KR20180059287A - A composition for antioxidating and blocking ultraviolet comprising extracts of jujube seed - Google Patents

Abstract

The present invention relates to a composition containing a jujube seed extract. The composition containing the jujube seed extract as an active ingredient of the present invention has an antioxidant effect due to excellent DPPH free radical, ABST radical cation, hydrogen peroxide and superoxide anion radical scavenging activity, and cells are not killed by irradiation of ultraviolet rays so that the composition can be effectively used as sunblock. Also, the jujube seed extract of the present invention has excellent antimicrobial activity and is free from cytotoxicity and skin side effects so that the composition can be safely used in cosmetic, pharmaceutical and food compositions.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for antioxidation and ultraviolet screening comprising a jujube extract as an active ingredient,

The present invention relates to a composition containing an extract of Jujube seed, and more specifically to a cosmetic, a pharmaceutical composition and a food composition for antioxidation and ultraviolet screening containing a jujube extract as an active ingredient.

The human skin consists of the epidermis, dermis, and connective tissue including the stratum corneum, and the stratum corneum is composed of a dead cell layer formed through the differentiation process of keratinocyte, the basal cell of the epidermis And is responsible for protecting the human body from the effects of the external environment. There are two major researches on the causes of skin aging. One is "internal aging", which is caused by changes in the function of cells, which are constitutive units of skin, with age, and the other is "external aging" Which can be divided into external environment such as ultraviolet rays, pollution, stress and the like.

As the cells of the skin are exposed to oxidative stress by reactive oxygen species generated in a continuous biochemical reaction for the necessary energy supply in the human body in order to supply necessary energy in vivo, the cellular components such as lipid, protein, It changes carbohydrate and DNA oxidative damage and enzymatic activity, causing various diseases such as skin cancer, and promotes aging.

The domestic sun protection products market is expected to grow steadily at W332bn in 2010 and reach W420bn in 2014. According to the Data Monitor research, the domestic sunscreen market has continued to grow at an annual average of 6.34%, from KRW332 billion in 2010 to KRW357 billion in 2011, KRW377 billion in 2012, and KRW398 billion in 2013. As the risk of photo-aging becomes known, sunscreen agents are becoming a necessity for the seasons, but they are still sold as a typical seasonal product with a strong summer season.

About 90% of ultraviolet rays reaching the earth surface are ultraviolet ray A, and less than 10% is ultraviolet ray B. Since the wavelength of light is inversely proportional to energy, ultraviolet B can have an effect on the cell's genes, since the absolute amount has at least a strong energy. Minimal erythema dose (MED) is the minimum amount of light that causes skin erythema by light of a certain wavelength. The minimum erythema of skin in Asian is 50-70 J / cm2 for ultraviolet A, 50-70 mJ / cm2 for ultraviolet B and 1 / 1,000 of ultraviolet A can cause erythema. The minimum erythema of ultraviolet B in hairless mice, which are widely used in skin barrier studies, varies from 20 to 80 mJ / cm ² .

Ultraviolet rays can cause skin cancer by changing DNA, and it is unpleasant for skin to reduce the number of cells that are responsible for immunity of the skin to suppress the skin immune response. Due to the increased awareness of ultraviolet rays, ultraviolet screening and absorption cosmetics are being developed in a variety of formulations and types.

Therefore, in the field of cosmetics, research and development on functional cosmetics having antioxidative and UV-blocking effects have been actively carried out, and studies using natural substances have been continued to prevent toxicity or irritation to the skin.

On the other hand, the jujube seed is the seed part in the jujube of jujube, which is the byproduct of jujube produced after the processing of jujube. The jujube seed by-products produced in the jujube process account for about 30% of the jujube raw materials. Since jujube has been added to sweeten the juice in many cases, the jujube has a pharmacological effect which improves symptoms of stomach cramps, insomnia, dyspepsia, colonic hemoglobin, cough, and hypersensitivity.

Nowadays, the jujube leaves and fruits are edible, but the seeds are reportedly limited to edible. The content of protein, minerals, dietary fiber, etc. Has high nutritional value. Despite these excellent nutritional and pharmacological values, most of the jujube seed resources are not used industrially and are discarded.

The components of the jujube seeds are mainly oleic acid, fatty oil consisting of unsaturated fatty acids of linoleic acid, saponin, eblin, and lactone. Betulin and betulicacid are known as medicinal ingredients. Various sterols, alkaloids, vitamins, saponins, serotonin, fatty acids, polyphenols and flavonoids are known as medicinal components of jujube. In particular, the cyclic-AMP content is about 1,000 times higher than that of ordinary plants, and other nutrients such as sugars and organic acids are known (Ministry of Food and Drug Safety, 2013).

