KR102112806B1 - A use of Pistacia weinmannifolia for skin whitening and wrinkle improvement - Google Patents

Abstract

The present invention is Pistacia Wayne Manifolia Weinmannifolia ) relates to a cosmetic composition comprising an extract or a fraction thereof as an active ingredient, a composition comprising a pistacia Wayne Manipolia extract or a fraction thereof as an active ingredient, inhibiting the activity of tyrosinase involved in melanin synthesis, It has the effect of inhibiting melanin production in melanocytes, protecting cells against UV rays, suppressing the expression of wrinkle-related genes MMP-1 and MMP-3, and promoting the expression of elastin genes, so it has excellent skin whitening effect and wrinkle improvement effect. .

Description

A use of Pistacia weinmannifolia for skin whitening and wrinkle improvement}

The present invention is Pistacia Wayne Manifolia Weinmannifolia ) relates to a cosmetic composition comprising an extract or a fraction thereof as an active ingredient, specifically, a cosmetic composition for skin whitening or wrinkle improvement comprising an extract or fraction of pistacia Wayne Manipolia as an active ingredient, an external preparation for skin It relates to a composition and health functional food.

With the development of science, human life is prolonged and interest in functional cosmetics is also increasing. Human skin is the largest immune organ that is the primary defense tissue that protects the body from various harmful environments, and in order to protect it, the development of functional cosmetics with effects such as whitening, UV protection, and wrinkle improvement is required.

A person's skin color is genetically determined according to the concentration and distribution of melanin in the skin, but is also influenced by environmental or physiological conditions such as sun UV rays, fatigue, and stress. The chemical action of melanin production, which determines the color of the skin, has been reported many times through several papers, and melanocytes, melanocytes, produce cells from ultraviolet rays in the intracellular organelles called melanosomes. After synthesizing melanin having a protective function, the melanin is known to migrate from melanocytes to keratinocytes, and it has been reported that melanin transferred to keratinocytes determines skin color.

The precursor required in the process of biosynthesis of melanin is an amino acid called tyrosine, which is converted to dihydroxyphenylalanine (DOPA), and then finally converted to melanin through a substance called dopaquinone. It is said to be. The melanin synthesized in this way makes the skin color darker, but studies on the skin whitening method to suppress the melanin synthesis and brighten the skin color due to health or beauty needs are actively being conducted.

As a commonly known whitening component, substances that inhibit tyrosinase enzyme activity, such as kojic acid or arbutin, hydroquinone, vitamin C (L-Ascorbic acid), or derivatives thereof and various There are plant extracts. By inhibiting the synthesis of melanin pigments, they can not only realize skin whitening by brightening the skin tone, but also improve skin hyperpigmentation, such as ultraviolet rays, hormones, or freckles caused by genetics. However, when applied to the skin, there is a problem in that the use amount is limited due to safety problems such as irritation and redness, or the effect is insignificant, so that a practical effect cannot be expected.

There are two mechanisms to confirm the anti-wrinkle effect. One is to confirm the synthesis ability of collagen and elastin, and the other is to confirm the inhibition of MMP (proteinase). Collagen is the main component of connective tissue, of which Type I and III play an important role in the formation of skin cells and cell membranes. Elastin is a fibrous, natural polymer that exists in the connective tissue of the skin and supports collagen like a spring to maintain the elasticity of the skin. MMP stands for Matrix metalloproteinase, MMP-1 is a collagen degrading enzyme, and MMP-3 is an enzyme that denatures collagen, fibronectin, gelatin, and laminin.

Collagen is a major matrix protein produced by fibroblasts in the skin. It is present in the extracellular epilepsy, and its important functions are mechanical firmness of the skin, resistance to connective tissue and tissue, and cell adhesion, cell division and differentiation. It is known to induce (at the time of growth or wound healing of an organism). Such collagen is reduced by age and photoaging by ultraviolet irradiation, and collagen reduction is promoted by collagenase enzyme activity that breaks down collagen. It is known that this is closely related to the formation of wrinkles on the skin.

In addition, elastin (Elastin) fibers form a cross-link with collagen and are important skin components for wrinkle formation, which are involved in skin elasticity. The deficiency and aggregation of elastin fibers and the marked increase in the activity of the elastin-degrading enzyme Elastase have been found to be one of the factors leading to skin wrinkle formation. Elastase is the only enzyme that can break down elastin, and its inhibition is known to fundamentally reduce skin wrinkle improvement.

In addition, collagen and elastin fibers are substrate proteins that play an important role in the retention of moisture in the dermal layer, and these substrate proteins adsorb moisture and increase the water retention capacity inside the structure that they make, so that the skin can maintain the state containing the proper moisture. It is known to be involved in maintaining the elasticity of the skin through this.

Currently, retinoids, adenosine, animal placenta-derived proteins, chlorella extracts, etc. are known as skin elasticity enhancement and wrinkle improvement cosmetics. The most well-known retinol is a substance that promotes collagen synthesis and inhibits the elastase enzyme, but is unstable, and when applied to the skin, it has limited use due to safety problems such as irritation and redness. It is known that it is difficult to expect an effect of improving elasticity and wrinkles.

Meanwhile, Pistacia Wayne Manifolia Weinmannifolia J. Poiss , Ex Franch) is a plant that grows widely in Yunnan Province, China, and has been used for headaches including dysentery, enteritis, and flu since ancient times. Previous studies have reported that two compounds isolated from this plant have the ability to remove free radicals (Zhao X. et al. , Biochim Biophys Acta , 1725, 103-110, 2005). Except for this report, however, there is no known skin whitening and wrinkle improvement activity of the pistacia Wayne Manipolia extract, its fraction or a compound isolated therefrom.

