried out under the conditions of concentrations at which no toxicity was observed.
1-1-4 inflammation radicalExperiment on expression
In order to confirm the effect of inhibiting the expression of the genes involved in inflammation in 14 plant extract samples, the expression of cytokines (IL-6 and IL-8) and NF-kB genes was confirmed by RT-PCR.
As a result, the expression inhibitory effect of the inflammation-related gene in the 14 plant extracts is shown in fig. 1. When the gene expression by UV was set to 100%, the group treated with eaglewood (AKS-7) showed inhibition rates of gene expression of 48% (IL-6), 49% (IL-8) and 54% (NF-kB), respectively, and thus it was found that the anti-inflammatory effect was exhibited by suppressing the expression of the inflammation-related gene.
1-2.14 antioxidant effect of plant extract
1-2-1. Determination of Total Polyphenol content
Polyphenols are one of the antioxidant substances that convert the active oxygen (harmful oxygen) in our body into harmless substances. For the measurement of the content of polyphenol contained in a sample, folin-Ciocalteu reagent (FCR) was used, which is a method for measuring the content of phenol (phenol) formed by the reduction of two proteins to a blue complex by a complex of phosphoric acid (phospholybdicacid) and Phosphotungstic acid (Phosphotungstic acid).
As a result, the polyphenol content of 14 plant extracts is shown in fig. 2. Green tea is known to have a large amount of polyphenols, and in view of the fact that green tea shows a content of 300mg/g, the balsamiferous wood extract (AKS-7) shows a total content of polyphenols of 333.5mg/g more than this. Therefore, it is known that the pistacia chinensis bunge extract has an excellent antioxidant effect.
1-2. DPPH radical scavenging Activity
DPPH (1, 1-diphenyl-2-picryl-hydrzyl, sigma) accepts electrons, hydrogen from antioxidant substances rather than reversibly forming stable molecules, so that when encountering substances with antioxidant activity, free radicals are eliminated. In this case, the electron donating ability was measured by calorimetric determination of the color difference, which has the characteristic of making bluish blue inherent to DPPH (Jeon et al, J Kor Soc Food sci Nutr.38 (1): 1-8, 2009).
Specifically, 90. Mu.l of a DPPH solution dissolved at 0.15mM in ethanol was dispensed into a 96-well plate, mixed with 10. Mu.l of each sample of 14 plant extracts, left at room temperature for 20 minutes, and then the absorbance was measured at 517nm using a microplate reader (SPARK 10). The group treated with DMSO was used as a negative control group to calculate the radical scavenging effect, and a synthetic antioxidant BHA and a natural antioxidant Quercetin (Quercetin) were used as a positive control group.
As a result, the DPPH radical scavenging activity of the 14 plant extracts is shown in FIGS. 2 and 3. In particular, the extract of pistacia stratiotes (AKS-7) showed DPPH radical scavenging activity similar to Ascorbic acid (Ascorbic acid) as an antioxidant substance, and thus was confirmed to have excellent antioxidant activity.
1-3.14 whitening effect of plant extract
1-3-1 Tyrosinase (Tyrosinase) retarding effect
Tyrosinase is an enzyme that catalyzes the change of two stages, L-tyrosine → DOPA (dihydroxyphenyl alanine) → DOPA quinone, with L-tyrosine (L-tyrosine) as a substrate in the process of melanin formation as a copper-containing oxidase (copper-containing oxidase). Tyrosinase acts in the early stages of melanin synthesis, and is generally most widely used in the whitening screening stage.
The results of the experiment show that the tyrosinase-inhibiting effect of the 14 plant extracts is shown in fig. 2. IC of Ascorbic acid (Ascorbic acid) control group 50 0.009ppm, and although there was no sample having an inhibitory activity higher than that, the obtained product showed an IC of not more than 0.1ppm for the obtained product (AKS-7) 50 Accordingly, it was confirmed that the whitening effect was excellent.