However, there has been no report on the antioxidative and UV-blocking effects of the jujube seeds.

Accordingly, the inventors of the present invention have continued to develop new natural substances having antioxidative and UV-blocking effects and fewer human adverse effects, and as a result, it has been found that the extract of Daicus japonica extract has an antioxidative effect as well as a UV- To complete the present invention.

Therefore, a technical problem to be solved by the present invention is to provide a substance having antioxidant and ultraviolet blocking effect and having excellent human stability.

In order to solve the above-mentioned technical problems, the present invention provides a composition for antioxidation and ultraviolet screening, which comprises a jujube extract as an active ingredient.

Preferably, the composition comprising the jujuba extract of the present invention may be a cosmetic composition, a pharmaceutical composition or a food composition.

The term "extract " of the present invention means a preparation which is obtained by squeezing a herbal medicine with an appropriate leaching solution and concentrating by evaporating the leaching solution, and is not limited thereto. The extract, the diluted solution, A dried product obtained by drying, a controlled preparation thereof, or a purified product thereof.

The jujube seed extract of the present invention can be extracted using a conventional extraction solvent known in the art, and the extracted liquid can be used in a liquid form or can be used by concentration and / or drying. The extraction solvent may be, for example, (a) water, (b) anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) Solvent, (d) acetone, (e) ethyl acetate, (f) chloroform or (g) 1,3-butylene glycol as the extraction solvent. Preferably, extraction is carried out using methanol, ethanol or butane. Depending on the extraction solvent, the degree of extraction and the degree of loss of the active ingredient of the extract may differ. Therefore, an appropriate extraction solvent should be selected and used. The extraction method is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, and reflux cooling extraction.

In addition, the jujube seed extract can be obtained through a conventional purification process in addition to the extraction method using the above-mentioned extraction solvent. For example, by separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity), and the like, The jujube seed extract can also be obtained through fractionation.

According to an embodiment of the present invention, the jujube seed extract is obtained by pulverizing jujube seeds and then using a solvent selected from the group consisting of water, an anhydrous or low-boiling alcohol having 1 to 3 carbon atoms, or a mixed solvent thereof as an extraction solvent And the amount of the extraction solvent may be 2 to 20 times by weight, preferably 5 to 15 times by weight, of the dry weight of the herbal medicine.

In one specific embodiment of the present invention, the jujube extract is immersed for 20 to 30 hours, preferably for 22 to 26 hours, using a 20 to 95% aqueous ethanol solution, preferably a 50 to 70% aqueous ethanol solution, , Followed by filtration with a filter paper and concentration to obtain a jujube extract.

The composition of the present invention may contain the jujube extract in an amount of 0.005 to 50% by weight, more preferably 0.01 to 30% by weight, and most preferably 0.1 to 10% by weight, based on the total weight of the composition. When the content of the jujube extract is less than 0.005% by weight, the antioxidant and ultraviolet screening effects, which are the object of the present invention, can not be obtained. When the content exceeds 50% by weight, the effect is not proportional to the content, There is a problem that the stability of the shape is not ensured.

The composition containing the jujuba extract of the present invention exhibits antioxidant and ultraviolet blocking effect, and has almost no cytotoxicity as a natural substance.

According to one embodiment of the present invention, there is provided a cosmetic composition comprising a jujube extract as an active ingredient.

The cosmetic composition according to one embodiment of the present invention may contain, in addition to a jujube extract as an active ingredient, conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and perfumes, The carrier may be further added.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be prepared as a nutritional cream, a convergent lotion, a soft lotion, a lotion, an essence, a nutritional gel or a massage cream.

When the formulation of the present invention is a paste, cream or gel, use is made of an animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide .

When the formulation of the present invention is a powder or a spray, tosse, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Castellulose, aluminum metahydroxide, bentonite, agar or tracert, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The pharmaceutical composition according to one embodiment of the present invention includes a pharmaceutically acceptable carrier other than the jujuba extract. The pharmaceutically acceptable carrier to be contained in the pharmaceutical composition of the present invention is one usually used at the time of formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably applied by parenteral administration, more preferably topical application by application.

A suitable dosage of the pharmaceutical composition of the present invention may vary depending on such factors as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg / kg on an adult basis. When the composition is an external preparation, it is preferably applied in an amount of 1.0 to 3.0 ml on an adult basis once to five times a day, and continued for 1 month or more. However, the dose is not intended to limit the scope of the present invention.