Under these backgrounds, the present inventors tried hard to solve the above problems, and as a result, the pistacia Wayne Manifolia extract or a fraction thereof did not show toxicity, and exhibited excellent cell protection against ultraviolet rays, MMP (Matrix Metalloprotease) expression The present invention was completed by confirming the effect of suppression, cytokine IL-8 gene expression suppression, and elastin gene expression increase.

One object of the present invention is to provide a cosmetic composition comprising an extract or a fraction thereof of Pistacia weinmannifolia as an active ingredient.

Another object of the present invention is to provide a composition for external application for skin, comprising an extract of Pistacia weinmannifolia or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a health functional food comprising an extract of Pistacia weinmannifolia or a fraction thereof as an active ingredient.

Specifically, it is as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the specific descriptions described below.

As one aspect for achieving the above object, the present invention is Pistacia Wayne Manipolia ( Pistacia Weinmannifolia ) provides a cosmetic composition comprising an extract or a fraction thereof as an active ingredient.

" Pistacia Wayne Manifolia weinmannifolia J. Poiss , Ex Franch) "is a plant that is widely inhabited near Yunnan, China, and has been used for headaches such as dysentery, enteritis, and flu since ancient times. In previous research reports, two compounds isolated from this plant It has been reported to have the ability to remove free radicals (Zhao X. et al. , Biochim Biophys Acta , 1725, 103-110, 2005). Except for this report, however, there is no known skin whitening and wrinkle improvement activity of the pistacia Wayne Manipolia extract, its fraction or a compound isolated therefrom.

In the present invention, the term "Pistacia Wayne Manifolia extract" is an extract obtained by extraction using a suitable solvent from the stem of Pistacia Wayne Manifolia, a diluent or concentrate of the extract, a dried product obtained by drying the extract, and these modulators Or it may contain any one or more of the purified.

The pistacia Wayne Manifolia extract of the present invention can be prepared using conventional extraction methods in the art such as ultrasonic extraction, filtration, and reflux extraction, and Pistacia Wayne Manifolia is purchased or used commercially. However, it can be used that is harvested or grown in nature.

The pistacia Wayne Manifolia extract according to the present invention can be separated using conventional solvents according to conventional methods known in the art for preparing extracts from natural products, i.e., under conditions of conventional temperature and pressure.

Water, C ₁ to C ₄ anhydrous or hydrous lower alcohols, or a mixed solvent thereof may be used in the preparation of the pistacia Wayne Manipolia extract according to the present invention, but is not limited thereto. Preferably, an extract of pistacia Wayne Manipolia can be prepared using ethanol.

In the present invention, the term "fraction" means a result obtained by a fractionation method for separating a specific component or a specific group from the pistacia Wayne manipolyia extract prepared as above.

Specifically, the fraction may be a solvent fraction of the pistacia Wayne Manipolia extract, and more specifically, the solvent fraction may be a solvent fraction fractionated with hexane, chloroform, ethyl acetate or butanol, and more specifically sugar. Conventional fractional solvents known in the industry, for example, polar solvents such as C1 to C4 anhydrous or hydrous lower alcohols such as water, ethanol, methanol, and non-polar solvents such as hexane, ethyl acetate, chloroform, dichloromethane, or the like. A mixed solvent may be used, but is not limited thereto.

The pistacia Wayne Manipolia fraction of the present invention may also include those obtained by additionally applying a purification process. For example, a fraction obtained by passing the pistacia Wayne Manifolia extract according to the present invention through an ultrafiltration membrane having a constant molecular weight cut-off value, various chromatography such as medium pressure liquid chromatograpy (MPLC) (size, charge, hydrophobicity or affinity) Column fractions obtained through various purification methods additionally carried out by separation, etc., produced for separation by sex) are also included in the pistacia Wayne Manipolia fraction of the present invention.

In addition, the extract or fraction of the present invention may be extracted or fractionated from the upper part of the pistacia Wayne Manifolia, and more specifically, may be extracted or fractionated from the leaves of the Pistacia Wayne Manifolia, but is not limited thereto. It is not.

In one embodiment of the present invention, the leaves of the pistacia Wayne Manifolia were collected, naturally dried and crushed, and methanol was added to the pulverized powder sample to prepare a Pistacia Wayne Manifolia terrestrial extract. In addition, hexane, chloroform, ethyl acetate, and butanol fractions were obtained using the extract (FIG. 6), and active fractions (Fr. 1 to 6) were obtained using medium pressure liquid chromatograpy (MPLC) (FIG. 7 and Table 1). ).

The cosmetic composition of the present invention may be for skin whitening or wrinkle improvement, and more specifically for skin whitening and wrinkle improvement.

The present invention is known to be effective for headaches including dysentery, enteritis, and flu, and is known to have the ability to remove free radicals, but it has no effect on the skin whitening and wrinkle improvement effect of pistacia Wayne Manipolia. Nothing is known, and was first identified by the inventors.

In one embodiment of the present invention, 14 different plant species show excellent tyrosinase inhibitory effect and melanin production inhibitory effect by the pistacia Wayne manipolia extract (Fig. 2), and protect the cells against UV rays Effects (FIG. 4 and FIG. 5), the expression of wrinkle-related gene suppression, elastin gene expression promoting effect is excellent (FIG. 4), it was confirmed that the wrinkle improvement effect is excellent.

In addition, in another embodiment of the present invention, as a result of examining the expression level of matrix metalloprotease (MMP) in order to confirm the anti-wrinkle effect by concentration of the extract, the pistacia Wayne manipolia extract is concentration-dependently MMP-1, MMP-3 And the expression of MMP-12 was inhibited, and the gene expression of cytokine IL-8 was also reduced (FIG. 22), confirming that the wrinkle improvement effect was excellent.