1-3-2 melanogenesis inhibitory effect
Melanin is a pigment which is produced in melanocytes and transferred to keratinocytes (keratinocytes) to determine the color of hair and skin. In order to confirm the whitening effect, the melanin biosynthesis amount of 14 plant extract samples was measured using B16F1 cells, and the production inhibition rate was confirmed with the melanin production amount of 100% in the negative control group, and Arbutin (Arbutin) was used in the positive control group.
As a result, the melanin production-inhibiting effect of the 14 plant extracts is shown in fig. 2. In particular, compared to IC 50 A value of 82.3ppm of melanin IC of positive control group Arbutin (Arbutin) and lignum Aquilariae Resinatum (AKS-7) 50 The value indicated 79.3ppm showed a more excellent melanin production-inhibiting effect. Thus, an excellent whitening effect of the balsamiferous wood was confirmed.
1-4.14 wrinkle-improving effects of plant extracts
1-4-1. Cytoprotective effect against ultraviolet rays
Ultraviolet rays are a main cause of various problems on the skin such as inflammation, wrinkles and coloring, and when ultraviolet rays are received, the expression of MMP, which is an enzyme that destroys collagen, elastin, and the like, is increased to generate wrinkles. The cell protective effect of the sample on ultraviolet rays was confirmed by coating 14 samples of plant extracts on fibroblasts (fibroplasts) and irradiating the cells with ultraviolet rays to confirm the survival rate of the cells.
As a result, the cell protective effect of 14 plant extracts against ultraviolet rays is shown in fig. 4 and 5. In the case of Ascorbic acid (Ascorbic acid) in the control group, the cytoprotective effect was 50% at 50ppm, whereas the pistachio extract (ASK-7) exhibited a cytoprotective effect of 89%, and thus was found to have an excellent cytoprotective effect against ultraviolet rays and thus a wrinkle-improving effect.
1-4-2. Investigation of wrinkle-associated Gene expression
The wrinkle-inhibiting effect of the sample was confirmed by examining the gene expression levels of MMP-1 and MMP-3, which change when fibroblasts (fibroplasts) are irradiated with ultraviolet light, and Elastin (Elastin).
As a result, the effect of the expression of the wrinkle-associated gene on the 14 plant extracts is shown in fig. 4. When the expression levels of MMP-1 and MMP-3 activated by ultraviolet irradiation were set to 100%, the group treated with eaglewood (AKS-7) showed expression levels of 48% and 18%, respectively, and showed an inhibitory effect of 50% or more. Moreover, the elastin gene treated with balsamifera showed a high expression rate of 321%.
Thus, it was found that the pistacia stratiotes inhibit the expression of MMP-1 and MMP-3, which are wrinkle-related genes, and significantly promote the expression of elastin gene, thereby having wrinkle prevention, improvement, and treatment effects.
Example 2: extract, isolate and compound preparation of aerial parts of lignum Aquilariae Resinatum
The anti-inflammatory, antioxidant, whitening and wrinkle-improving effects of the balsamiferous wood, which showed a significant effect among the 14 plants confirmed in example 1 above, will be described in more detail.
2-1 preparation of extract of aerial parts of pistacia weinmannifolia
Methanol extract of Pistacia weinmannifolia j. Poiss. Ex Franch) was purchased from overseas plant extract bank of the overseas biosource herbal medicine center of the korean institute for life engineering.
The extraction process is as follows: 20kg of leaves of Pistacia lentiscus were collected, dried naturally (dried in the shade) using a dryer (50 to 55), ground after removing water, extracted at normal temperature after adding 30L of methanol based on the dry weight of the ground powder sample, and then filtered and concentrated under reduced pressure to obtain 564.7g of Pistacia lentiscus methanol extract. The methanol extract of pistacia weinmannifolia obtained in the subsequent experiments was called total (total) extract.