The pharmaceutical composition of the present invention may be prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art, Into the container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in an oil or aqueous medium, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

In addition, the food composition according to one embodiment of the present invention may contain not only the extract of the jujubu as an active ingredient but also the components normally added in the manufacture of food such as protein, carbohydrate, fat, nutrients, May be further included.

Examples of such carbohydrates are monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings.

For example, when the food composition of the present invention is prepared as a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract, .

On the other hand, the jujube extract of the present invention is a natural substance and is harmless to the human body. Since it has little toxicity and side effects, it can be safely used for a long period of use and can be safely applied to cosmetic, pharmaceutical and food compositions as described above .

As described above, the composition comprising the jujuba extract of the present invention as an active ingredient has an antioxidative effect because it is excellent in DPPH free radical, ABST radical cation, hydrogen peroxide and superoxide anion radical scavenging activity, and the cells are not killed by irradiation with ultraviolet rays Can be usefully used as an ultraviolet screening agent. Also, the jujube extract of the present invention has excellent antimicrobial activity, has no cytotoxicity and skin side effects, and can be used safely in cosmetic, pharmaceutical and food compositions.

1 is a graph showing the DPPH electron donating ability according to the concentration of ethanol as an extraction solvent of the jujube seed extract.
2 is a graph showing the ABTS radical scavenging ability according to the concentration of ethanol as an extraction solvent of the jujube extract.
FIG. 3 is a graph showing hydrogen peroxide (H ₂ O ₂ ) scavenging activity according to the concentration of ethanol as an extraction solvent of the jujube seed extract.
FIG. 4 is a graph showing the superoxide anion radical scavenging ability according to the concentration of ethanol as an extraction solvent of the jujube extract.
5 and 6 are photographs showing antimicrobial activity of the jujube extract against microorganisms.
Fig. 7 shows the cell viability of the jujube extract.
8 is a graph showing the protective effect of jujube seed extract against cell death due to UVB in HaCaT cells.
9 is a graph showing the effect of the jujube extract on the expression of MMP-2 and MMP-9.
10 is a graph showing the effect of the jujube extract on the UV-irradiated Pro-Collagen Type 1, MMP-2 and MMP-9 expression.
11 is a graph showing the change in pH of sunscreen formulations according to the content of jujube seed extract.
FIG. 12 is a graph showing changes in viscosity of sunscreen formulations according to the content of jujube seed extract.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

<Example 1> Preparation of jujube seed extract

The jujube extracts used in this experiment were obtained from Maenam Agricultural Products (Qingdao, Gyeongbuk) and used for grinding. 1000 ml of ethanol (20%, 40%, 60%, 80%, and 95%) were immersed in 1 kg of the sample for 24 hours and collected three times. 2 filter paper, and the filtrate was concentrated using a vacuum rotary evaporator, lyophilized, and stored at -20 ° C.

<Test Example 1> Measurement of antioxidant effect

(1) Measurement of DPPH radical scavenging ability

The electron donating ability (EDA) was measured according to Blois's method (1958). 1 mL of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was added to 2 mL of each sample solution, and the mixture was left to stand for 30 minutes and absorbance was measured at 517 nm. The electron donating ability was expressed by the absorbance reduction rate of the sample solution added group and the no added group.

The measurement of DPPH radical scavenging ability is a method of measuring electron donating ability as a strong antioxidant as the reducing power becomes larger. Therefore, the reducing power and the antioxidant power of the test substance can be measured based on the reduction degree of DPPH. DPPH electron donating ability which inhibits oxidation by donating electrons of free radical in association with lipid peroxidation chain reaction was measured by controlling and adding 10 ~ 1,000 ug / ml of jujube extract.

The results are shown in Fig. 1 is a graph showing the DPPH electron donating ability according to the concentration of ethanol as an extraction solvent of the jujube seed extract. Here, J20 is a jujube extract extracted with 20% ethanol, J40 is a jujube extract extracted with 40% ethanol, J60 is a jujube extract extracted with 60% ethanol, and J80 is a jujube extract extracted with 80% ethanol , J95 is a jujube extract extracted with 95% ethanol, and BHA is a butyric hydroxyanisole as a positive control.

As shown in FIG. 1, it was confirmed that the jujube extract of 60% ethanol showed higher DPPH radical scavenging ability than the other extracts.

(2) ABTS radical cat ion scavenging ability measurement

The ABTS radical cation scavenging activity is due to the reaction of 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) with potassium persulfate. It is a method to measure both hydrogen donating antioxidant and chain breaking antioxidant by measuring decolorization by light green as added.