Therefore, it was confirmed that the composition comprising the pistacia Wayne Manipolia extract or a fraction thereof as an active ingredient can be usefully used as a cosmetic composition for skin whitening or wrinkle improvement.

In addition, the cosmetic composition of the present invention may be an anti-inflammatory or antioxidant cosmetic composition.

In one embodiment of the present invention, it was confirmed that the extract of Pistacia Wayne Manifolia from 14 different plant species contains a significant amount of flavonoids, exhibits LO (lipoxygenase) inhibitory activity, and inhibits expression of inflammation-related genes. (Fig. 1), it was confirmed that the excellent anti-inflammatory effect. In addition, it was confirmed that the extract of Pistacia Wayne Manifolia contained more polyphenols than green tea, and exhibited DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging activity (FIGS. 2 and 3). , It was confirmed excellent antioxidant effect.

In another embodiment of the present invention, the pistacia Wayne Manifolia extract or a fraction thereof exhibits nitric oxide (NO) inhibitory activity (FIG. 12) and cytokine inhibitory activity (FIGS. 13 to 17) and has excellent anti-inflammatory effects. And DPPH free radical scavenging activity (Figs. 18 and 19), ABST free radical scavenging activity (Figs. 18 and 20), and SOD (Superoxide dismutase) activity (Fig. 18), confirming that it has excellent antioxidant effect .

Therefore, it was confirmed that the composition comprising the pistacia Wayne Manipolia extract or a fraction thereof as an active ingredient can be usefully used as an anti-inflammatory or antioxidant cosmetic composition.

As another aspect for achieving the above object, the present invention is Pistacia Wayne Manifolia ( Pistacia Weinmannifolia ) provides a composition for external application for skin comprising an extract or a fraction thereof as an active ingredient.

In the composition for external application for skin, pistacia Wayne manipolia, extract, and fraction are as described above.

The term “external skin preparation” of the present invention is a comprehensive concept including generally used substances for external application of skin. Non-limiting examples of external preparations for skin include PLASTERS, LOTIONS, and linement. (LINIMENTS), LIQUIDS AND SOLUTIONS, Aerosols, AEROSOLS, EXTRACTS, Ointments, FLUIDEXTRACTS, Emulsions, Suspension Agents, Capsules (CAPSULES) , Creams (CREAMS), soft, hard gelatin capsules, patch, or sustained release agents.

The composition for external application for skin of the present invention may be for skin whitening or wrinkle improvement, and more specifically, for skin whitening and wrinkle improvement.

In one embodiment of the present invention, 14 different plant species show excellent tyrosinase inhibitory effect and melanin production inhibitory effect by the pistacia Wayne manipolia extract (Fig. 2), and protect the cells against UV rays Effects (FIG. 4 and FIG. 5), the expression of wrinkle-related gene suppression, elastin gene expression promoting effect is excellent (FIG. 4), it was confirmed that the wrinkle improvement effect is excellent.

In addition, in another embodiment of the present invention, as a result of examining the expression level of matrix metalloprotease (MMP) in order to confirm the anti-wrinkle effect by concentration of the extract, the pistacia Wayne manipolia extract is concentration-dependently MMP-1, MMP-3 And MMP-12 were inhibited, and the cytokine IL-8 gene expression was also reduced (FIG. 22), confirming that the wrinkle improvement effect was excellent.

Therefore, it was confirmed that the composition comprising the pistacia Wayne Manipolia extract or a fraction thereof as an active ingredient can be usefully used as an external composition for skin whitening or wrinkle improvement.

In addition, the composition for external application for skin of the present invention may be a composition for external application for skin for anti-inflammatory or antioxidant properties.

In one embodiment of the present invention, it was confirmed that the extract of Pistacia Wayne Manifolia from 14 different plant species contains a significant amount of flavonoids, exhibits LO (lipoxygenase) inhibitory activity, and inhibits expression of inflammation-related genes. (Fig. 1), it was confirmed that the excellent anti-inflammatory effect. In addition, it was confirmed that the extract of Pistacia Wayne Manifolia contained more polyphenols than green tea, and exhibited DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging activity (FIGS. 2 and 3). , It was confirmed excellent antioxidant effect.

In another embodiment of the present invention, the pistacia Wayne Manifolia extract or a fraction thereof exhibits nitric oxide (NO) inhibitory activity (FIG. 12) and cytokine inhibitory activity (FIGS. 13 to 17) and has excellent anti-inflammatory effects. And DPPH free radical scavenging activity (Figs. 18 and 19), ABST free radical scavenging activity (Figs. 18 and 20), and SOD (Superoxide dismutase) activity (Fig. 18), confirming that it has excellent antioxidant effect .

Accordingly, it was confirmed that the composition comprising the pistacia Wayne Manipolia extract or a fraction thereof as an active ingredient can be usefully used as an external composition for anti-inflammatory or antioxidant skin.

As another aspect for achieving the above object, the present invention is Pistacia Wayne Manifolia ( Pistacia Weinmannifolia ) provides a health functional food containing an extract or a fraction thereof.

Pistacia Wayne Manifolia, extract, and fractions in the dietary supplement are as described above.

The term "health functional food" of the present invention refers to food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using ingredients or ingredients having useful functionality for the human body. Here, the term "functionality" means to obtain a useful effect for health use such as adjusting nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be manufactured by a method conventionally used in the art, and at the time of manufacture, it may be prepared by adding raw materials and ingredients that are conventionally added in the art. In addition, unlike general drugs, there is an advantage that there is no side effect, etc. that may occur when taking the drug for a long time by using food as a raw material, and may be excellent in portability.