2-2. Preparation of solvent isolate of aerial parts of aquilaria sinensis
564.7g of the total extract of aerial parts of Sasa Veitchii obtained in example 2-1 was suspended by adding 5L of water, and an equal amount of hexane was added to separate the aqueous layer and the hexane layer. This procedure was repeated 3 more times in the same manner, followed by filtration and concentration under reduced pressure to isolate a cyclohexane isolate (39.2 g). The hexane layer was separated in the same manner, and the remaining aqueous layer was added with the same amount of chloroform, followed by separation in the same manner as above to obtain a chloroform fraction (17.8 g). The chloroform layer was separated in the same manner, and an equal amount of ethyl acetate was added to the remaining aqueous layer, followed by separation in the same manner as described above to obtain an ethyl acetate isolate (68.4 g). The ethyl acetate layer was separated in the same manner, and an equal amount of butanol was added to the remaining aqueous layer, followed by separation in the same manner as described above to obtain a butanol isolate (111.0 g). The remaining aqueous layer was concentrated to give an aqueous isolate (109.0 g) (FIG. 6).
2-3. Preparation of aerial column isolate of Aquilaria sinensis
For MPLC analysis, after mounting a column (20 mm. Times.250mm resin YMC-Pack ODS AQ-HG,10 μm) in an MPLC instrument (Armen Spot prep, gilson, fran ce), methanol extract was repeatedly added in an amount of 1 g. At this time, methanol/water [0-10min,0% MeOH was used as a solvent; 10-70min,0-100% MeOH;70-90min,100% MeOH ], an elution rate of 20 ml/min, and detection at a wavelength of UV200-400mm to obtain active isolates (Fr.1-6) (FIG. 7 and Table 1).
TABLE 1
2-4 analysis of the extract and isolate of the pistacia weinmannifolia
In order to examine the active isolate of the ethyl acetate isolate of the pistacia chinensis bunge obtained in the above example 1-2, it was analyzed by UPLC (ultra performance liquid chromatography) as a liquid chromatography.
First, for UPLC analysis, the methanol extract, the solvent isolate, and the column isolate were filtered 1 time with a 0.25mm membrane filter by UPLC. After the installation of a column (Waters BEH C18 column, 2.1X 100mm,1.7 mm) on a UPLC apparatus (Waters UPLC-Q-TOF), the filtered isolates were added in an amount of 0.3. Mu.l (loading).
In this case, acetonitrile +0.1% formic acid/water +0.1% formic acid [ 10- > 90 (v/v) ], which was used as a solvent for UPLC analysis, was used, and the elution rate was set to 0.4 ml/min. The degrees of separation of the effective isolates and the isolated substances were confirmed chromatographically by UPLC using a detector with a wavelength of UV200 to 400nm, CAD (charged aerosol detection), MS (Mass spectrometry) (fig. 8 to 10 and table 2).
TABLE 2
Example 3: activity confirmation of Sasa Veitchii Rehder extract and isolate
3-1 anti-inflammatory Activity of extracts and isolates of pistacia weinmannifolia
3-1-1. Determination of cytotoxicity of P.amethystoides sample
For cytotoxicity assay of the P.deodara sample, mouse macrophage RAW264.7 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, gibco) Medium supplemented with 5% Fetal Bovine Serum (Total Bovine Serum) at 1X 10 5 The suspension was suspended in a concentration of 100. Mu.l each time and seeded on a 96-well plate (96 well plate) to adhere. After 4 hours, the sample of balsamiferous wood was treated to a concentration of 20. Mu.g/ml. After 24 hours of incubation, 10. Mu.l of 5mg/ml MTT solution was added to each well, and after 4 hours of incubation, the supernatant was removed, 100. Mu.l each of DMSO was added, the absorbance at 570nm was measured, and the cell viability was calculated according to the following equation with the negative control group treated with DMSO at 0.25% set as 100%.
As a result, the cell viability in the case of treatment with the extract of pistacia weinmannifolia and each isolate is shown in fig. 11.