Antioxidant activity using ABTS radical was measured by ABTS + cation decolorization assay (Roberta, et al., 1999). After incubation at room temperature for 24 hours, ABTS + was formed and diluted with ethanol, and the sample was added to ABTS + 0.1 mL. 0.1 mL was added, and the mixture was allowed to stand for 1 minute, and the absorbance was measured at 732 nm.

2 is a graph showing the ABTS radical scavenging ability according to the concentration of ethanol as an extraction solvent of the jujube extract. Here, J20 is a jujube extract extracted with 20% ethanol, J40 is a jujube extract extracted with 40% ethanol, J60 is a jujube extract extracted with 60% ethanol, and J80 is a jujube extract extracted with 80% ethanol , J95 is a jujube extract extracted with 95% ethanol, and BHA is a butyric hydroxyanisole as a positive control.

As shown in FIG. 2, the ABTS radical cation scavenging activity of the jujube seeds was measured to be 44.56% at 100 μg / ml and 99.38% at 500 μg / ml. This result is similar to that of the control group, BHA, and thus it is highly likely that the DPPH effect is applied as a natural antioxidant.

(3) Measurement of scavenging ability of hydrogen peroxide

Hydrogen peroxide (H ₂ O ₂ ) inhibitory activity was measured by hydrogen peroxide analysis (Jayaprakasha et al., 2004). 1.0 mL of each sample solution and 2.0 mL of 40 mM hydrogen peroxide solution dissolved in phosphate-buffered saline (PBS, pH 7.4) were reacted at 37 ° C for 10 minutes. The amount of hydrogen peroxide inhibited in the reaction solution was measured at 230 nm Respectively. We compared the activity with BHA, a synthetic antioxidant.

Measurement of hydrogen peroxide inhibition activity is one of the most useful methods for measuring the ability of antioxidants to reduce the content of precursor oxidizing agents such as hydrogen peroxide. Hydrogen peroxide is known to oxidize the thiol group, which is an essential element of the enzyme, as a weak oxidizing agent, thereby directly reducing the activity of some enzymes. Hydrogen peroxide is also very important because it is a reactive non-radical material that can pass directly through the biological membrane. Hydrogen peroxide is converted to singlet oxygen and hydroxyl radicals through a variety of reactions when it passes through biological membranes, leading to lipid peroxidation or intracellular toxicity.

FIG. 3 is a graph showing hydrogen peroxide (H ₂ O ₂ ) scavenging activity according to the concentration of ethanol as an extraction solvent of the jujube seed extract. Here, J20 is a jujube extract extracted with 20% ethanol, J40 is a jujube extract extracted with 40% ethanol, J60 is a jujube extract extracted with 60% ethanol, and J80 is a jujube extract extracted with 80% ethanol , J95 is a jujube extract extracted with 95% ethanol, and BHA is a butyric hydroxyanisole as a positive control.

As shown in FIG. 3, the inhibitory activity against the hydrogen peroxide of the jujube extract was increased in a concentration-dependent manner, and 60% ethanol extract showed similar activity to the positive control.

(4) Superoxide anion radical scavenging ability measurement

The superoxide anion radical scavenging activity was measured by the NBT reduction method according to the method of Fridovich. Add 0.1 ml of each sample solution and 0.4 ml of 0.1 M potassium phosphate buffer (pH 7.5), add 1 ml of substrate solution in which xanthine (0.4 mM) and NBT (0.24 mM) were dissolved, add 1 ml of xanthine oxidase (0.2 U / ml) After reacting at 37 ° C for 20 minutes, 1 ml of 1 N HCl was added to terminate the reaction, and the amount of superoxide anion radical produced in the reaction solution was measured at 560 nm.

Since the superoxide anion radical acts as a precursor of other harmful active oxygen, the measurement of scavenging activity on this substance is known to be an effective method for detecting the antioxidative activity of the sample.

FIG. 4 is a graph showing the superoxide anion radical scavenging ability according to the concentration of ethanol as an extraction solvent of the jujube extract. Here, J20 is a jujube extract extracted with 20% ethanol, J40 is a jujube extract extracted with 40% ethanol, J60 is a jujube extract extracted with 60% ethanol, and J80 is a jujube extract extracted with 80% ethanol , J95 is a jujube extract extracted with 95% ethanol, and BHA is a butyric hydroxyanisole as a positive control.

As shown in Fig. 4, the jujube extract showed about 20% of superoxide anion radical scavenging activity at all concentrations.