The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, the pistacia Wayne Manifolia extract or fractions thereof of the present invention in the manufacture of food is added in an amount of 1 to 10% by weight, preferably 5 to 10% by weight of the raw material composition. However, in the case of long-term intake for health and hygiene purposes or for health control purposes, the amount may be used below the above range.

The health functional food of the present invention may be for skin whitening or wrinkle improvement, and more specifically for skin whitening and wrinkle improvement.

In one embodiment of the present invention, 14 different plant species show excellent tyrosinase inhibitory effect and melanin production inhibitory effect by the pistacia Wayne manipolia extract (Fig. 2), and protect the cells against UV rays Effects (FIG. 4 and FIG. 5), the expression of wrinkle-related gene suppression, elastin gene expression promoting effect is excellent (FIG. 4), it was confirmed that the wrinkle improvement effect is excellent.

In addition, in another embodiment of the present invention, as a result of examining the expression level of matrix metalloprotease (MMP) in order to confirm the anti-wrinkle effect by concentration of the extract, the pistacia Wayne manipolia extract is concentration-dependently MMP-1, MMP-3 And MMP-12 were inhibited, and the cytokine IL-8 gene expression was also reduced (FIG. 22), confirming that the wrinkle improvement effect was excellent.

Accordingly, it was confirmed that the composition comprising the pistacia Wayne Manipolia extract or a fraction thereof as an active ingredient can be usefully used as a health functional food for skin whitening or wrinkle improvement.

In addition, the health functional food of the present invention may be a health functional food for anti-inflammatory or antioxidant.

In one embodiment of the present invention, it was confirmed that the extract of Pistacia Wayne Manifolia from 14 different plant species contains a significant amount of flavonoids, exhibits LO (lipoxygenase) inhibitory activity, and inhibits expression of inflammation-related genes. (Fig. 1), it was confirmed that the excellent anti-inflammatory effect. In addition, it was confirmed that the extract of Pistacia Wayne Manifolia contained more polyphenols than green tea, and exhibited DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging activity (FIGS. 2 and 3). , It was confirmed excellent antioxidant effect.

In another embodiment of the present invention, the pistacia Wayne Manifolia extract or a fraction thereof exhibits nitric oxide (NO) inhibitory activity (FIG. 12) and cytokine inhibitory activity (FIGS. 13 to 17) and has excellent anti-inflammatory effects. And DPPH free radical scavenging activity (Figs. 18 and 19), ABST free radical scavenging activity (Figs. 18 and 20), and SOD (Superoxide dismutase) activity (Fig. 18), confirming that it has excellent antioxidant effect .

Therefore, it was confirmed that the composition comprising the pistacia Wayne Manipolia extract or a fraction thereof as an active ingredient can be usefully used as an anti-inflammatory or antioxidant health functional food.

The composition comprising the pistacia Wayne Manipolia extract or a fraction thereof as an active ingredient of the present invention inhibits the activity of tyrosinase involved in melanin synthesis, inhibits melanin production in melanocytes, protects cells against UV rays, and MMP, a wrinkle-related gene It has the effect of suppressing the expression of -1 and MMP-3 and promoting the expression of the elastin gene, and thus has an excellent skin whitening effect and wrinkle improvement effect.

1 is a table showing the effect of gene expression of IL-6, IL-8 and N-kB1, total flavonoid content of 14 plant extracts, LO inhibitory activity, concentration indicating cytotoxicity.
Figure 2 is a table showing the total polyphenol content of 14 plant extracts, DPPH free radical scavenging activity, tyrosinase inhibitory activity, and melanin synthesis inhibitory activity.
3 is a graph showing DPPH free radical scavenging activity of 14 kinds of plant extracts.
4 is a table showing the effect of 14 kinds of plant extracts on cell protection against UV light, MMP-1 and MMP-3 expression levels, and elastin gene expression levels.
5 is a graph showing the cell protection effect of 14 kinds of plant extracts against ultraviolet rays.
Figure 6 shows the respective solvent fractions obtained from the extract of Pistacia Wayne Manifolia terrestrial extract.
FIG. 7 shows the MPLC results for each active fraction (Fr. 1 to 6) obtained from the extract of Pistacia Wayne Manifolia above ground.
FIG. 8 shows the results of UPLC (ultra performance liquid chromatography) analysis using a 254 nm wavelength for each column fraction of pistacia Wayne Manipolia extract.
FIG. 9 shows the results of ULLC (ultra performance liquid chromatography) analysis using charged aerosol detection (CAD) for each column fraction of pistacia Wayne Manipolia extract.
FIG. 10 shows the results of ultra performance liquid chromatography (UPLC) analysis using Mass Spectrometry (MS) for each column fraction of pistacia Wayne Manipolia extract.
11 is a graph showing the cell viability in the case of treating the extract of pistacia Wayne Manipolia and each solvent fraction.
12 is a graph showing NO inhibitory activity in the case of treating the extract of pistacia Wayne Manipolia and each solvent fraction.
13 is a graph showing the NO inhibitory activity and the degree of cytotoxicity when the extract of Pistacia Wayne Manipolia and each solvent fraction are treated.
14 is a graph showing the degree of cytotoxicity of extracts and column fractions of Pistacia Wayne Manifolia.
15 is a graph showing the TNF-alpha inhibitory activity of extracts and column fractions of Pistacia Wayne Manifolia.
16 is a graph showing NO inhibitory activity of extracts and column fractions of Pistacia Wayne Manipoli.
17 is a graph showing IL-6 inhibitory activity of extracts and column fractions of pistacia Wayne manipolia.
FIG. 18 is a table showing DPPH free radical scavenging activity, ABTS radical scavenging activity, and superoxide scavenging activity of extracts and column fractions of pistacia Wayne manipolia.
19 is a table showing DPPH free radical scavenging activity of extracts and column fractions of Pistacia Wayne Manipoli.
FIG. 20 is a table showing ABTS radical scavenging activity of extracts and column fractions of Pistacia Wayne Manifolia.
21 is a graph showing cell survival rates when the pistacia Wayne Manipolia extract was treated by concentration.
FIG. 22 is an electrical diagram showing the inhibitory effects on gene expression of MMP-1, MMP-3 and MMP-12, and cytokine IL-8 gene expression when the Pistacia Wayne Manifolia extract is treated by concentration in BJ cells. Yeongdong image.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: 14 types  Plant cosmetic material screening, and Pistacia Wayne Manipoli  activation

In order to select cosmetic materials, experiments were conducted on 14 kinds of plant extracts for anti-inflammatory, antioxidant, whitening, skin protection and wrinkle improvement effects.