3-1-2. Nitric oxide inhibiting Activity of Aquilaria sinensis
In order to observe the inhibitory effect of inflammation induced artificially by treating RAW264.7 cells with Lipopolysaccharide (LPS), the amount of induced nitric oxide production was measured. To DMEM (Dulbecco's Modified Eagle Medium, gibco Co.) to which phenol Red (phenol-Red) was not added, 5% Fetal Bovine Serum (Total Bovine Serum) was added, and the cells were cultured at 2.5X 10 5 The concentration of the suspension is 200. Mu.l each time in 96well plates (96 well plate) were inoculated. After allowing them to adhere for 4 hours, they were treated with a fresh lignum Aquilariae Resinatum sample, incubated for 1 hour, then treated with 0.5. Mu.g/ml lipopolysaccharide (LPS, lipo polysaccharide, sigma Co.) and incubated for 24 hours. Then, 100. Mu.l of the supernatant was collected and placed in a new 96-well plate, an equal amount of Griess reagent (Sigma) was added thereto, and the mixture was reacted at room temperature for 10 minutes, followed by measuring absorbance at a wavelength of 540nm using a microplate reader (Bio-Rad). A calibration curve was prepared using sodium nitrite (sodium nitrite), and the amount of sodium nitrite produced in the culture solution was determined based on the calibration curve (FIG. 12).
3-1-3 cytokine inhibitory Activity of Artocarpus communis
To confirm the inflammation inhibitory effect, the amounts of cytokines produced were measured using IL-6, TNF-alpha ELISA kit (mouse IL-6 Enzyme Linked immunological Assay kit BD bioscience) according to the manufacturer's protocol, respectively.
The anti-inflammatory activity was measured, and as a result, it was confirmed that: for the methanol (MeOH), hexane (Hexane), ethyl acetate (EtOAc) isolates of the leaves and stems of pistacia weinmannifolia, the increase in NO blocking activity was dependent on the concentration of the isolate. In particular, ethyl acetate isolate was found to have low toxicity and high NO inhibitory activity (fig. 13).
In addition, the anti-inflammatory activity of the column isolate of pistacia weinmannifolia was measured, and as a result, it was confirmed that: PWL was not toxic in the test sample, showed a weak NO inhibitory effect (FIGS. 13 and 14), and TNF-alpha inhibitory activity was also dependent on the concentration (FIG. 15).
In particular, it was confirmed that: fr.2, 3, 4 and 6 showed weak toxicity at high concentrations, NO inhibitory activity was dependent on concentration (FIG. 16), fr.5 showed NO toxicity, and IL-6 inhibitory activity was dependent on concentration (FIG. 17).
3-2 antioxidant activity of aromatic wood extract and isolate
3-2-1.DPPH radical scavenging Activity
The extract and column isolate of the aerial part of the pistacia weinmannifolia were subjected to the same experimental procedure as in example 1, and the radical scavenging effect was calculated using the group treated with DMSO as a negative control group, and the synthetic antioxidant BHA and the natural antioxidant Quercetin (Quercetin) were used as positive control groups.
As a result, it was confirmed that the extract and the isolate showed high DPPH radical scavenging activity, and particularly in the case of isolate No. 3, IC was observed 50 The value was 3.180. + -. 0.137. Mu.g/ml, showing higher antioxidant activity than the positive control group (FIGS. 18 and 19).
Therefore, it can be known that the extract of pistacia weinmannifolia and the isolate thereof have excellent antioxidant activity.
3-2. ABST free radical scavenging Activity
7mM of 2, 2-azabicyclo (3-ethylbenzothiazoline-6-sulfonic acid) (2, 2-azinobis- (3-ethyllbenzothiazoline-6-sulfonic acid) and 2.45mM of potassium persulfate (potassium persulfate) were mixed and left in the dark for 12 hours to prepare an ABTS solution, the ABTS solution was diluted with distilled water until the absorbance at 734nm became 0.7, and the solution was dispensed in an amount of 190. Mu.l each time to a 96-well plate, 10. Mu.l of a sample was mixed therein and allowed to react in the dark at room temperature for 7 minutes, the absorbance was measured by a microplate reader at absorbance 734nm, the group treated with DMSO was used as a negative control group to calculate the radical scavenging ability, and a synthetic antioxidant BHA and a natural antioxidant Quercetin (Quercetin) were used as a positive control group.