&Lt; Test Example 2 >

(1) Target strain

Nutrient broth (NB) was used as a liquid medium for Escherichia coli (KCTC 1039) and Staphylococcus epidermidis (KCTC 1917), and YM broth (YMB) was used as a liquid medium for culturing Candida albicans (KCTC 7965) Tryptic soy broth (TSB) was used as a liquid medium for Staphylococcus aureus (KCTC 1621), GAM was used for Propionibacterium acnes (KCTC 3314), and agar was added to the liquid medium for the solid medium. The strains were cultured in BOD incubator at different growth temperature conditions.

(2) Disk diffusion method for susceptibility test

The antimicrobial activity was measured by paper disc method. In other words, each strain cultivated on a plate culture medium was plated with 1 pl of 10 ml of liquid medium for 18-24 hours, and the mixture was spread in 0.85% saline, mixed with 0.5 McFarland turbidity (530 nm) ⁶ CFU / mL. The fungus was smoothed evenly on MH-GMB medium using a disinfected cotton swab, and a disk and an antibiotic (control) disk were prepared, each of which was prepared in a concentration dependent manner. The diameter of the clear zone (mm) around the disc was measured by incubating at 37 ° C for 18-24 hours. The results are shown in Fig. 5 and Fig.

5 and 6 are photographs showing antimicrobial activity of the jujube extract against microorganisms. Here, J20 is a jujube extract extracted with 20% ethanol, J40 is a jujube extract extracted with 40% ethanol, J60 is a jujube extract extracted with 60% ethanol, and J80 is a jujube extract extracted with 80% ethanol , J95 is a jujube extract extracted with 95% ethanol. As a result, the jujube extract showed no antimicrobial activity against gram positive, negative, and fungi.

&Lt; Test Example 3 > Cell regeneration measurement

(1) Cell culture

CCRF S-180 Ⅱ (mouse skin fibroblast cell line, KCLB No. 10008) was purchased from Korean Cell Line Bank (Seoul, Korea). The cell culture medium was Dulbecco's modified Eagle's medium (DMEM). 10% fetal bovine serum (FBS), 100 U / ml penicillin (100 U / ml), and 100 mg / ml streptomycin (100 mg / ml) were added. For the subculture, 0.05% trypsin and 0.53 mM EDTA were used.

(2) Evaluation of cell viability by MTT assay

The toxicity of jujube seeds to cells was analyzed by MTT assay. The cells were treated with 1x10 ⁴ cells / well of CCRF S-180 Ⅱ (mouse skin fibroblast cell) in a 96-well plate for 60 min. To 1000 ug / ml) and cultured for 24 hours. 20 μl of MTT solution per well was added and incubated at 37 ° C in 5% CO ₂ for 4 hours. After incubation for 4 hours, the absorbance at 490 nm was measured using a microplate reader, and the cell viability of the control group was expressed as a percentage.

Based on in vitro experiments, the cell viability of CCRF S-180 Ⅱ (mouse fibroblast cell) at a concentration of 50-200 ug / ml was measured using J60, which had the best inhibitory effect.

Figure 7 shows the cell viability of the jujube extract. As shown here, the cell survival rate was 97.9% at a concentration of 200 ug / ml.

(3) Measurement of cytotoxic effect of J60 against UVB

Ultraviolet rays are directly or indirectly damaged when irradiated to skin cells, which directly induces changes in DNA structure or damages cellular constituents through oxidation reaction.

UVB was irradiated to the cells to kill the cells, and UVB was irradiated and MTT reagent was applied to confirm the extent of cell death by using the sample.

HaCaT cells (Human Keratynocyte Cell) were seeded in a 96-well plate for 24 hours, and the medium was changed to serum free. After 24 hours, UVB (250 mJ / cm ² ) (DMSO) solution. The cells were treated with various concentrations of J60 (60% ethanol extracts of Jujube seed) and incubated for 24 hours. MTT (Thiazolyl Blue Tetra-zolium Bromide) 1: 1 solution was applied to each well and absorbance was observed at 540 nm.

8 is a graph showing the protective effect of jujube seed extract against cell death due to UVB in HaCaT cells. As shown here, the addition of Daechu Seed Extract after UV irradiation increases cell viability. This shows that Daechu Seed Extract is effective in inhibiting the aging process.

(4) Gelatin Zymography

Gelatin zymography was performed to investigate the effects of MMP-2 and MMP-9 on the activity of MMP-2 and MMP-9 in the jujube extract. The gelatin zymography method was used to investigate the effect of the jujube on the expression of MMP-2 and MMP-9. The medium and sample were quantitated in 10% SDS-polyacrylamide gel (30% acrylamide / 0.8% bis-acrylamide, 1.5M Tris-HCl (pH 8.8), 10% SDS, 10% APS, TEMED, 0.2% Gelatin, DW) buffer was mixed with 1: 1 and electrophoresed. After shaking for 30 minutes in a renaturing buffer (Triton X-1002% in DW) to remove SDS, it was added to developing buffer (50 mM Tris-HCl, 0.2 M NaCl, 5 mM CaCl ₂ , 1% TritonX-100) And reacted for 12 hours. Staining solution was stained in 0.5% coomassie blue for 1 hour and decolorized in Destaining Solution (Methanol: Acetic acid: DW = 5: 1: 4) for 1 hour.