1-1. 14 kinds of plants Anti-inflammatory effect of extract

1-1-1. Flavonoid content measurement

Flavonoids have antibacterial, anticancer, antiviral and anti-inflammatory activities and are known to have antioxidant activity in vivo. To measure the amount of flavonoids, the absorbance of the yellow flavan or water-soluble flavonol glycosides produced by treating the sample with alkali was measured. The content of total flavonoids in 14 plant extract samples was calculated from the standard curve.

As a result, the total flavonoid content in 14 plant extracts is shown in FIG. 1. It was confirmed that the pistacia Wayne Manipolia extract (AKS-7) contained 0.41 mg / g of flavonoids, and was expected to exhibit antibacterial, anticancer, antiviral and anti-inflammatory activities.

1-1-2. LO ( lipoxygenase ) Inhibitory activity

Lipoxygenase Inhibitor Screening Assay kit (Cayman, MI, USA) was used to measure the LO inhibitory activity of 14 plant extracts, and was performed according to the manufacturer's protocol.

As a result, LO inhibitory activity of 14 types of plant extracts is shown in FIG. 1. In particular, the positive control indomethacin showed an inhibitory effect of about 22%, while the pistacia Wayne Manipolia extract (AKS-7) showed an inhibitory effect of 53%, indicating that it has an anti-inflammatory effect.

1-1-3. Cell viability measurement

Dark blue formazan in which yellow MTT (3- [4,5-Dimethylthiazol-2-yl] -2, 5-diphenyltetrazolium bromide) is oxidized by the mitochondria of cells to confirm the toxicity of 14 plant extract samples Was dissolved in DMSO, and absorbance was measured at 570 nm with a microplate.

As a result, the toxicity of 14 types of plant extracts is shown in FIG. 1. Particularly, since it was confirmed that the pistacia Wayne Manipolia extract (AKS-7) had no toxicity below 125 ppm, the following anti-inflammatory effect experiment was conducted at a concentration that did not show toxicity.

1-1-4. Inflammation gene expression experiment

In order to confirm the effect of suppressing the expression of inflammation-related genes in 14 plant extract samples, gene expression of cytokines (IL-6, IL-8) and NF-kB was confirmed through RT-PCR.

As a result, the effect of suppressing the expression of inflammation-related genes of 14 plant extracts is shown in FIG. 1. When the gene expression by UV was 100%, the group treated with pistacia Wayne manipolia (AKS-7) was 48% (IL-6), 49% (IL-8) and 54% (NF-), respectively. kB), and it was found that the anti-inflammatory effect was exhibited by suppressing the expression of inflammation-related genes.

1-2. 14 kinds of plants Antioxidant effect of extract

1-2-1. Total polyphenol content measurement

Polyphenols are one of the antioxidants that turn free radicals (harmful oxygen) in our body into harmless substances. Folin-Ciocalteu reagent (FCR) was used to measure the content of polyphenols in the sample, which is a complex of Phosphomolybdic acid and Phosphotungstic acid. to be.

As a result, the polyphenol content of 14 kinds of plant extracts is shown in FIG. 2. Considering that the green tea, which is known to have a lot of polyphenols, exhibits a content of 300 mg / g, the pistacia Wayne Manipolia extract (AKS-7) showed more total polyphenol content of 333.5 mg / g than this. Therefore, it was found that the pistacia Wayne Manipolia extract has an excellent antioxidant effect.

1-2-2. DPPH  Free radical scavenging activity

DPPH (1,1-diphenyl-2-picryl-hydrazyl, Sigma) receives electrons or hydrogen from antioxidants to form irreversibly stable molecules, so free radicals are eliminated when it encounters substances with antioxidant activity. At this time, DPPH's unique blue-blue color has a characteristic of being pale, and the color difference is non-color-quantized to measure the electron donating ability (Jeon et al., J Kor Soc Food sci Nutr. 38 (1): 1-8, 2009)

Specifically, 90 μl of DPPH solution dissolved in 0.15 mM in ethanol was dispensed into a 96-well plate, mixed with 10 μl of each sample of 14 plant extracts, and allowed to stand at room temperature for 20 minutes, followed by microplate reader (SPARK10) ) Was measured at 517 nm. The free radical scavenging effect was calculated using the DMSO-treated group as a negative control, and the synthetic antioxidant BHA and the natural antioxidant Quercetin were used as positive controls.

As a result, DPPH free radical scavenging activity of 14 kinds of plant extracts is shown in FIGS. 2 and 3. In particular, the pistacia Wayne Manipolia extract (AKS-7) exhibited DPPH free radical scavenging activity similar to Ascorbic acid, known as an antioxidant, and confirmed that it has excellent antioxidant activity.

1-3. 14 kinds of plants Whitening effect of extract

1-3-1. Tyrosinase ( Tyrosinase ) Inhibitory effect

Tyrosinase is an enzyme that catalyzes the conversion of two steps from L-tyrosine to DOPA (dihydroxyphenylalanine) → DOPA quinone, using L-tyrosine as a substrate during melanin formation as a copper containing oxidase. Tyrosinase acts early in the process of synthesizing melanin, and is most commonly used in the whitening screening phase.