As a result, it was confirmed that the extract and the isolate showed high ABTS radical scavenging activity, and particularly in the case of isolate Nos. 2 and 3, IC was 50 The values were 1.974. + -. 0.237. Mu.g/ml and 2.236. + -. 0.279. Mu.g/ml, respectively, and showed similar activities to those of the positive control group (FIGS. 18 and 20).
3-2-3.SOD (Superoxide dismutase) Activity assay
Superoxide (Superoxide) radical scavenging activity was measured by the photosensitization reaction (photosensize reaction) of riboflavin. After dispensing 150. Mu.l containing 50mM potassium phosphate buffer (pH 7.8), riboflavin (riboflavin) (0.03 mM), EDTA (1 mM), methionine (0.6 mM), NBT (0.03 mM) in a 96-well plate (96-well plate), 10. Mu.l of a sample dissolved in DMSO was added, and the activity of scavenging radicals (radial) generated by photoreaction was measured. In the photoreaction for generating radicals, the light intensity was adjusted to 1000lux using an LED lamp provided in an illumination box (irradiation box), a 96well plate (96 well plate) to which a sample was added was irradiated with light at 25 ℃ for 6 minutes to react, and then the change in absorbance at 560nm was measured with a microplate reader. In this case, DMSO was added instead of the sample to the control group, and the absorbance during the reaction was set to 0.4 by irradiating with light for 6 minutes. The antioxidant BHA and the natural antioxidant Quercetin (Quercetin) were used as positive control groups.
As a result, it was confirmed that the column isolate showed high activity, and particularly in the case of isolate Nos. 2 and 4, IC was exhibited 50 The values were 3.1. Mu.g/ml and 3.9. Mu.g/ml, respectively, and showed similar activities to those of the positive control group (FIG. 18)
Therefore, it is known that the extract and isolate of pistacia weinmannifolia have excellent antioxidant activity.
3-3. Wrinkle inhibitory Activity of the extract and isolate of Aquilaria sinensis
In order to confirm the wrinkle-suppressing effect of each concentration of the extract, the amount of MMP (Matrix metalloproteinase) expression was examined.
The experimental method is as follows: BJ cells (ATCC, USA) as human fibroblasts were cultured in EMEM (Welgene, korea) supplemented with 10% FBS, and first, in order to confirm the non-toxicity of the extract, the cell viability was confirmed by treating the extract and the isolate of pistacia weinmannifolia at different concentrations. As a result, it was confirmed that the extract of Sasa Veitchii was not cytotoxic at a concentration of 2.5 to 20ug/ml (FIG. 21).
BJ cells were distributed in 6-well plates, allowed to adhere for one day, then pre-treated with different concentrations of extract for 1 hour and treated with 10ng/ml TNF for 24 hours. After washing the cells with 1 XPBS, RNA was extracted using Trizol, and cDNA was synthesized from the quantified RNA using Qiagen Omniscript RT-PC R kit. The cDNAs were mixed with the respective primers and PCR premix (Fermenta s) to carry out PCR, and the PCR products were loaded into 1.5% agarose (agarose Gel) and subjected to electrophoresis, and the results were visualized with Gel-Doc.
As a result, it was confirmed that: the extract of Pimpinella saxicola prevented gene expression of MMP-1, MMP-3, and MMP-12 depending on concentration, and reduced gene expression of cytokine IL-8 (FIG. 22).
Therefore, it is found that the extract of pistacia weinmannifolia and the isolate thereof have excellent wrinkle preventing, improving and treating effects.
From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, it should also be understood that the above-described embodiments are merely illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and range of the claimed scope of the present invention and all modifications or variations derived from the concept equivalent thereto.
Claims (2)
1. Use of a composition comprising as an active ingredient an extract of Pistacia weinmannifolia as an active ingredient for the manufacture of a skin external preparation for skin whitening or wrinkle improvement;
wherein the extract is obtained by using C 1 Alcohol extraction.
2. Use of a composition comprising an extract of Pistacia weinmannifolia as an active ingredient for the manufacture of a functional health food for skin whitening or wrinkle improvement,
wherein the extract is obtained by using C 1 Alcohol extraction.
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