9 is a graph showing the effect of the jujube extract on the expression of MMP-2 and MMP-9. As shown here, the expression levels of MMP-2 and MMP-9 are increased in the UVB-treated region (50 mJ / cm ² ). The concentration of MMP-2 and MMP-2 in the 100ug / ml and 200ug / ml-treated groups, respectively, did not show any change in the expression level when 50ug / ml of the extract of 60% 9 expression was decreased.

(5) Western blot

To investigate the effect of UVB on human keratinocytes, we examined the effect of UVB (50 mJ / cm ² ) on HaCaT cells on pro-Collagen Type 1, the precursor of collagen, and the activation of kinases related to the expression of MMPs The amount of protein expression was measured by Western blot method.

After irradiation the UVB 50mJ / cm ² and then washed with PBS and processed for jujube seed extract at different concentrations and the cells collected were dissolved for 1 hour in RIPA buffer (M.biotek) for 10 min centrifugation at 12,000rpm, 4 ℃ supernatant Respectively. The recovered supernatant was denatured at 90 ° C for 10 minutes by adding SDS sample buffer (M.biotek), and proteins were separated by molecular weight by SDS-PAGE. The separated proteins were transferred to nitrocellulose membrane (Whatman, UK) at 100 V for 1 hour, blocked with 0.1% BSA solution, and immersed in Antibody solution at 4 ° C for 18 hours. And washed three times with TBST (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.2% Tween 20) for 10 minutes each. The washed membrane was incubated for 2 hours with a horse radish peroxidase (HRP), which is capable of generating chemiluminescence, followed by chemiluminescence imaging (ATTO).

10 is a graph showing the effect of the jujube extract on the UV-irradiated Pro-Collagen Type 1, MMP-2 and MMP-9 expression. As shown here, the expression level of Pro-Collagen Type 1 was found to be decreased by UVB irradiation, and the positive control and J60 were increased in a concentration-dependent manner. In the case of MMP-2 and MMP-9, the inhibition of the expression amount of MMP-9 was confirmed.

<Formulation Example> Development of sunscreen formulation according to the content of Daechu seed extract

As shown in the following Table 1, sunscreen formulations according to the content of Daechu Seed Extract (hereinafter referred to as "HC-Phytoju") were prepared.

Phase Trade Name HC-A HC-B HC-C A phase D.I-water up to 100 up to 100 up to 100 Disodium EDTA Q.S Q.S Q.S 1.2-Hexanediol Q.S Q.S Q.S MgSO4 Q.S Q.S Q.S 1,3-BG Q.S Q.S Q.S D.P.G Q.S Q.S Q.S HC-Phytoju 1.00 3.00 5.00 B phase Parsol MCX Q.S Q.S Q.S Parsol EHS Q.S Q.S Q.S Neoheliopan E1000 Q.S Q.S Q.S HallBrite BHB Q.S Q.S Q.S Finoslov TN Q.S Q.S Q.S Vit.E Acetate Q.S Q.S Q.S C phase KF 995 Q.S Q.S Q.S Bentone 38V Q.S Q.S Q.S D phase KF 995 Q.S Q.S Q.S DC 200 / 6cs Q.S Q.S Q.S Abil EM 90 Q.S Q.S Q.S E phase Silica 67 SDM Q.S Q.S Q.S Micro TiO2 035 AS Q.S Q.S Q.S Z Cote HP-1 Q.S Q.S Q.S F phase Frag-80890 Q.S Q.S Q.S Q.S. (Quantum sufficient: Proper quantity)

&Lt; Test Example 4 >

(1) pH measurement

pH was measured using Ohaus (Starter 3100F) pH meter. Before measurement, the glass electrode was immersed in basic buffer or distilled water for several hours, and the pH meter was connected to a power source for more than 10 minutes before use. The detector was washed with water and lightly wiped off.

11 is a graph showing the change in pH of sunscreen formulations according to the content of jujube seed extract. As shown here, the pH of the sunscreen stored at 25 ° C for 12 weeks at 0, 1, 3, 5, 7, 14, 28, During the storage period in C formulation, slight difference was observed in the pH value depending on the content of jujube seed extract.