As a result of the experiment, tyrosinase inhibitory effects of 14 plant extracts are shown in FIG. 2. IC ₅₀ of the control group, Ascorbic acid, was 0.009 ppm, and no sample had a higher inhibitory activity, but Pistacia Wayne manipolia (AKS-7) exhibited an IC ₅₀ value of 0.1 ppm or less, so it has excellent whitening effect. Was confirmed.

1-3-2. Melanin production inhibitory effect

Melanin is a pigment that is produced by melanocytes and transmitted to keratinocytes to determine the color of hair or skin. In order to confirm the whitening efficacy, the melanin biosynthesis of 14 kinds of plant extract samples was measured using B16F1 cells, and the production inhibition rate was confirmed by setting the melanin production amount of the negative control group to 100%, and arbutin as the positive control group. Was used.

As a result, the effect of inhibiting melanin production of 14 kinds of plant extracts is shown in FIG. 2. In particular, showed a blood star cyano Wayne Mani polyamic excellent melanogenesis inhibitory than the IC ₅₀ value 82.3ppm of arbutin (Arbutin) melanin IC ₅₀ value of the positive control of the 79.3ppm (AKS-7) effect. Therefore, the excellent whitening effect of Pistacia Wayne Manipoli was confirmed.

1-4. Wrinkle improvement efficacy of 14 kinds of plant extracts

1-4-1. Cell protection effect against UV rays

Ultraviolet rays are a major cause of various problems in the skin such as inflammation, wrinkles, and pigmentation. When UV rays are received, wrinkles are generated by increasing the expression of MMP, an enzyme that destroys collagen or elastin. After applying 14 kinds of plant extract samples to Fibroblast cells and irradiating them with ultraviolet rays, the cell viability of the samples was confirmed by ultraviolet light by confirming the cell viability.

As a result, the cell protection effect of 14 kinds of plant extracts against ultraviolet rays is shown in FIGS. 4 and 5. In the case of the control group, Ascorbic acid, the cell protective effect was 50% at 50 ppm, whereas the pistacia Wayne Manipolia extract (ASK-7) showed a cell protective effect of 89%, thereby providing excellent cell protection against UV rays and thus It can be seen that it has a wrinkle improvement effect.

1-4-2. Wrinkle-related gene expression investigation

By examining the gene expression levels of MMP-1, MMP-3, and elastin, which are changed by irradiating ultraviolet rays to the fibroblast, it was intended to confirm the anti-wrinkle effect of the sample.

As a result, the effect of 14 kinds of plant extracts on wrinkle-related gene expression is shown in FIG. 4. When the expression of MMP-1 and MMP-3 activated by UV irradiation was 100%, the group treated with Pistacia Wayne Manipolia (AKS-7) showed expression levels of 48% and 18%, respectively, and 50%. It showed the above-mentioned inhibitory effect. In addition, the elastin gene exhibited a high expression rate of 321% by treatment with Pistacia Wayne Manipolia.

Therefore, it can be seen that pistacia Wayne manipolia significantly suppresses the expression of the wrinkle-related genes MMP-1 and MMP-3, while significantly promoting the expression of the elastin gene, thereby preventing, improving and treating wrinkles.

Example  2: Pistacia Wayne Manipoli  Aboveground extract, Fraction  And compound preparation

The anti-inflammatory, anti-oxidation, whitening and anti-wrinkle effects of pistacia Wayne Manifolia, which showed a remarkable effect among the 14 plants identified in Example 1, were intended to be more specifically confirmed.

2-1. Pistacia Wayne Manipoli  Preparation of above-ground extract

Pistacia Wayne Manifolia weinmannifolia J. Poiss. Ex Franch) 's methanol extract was purchased from the Overseas Plant Extract Bank of the Korea Research Institute of Bioscience and Biotechnology's Biomaterials Hub Center.

Looking at the extraction process, 20 kg of leaves of Pistacia Wayne Manifolia were collected, dried by using a dryer (50 to 55) (shaded) to remove moisture, and then crushed. Based on the dry weight of the pulverized powder sample, 30 l of methanol was added and extracted at room temperature. Then, after filtration and concentration under reduced pressure, 564.7 g of a pistacia Wayne manipolyia methanol extract was obtained. In the subsequent experiments, the obtained pistacia Wayne Manipolia methanol extract was referred to as a total extract.

2-2. Pistacia Wayne Manipoli  Above-ground solvent Fraction  Produce

54.7 L of water was added to 564.7 g of the total extract of Pistacia Wayne Manifolia supernatant obtained in Example 2-1 and suspended, and the same amount of hexane was added to separate the water layer and the hexane layer. This process was repeated three more times in the same manner, filtered and concentrated under reduced pressure to separate the hexane fraction (39.2 g). The hexane layer was separated in the same manner, and the same amount of chloroform was added to the remaining water layer, followed by separation in the same manner to obtain a chloroform fraction (17.8 g). In the same manner, the chloroform layer was separated, and the same amount of ethyl acetate was added to the remaining water layer, followed by separation in the same manner to obtain an ethyl acetate fraction (68.4 g). The ethyl acetate layer was separated in the same manner, and the same amount of butanol was added to the remaining water layer to separate it in the same manner to obtain a butanol fraction (111.0 g). The remaining water layer was concentrated to give a water fraction (109.0 g) (Figure 6).