(2) Measurement of viscosity change with time

Viscosity measurements were made using a Brookfield Viscometer (Brookfield LV-II +, Brookfield Engineering Laboratories Inc., Middleboro, MA, USA). In order to reduce the influence of bubbles, we measured the factors that could affect the viscosity value by minimizing the defoaming during manufacturing. The spindle No. 4 was selected for the sunscreen stored at 25 ° C and the viscosity was measured at 12 rpm for 1 minute. The viscosity was measured for 12 weeks at 0, 1, 3, 5, 7, 15, 30, 60, and 90 days.

FIG. 12 is a graph showing changes in viscosity of sunscreen formulations according to the content of jujube seed extract. As shown here, the viscosity of the sunscreen stored at 25 ° C for 12 weeks at 0, 1, 3, 5, 7, 14, 28, C showed a viscosity difference of 26,000-17,500 cp for 3 months in all the formulations. In particular, it was confirmed that the viscosity of HC-B and HC-C formulations decreased with storage period.

(3) Observation of the stability over time at each temperature condition (0 ℃, 25 ℃ and 45 ℃)

The sunscreen is stored in a transparent container and stored at 0 ° C, 25 ° C and 45 ° C for 12 weeks, respectively, and the stability by temperature is evaluated at 0, 1, 3, 5, 7, 15, 30, 60 and 90 days. The temperature stability of the sunscreen was evaluated to investigate the chemical and physical changes caused by the spontaneous aging deterioration. In the high temperature (45 ℃), the appearance of rust, discoloration, redness, floatation, precipitation and separation were mainly observed and chemical and physical changes such as solidification, precipitation and separation were observed at low temperature.

 The sunscreen prepared according to each prescription was placed in a transparent container and stored at 0 ° C, 25 ° C, and 45 ° C for 12 weeks, respectively, and the stability was evaluated according to the temperature by 1, 3, 5, 7, 14, 28, (0 ° C), Table 3 (25 ° C) and Table 4 (45 ° C), respectively.

Sample
Day HC-A HC-B HC-C One O O O 3 O O O 5 O O O 7 O O O 14 O O O 28 O O O 60 O O O 90 O O O O: Stable X: Unstable

Sample
Day HC-A HC-B HC-C One O O O 3 O O O 5 O O O 7 O O O 14 O O X 28 O X X 60 O X X 90 O X X O: Stable X: Unstable

Sample
Day HC-A HC-B HC-C One O O O 3 O O O 5 O O O 7 O O X 14 O X X 28 O X X 60 O X X 90 O X X O: Stable X: Unstable

As shown in Table 2, the sunscreen (HC-A, HC-B, and HC-C) stored at 0 ° C was stable for 28 days without development of creaming or aggregation. As shown in Table 3, in the case of the sunscreen stored at 45 ° C, B is 14 days, C is 7 days, and C is 14 days. In the case of a sunscreen, B is 28 days and C is 14 days. And it was observed that the stability was unstable.

(4) Stability test according to cycle chamber

The sunscreen was stored in a transparent container at -5 ° C, 25 ° C, and 50 ° C for 8 hours and then repeated 14 times with 1 dah / 1 cycle. After 2 weeks at 0, 1, 3, 5, Were evaluated for stability with temperature cycling. The stability test according to the cycle chamber is carried out in order to observe the change of physical properties according to the storage condition of the low temperature and the high temperature of cosmetics in four seasons of Korea and the change of physical properties in emulsification and solubilization . Most of the products are hydrolyzed at high temperatures and other phenomena occur rapidly. At low temperatures, volume expansion, interfacial membrane damage due to solubility difference, and precipitation occur. The stability test according to the cycle chamber can check this phenomenon in a short period of time. The results are shown in Table 5 below.

Sample
Day HC-A HC-B HC-C One O O O 3 O O X 5 O X X 7 O X X 14 O X X O: Stable X: Unstable

As shown in Table 5, changes in physical properties such as creaming, coagulation, and separation were observed in sunscreen HC-B and HC-C, and HC-A was stable without separation, discoloration, Respectively.

&Lt; Test Example 5 > Evaluation of effectiveness (SPF, PA)

This test was conducted in accordance with the provisions of Article 9 of the Cosmetics Act, "Functional Cosmetics for Functional Cosmetics that Helps Skin Lightly Protect or Protect Skin from Ultraviolet Light" (Regulation on Functional Cosmetics Examination, Article 2015-14 of the Food and Drug Administration Notice) &Lt; / RTI &gt; &lt; RTI ID = 0.0 &gt; UV &lt; / RTI &gt;

(1) Test site

The test was conducted on subjects. The test site was selected to avoid areas of particular skin damage, excessive hair or hue, and was clean and dry.

(2) Method of applying test substance

The test specimens were applied to the test area at a dose of 2.0 mg / cm 2 (total 70.0 mg) with 35 cm 2 of the standard sample and the test sample application area on the back of the subjects except for the spinal part. A micropipette was used to apply the sample in the correct amount. The samples were uniformly applied to the surface of the skin several times uniformly using a finger thimble or a spatula.

Compartment of ultraviolet irradiation site

Six square irradiated regions having an area of 0.8 cm x 0.8 cm were defined with the product application area of 35 cm 2.

(3) Equipment used

(A) Multiport solar simulator 601, V2.5 Solar Light, USA)

The equipment is equipped with a 300 watt Xenon arc lamp, which has a continuous emission spectrum similar to sunlight and does not show a specific peak, and the ultraviolet light emitted from the lamp is reflected by a dichroic mirror and a collimating lens through a collimating lens and then through a liquid light guide to a square of 0.8 cm x 0.8 cm. A special filter is used to eliminate wavelengths in the ultraviolet region below 290 nm, which is particularly harmful to the human body.

(B) Photometer (PMA-2100, Solar Light, USA)

㎼ / cm ² , which is measured by combining an SUV probe at the end of each light guide, and confirms that the measured values are maintained before and after UV irradiation.

(C) Standard sample

Based on the "Method and criteria for evaluating the ultraviolet blocking effect" of the "Regulations for Screening of Functional Cosmetics" (No. 2015-14 of the Food and Drug Administration Notice), a standard sample of high ultraviolet barrier index is prepared and used.

(4) SPF measurement result

A total of 12 subjects, including 2 preliminary subjects, who had sunk as a subject of this research project in the Korea Research Institute of Dermatology and Cosmetic Clinical Trial, . In order to ensure the reliability of the test, standard samples were prepared according to the test methods proposed by the Food and Drug Administration and tests were conducted at the same time. The average UV blocking index of 15.3 ± 1.3 was confirmed. ] Was within the range of 15.5 ± 3.0. The 95% confidence interval of the UV index of each of the standard and test samples measured by this test was within ± 20% of the measured value, and the reliability of the measurement was verified. In conclusion, the product is considered to have ultraviolet power equivalent to SPF 50.5 ± 2.3.

(5) PA measurement result

A total of 12 subjects, including 2 preliminary test subjects, were tested on the sunscreen performed by the Korea Research Institute of Dermatology, a clinical testing institute of cosmetics. The test was conducted to evaluate the ultraviolet A blocking index of the product. As a result, an average of 8.77 ± 0.71 ultraviolet A I could confirm the index. In order to ensure the reliability of the test, standard samples were prepared according to the test methods proposed by the Food and Drug Administration and tests were conducted at the same time. An average of 4.04 ± 0.41 ultraviolet A blocking index was confirmed. [Food Safety Management Notice 2015-14 Was within the range of 3.75 ± 1.01. The tolerance of the ultraviolet A blocking index of each of the standard and test samples measured by this test was confirmed to be within ± 10% of the measured value, and the reliability of the measurement was verified. In conclusion, the product is considered to have ultraviolet A blocking power equivalent to PFA 8.77 ± 0.71, PA +++.

As described above, the composition comprising the jujuba extract of the present invention as an active ingredient has an antioxidative effect because it is excellent in DPPH free radical, ABST radical cation, hydrogen peroxide and superoxide anion radical scavenging activity, and the cells are not killed by irradiation with ultraviolet rays Can be usefully used as an ultraviolet screening agent. Also, the jujube extract of the present invention has excellent antimicrobial activity, has no cytotoxicity and skin side effects, and can be used safely in cosmetic, pharmaceutical and food compositions.

Claims (5)

A cosmetic composition for antioxidative and UV-blocking comprising an extract of Jujube seed as an active ingredient. A pharmaceutical composition for antioxidant and ultraviolet screening comprising a jujube extract as an active ingredient. A food composition for antioxidant and sunscreen containing an extract of Daechu as an active ingredient. 4. The method according to any one of claims 1 to 3,
Wherein the jujube seed extract is obtained by pulverizing the jujube seeds, immersing in 20 to 95% ethanol aqueous solution for 20 to 30 hours, recovering the jujuba extract, filtering it with a filter paper, and concentrating the extract. 4. The method according to any one of claims 1 to 3,
Wherein the jujuba extract is contained in an amount of 0.005 to 50% by weight based on the total weight of the composition.

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