2-3. Pistacia Wayne Manipoli  Above ground column Fraction  Produce

For MPLC analysis, the MPLC instrument (Armen Spot prep, Gilson, France) was equipped with a calp (20 mm x 250 mm; Resin; YMC-Pack ODS AQ-HG, 10 μm), and then the methanol extract was repeated in an amount of 1 g. It was loaded. At this time, as a solvent, methanol / water [0-10 min, 0% MeOH; 10-70 min, 0-100% MeOH; 70-90 min, 100% MeOH] was used, the elution rate was 20 ml / min, and detected at a wavelength of UV 200-400 mm to obtain active fractions (Fr. 1 to 6) (FIG. 7 and Table 1). ).

2-4. Pistacia Wayne Manipoli  Extract and Fraction  analysis

In order to investigate the active fraction of the ethyl acetate fraction of the pistacia Wayne Manipolia obtained in Example 1-2, they were analyzed by liquid chromatography (UPLC) (ultra performance liquid chromatography).

First, for UPLC analysis, methanol extract, solvent fractions, and column fractions were filtered once with a 0.25 mm membrane filter for UPLC. After mounting a column (Waters BEH C18 column, 2.1 × 100 mm, 1.7 mm) to a UPLC instrument (Waters UPLC-Q-TOF), each filtered fraction was loaded in an amount of 0.3 μl.

At this time, acetonitrile + 0.1% formic acid / water + 0.1% formic acid [10: 90-> 100: 0 (v / v)] was used as the solvent used for the UPLC analysis, and the elution rate was 0.4 ml / min. . The effective fractions and separated substances from UPLC were chromatographed using a wavelength of 200-400 nm as a detector, charged aerosol detection (CAD), and mass spectrometry (MS) to confirm the separation of the substances (FIGS. 8 to 10). , And Table 2).

Example  3: Pistacia Wayne Manipoli  Extract and Fraction  Active check

3-1. Pistacia Wayne Manipoli  Extract and Fraction  Anti-inflammatory activity

3-1-1. Pistacia Wayne Manipoli  Cytotoxicity measurement of samples

1 × 10 in DMEM (Dulbecco's Modified Eagle Medium, Gibco) medium with 5% Fetal Bovine Serum 5% RAW264.7 cells, mouse macrophages, to measure cytotoxicity of Pistacia Wayne Manipolia samples ^It was suspended at a concentration of ⁵ / mL and inoculated in 96-well plates at 100 µl each. After 4 hours, a sample of Pistacia Wayne Manipolia was treated at a concentration of 20 μg / ml. After incubation for 24 hours, 5 μl / ml MTT solution was added at 10 μl per well, followed by further incubation for 4 hours, supernatant was removed, DMSO was added at 100 μl, and absorbance was measured at 570 nm. Cell viability was calculated according to the following equation using the negative control treated with 0.25% DMSO as 100%.

As a result, the cell viability in the case of treating the extract and each fraction of pistacia Wayne Manipoli was shown as shown in FIG. 11.

3-1-2. Pistacia Wayne Manipoli Nitric oxide  Inhibitory activity

RAW264.7 cells were treated with lipopolysaccharide (LPS) to determine the inhibitory effect on artificially induced inflammation. Fetal Bovine Serum (5%) was added to DMEM (Dulbecco's Modified Eagle Medium, Gibco) medium without phenol-red, and the cells were suspended at a concentration of 2.5 × 10 ⁵ / ml to 96 Well plates (96 well plates) were inoculated with 200 μl. After attaching for 4 hours, After treating the Pistacia Wayne Manipolia sample and incubating for 1 hour, 0.5 µg / ml lipopolysaccharide (LPS, lipopolysaccharide, Sigma) was treated and incubated for 24 hours. Thereafter, 100 µl of the supernatant was collected, placed in a new 96-well plate, and the same amount of grease reagent (Griess reagent, Sigma) was added, reacted at room temperature for 10 minutes, and then a microplate reader (microplate reader, Bio-Rad) Absorbance was measured at a wavelength of 540 nm. A calibration curve was prepared using sodium nitrite, and the amount of nitric oxide in the culture was determined based on this (FIG. 12).

3-1-3. Pistacia Wayne Manipoli  Cytokine inhibitory activity

To confirm the anti-inflammatory effect, cytokine production was measured using IL-6 and TNF-alpha ELISA kits (mouse IL-6 Enzyme Linked Immunosorbent Assay kit; BD bioscience), respectively.

As a result of measuring the anti-inflammatory activity, it was confirmed that the NO inhibitory activity was increased depending on the concentration of methanol (MeOH), hexane (Hexane), and ethyl acetate (EtOAc) fractions of the pistacia Wayne Manifolia leaf and stem. In particular, it was confirmed that the ethyl acetate fraction has weak toxicity and strong NO inhibitory activity (FIG. 13).

In addition, as a result of measuring the anti-inflammatory activity of the column fraction of Pistacia Wayne Manifolia, PWL was not toxic in the sample, showed a weak NO inhibitory effect (FIGS. 13 and 14), and TNF-alpha inhibitory activity concentration. It was confirmed to appear dependent (FIG. 15).

Particularly, Fr.2, 3, 4, and 6 exhibited weak toxicity at high concentrations, and the NO inhibitory activity was concentration-dependent (FIG. 16), and Fr.5 showed IL-6 inhibitory activity without showing toxicity. It was confirmed to appear dependent (FIG. 17).

3-2. Pistacia Wayne Manipoli  Extract and Fraction  Antioxidant activity

3-2-1. DPPH  Free radical scavenging activity

In the same experimental method as in Example 1, the extracts and column fractions of Pistacia Wayne Manifolia were added, and the free radical scavenging effect was calculated using the DMSO-treated group as a negative control, and the synthetic antioxidant BHA. Quercetin, a natural antioxidant, was used as a positive control.

As a result, it was confirmed that the DPPH free radical scavenging activity was high in the extracts and fractions. In particular, in the case of fraction 3, the IC ₅₀ value was 3.180 ± 0.137 µg / ml, which showed higher antioxidant activity than the positive control (FIG. 18). And Fig. 19)

Therefore, it can be seen that the extract of pistacia Wayne Manipoli and its fraction have excellent antioxidant activity.

3-2-2. ABST  Free radical scavenging activity

7 mM 2,2-azinobis- (3-ethylbenzothiazoline-6-sulphonic acid) (2,2-azinobis- (3-ethylbenzothiazoline-6-sulphonic acid) and 2.45 mM potassium persulfate (potassium) persulfate) was mixed, and the ABTS solution prepared by leaving it in the cow for 12 hours was diluted with distilled water to have an absorbance of 0.7 at 734 nm, and then dispensed into each 96 µl 96-well plate. The reaction was measured for 7 min.Absorbance was measured with a microplate measuring instrument at 734 nm, and the radical scavenging ability was calculated using the DMSO-treated group as a negative control, and the synthetic antioxidant BHA and the natural antioxidant Quercetin were used as a positive control.

As a result, it was confirmed that the extracts and fractions showed high ABTS free radical scavenging activity. In particular, for fractions 2 and 3, IC ₅₀ values were 1.974 ± 0.237µg / ml and 2.236 ± 0.279µg / ml, respectively, and were positive. It showed similar activity to the control group (Fig. 18 and Fig. 20).

3-2-3. SOD ( Superoxide dismutase ) Activity measurement

The superoxide radical scavenging activity was measured using a photoensitize reaction of riboflavin. 150 μl including 50 mM potassium phosphate buffer (pH 7.8), riboflavin (0.03 mM), EDTA (1 mM), methionine (0.6 mM), NBT (0.03 mM) To After dispensing in 96-well plate, 10 μl of the sample dissolved in DMSO After adding, the activity of scavenging radicals generated by the photochemical reaction was measured. For the photochemical reaction for radical generation, the intensity of light was adjusted to 1000 lux using the LED lamp installed in the illumination box, and the 96-well plate to which the sample was added was reacted by dividing light for 25 to 6 minutes, and then absorbed the light at 560 nm The degree change was measured with a microplate reader. At this time, DMSO was added to the control instead of the sample, and when absorbed by reacting with light for 6 minutes, the absorbance was set to 0.4. Antioxidant BHA and natural antioxidant Quercetin were used as positive controls.

As a result, it was confirmed that it showed high activity in the column fraction, and in particular, in the case of fractions 2 and 4, IC ₅₀ values were 3.1 μg / ml and 3.9 μg / ml, respectively, and showed similar activity to the positive control (FIG. 18)

Therefore, it can be seen that the pistacia Wayne Manipolia extract and fraction have excellent antioxidant activity.

3-2. Pistacia Wayne Manipoli  Extract and Fraction  Anti-wrinkle activity

MMP (Matrix Metalloprotease) expression level was examined to confirm the anti-wrinkle effect by concentration of the extract.

As an experimental method, human fibroblasts BJ cells (ATCC, USA) were cultured in EMEM (Welgene, Korea) with 10% FBS added, and first, to check the toxicity of the extract, the pistacia Wayne Manipolia extract and Cell viability was confirmed by treating the fractions by concentration. As a result, it was confirmed that there was no cytotoxicity at a concentration of 2.5 to 20 ug / ml of Pistacia Wayne Manipolia extract (FIG. 21).

After dispensing BJ cells in a 6-well plate for 1 day, the extract was pre-treated for 1 hour by concentration and treated with 10 ng / ml of TNF for 24 hours. After washing the cells with 1X PBS, RNA was extracted using Trizol, and cDNA was synthesized using Qiagen Omniscript RT-PCR kit for quantified RNA. PCR was performed by mixing cDNA with each primer and PCR premix (Fermentas), and the PCR product was loaded on a 1.5% agarose gel and subjected to electrophoresis, and the results were imaged with Gel-Doc.

As a result, it was confirmed that the extract of Pistacia Wayne manipolia inhibited the gene expression of MMP-1, MMP-3 and MMP-12 in a concentration-dependent manner, and also reduced the gene expression of cytokine IL-8 (FIG. 22). .

Therefore, it can be seen that the pistacia Wayne Manipolia extract and its fractions are excellent in preventing, improving and treating wrinkles.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed to include all changes or modifications derived from the meaning and scope of the following claims rather than the above detailed description and equivalent concepts thereof.

Claims (12)

Pistacia Wayne Manifolia cosmetic composition comprising an extract of weinmannifolia or a fraction thereof as an active ingredient.
The cosmetic composition according to claim 1, wherein the cosmetic composition is for skin whitening.
The cosmetic composition according to claim 1, wherein the cosmetic composition is for wrinkle improvement.
The cosmetic composition according to claim 1, wherein the extract is extracted using water, an alcohol of C1 to C4, or a mixed solvent thereof.
The cosmetic composition according to claim 1, wherein the fraction is a solvent fraction fractionated with hexane, chloroform, ethyl acetate or butanol.
The cosmetic composition according to claim 1, wherein the fraction is a column fraction fractionated by medium pressure liquid chromatograpy (MPLC).
The cosmetic composition according to claim 1, wherein the extract or fraction is extracted or fractionated from the upper part of the pistacia Wayne Manifolia.
The cosmetic composition according to claim 1, wherein the extract or fraction is extracted or fractionated from the leaves of Pistacia Wayne Manifolia.
delete delete Pistacia Wayne Manifolia weinmannifolia ), a health functional food comprising an extract or a fraction thereof.
The health functional food according to claim 11, wherein the health functional food is for skin whitening or wrinkle improvement.

Images