WO2018097388A1 - Composition for skin whitening, wrinkle alleviation, antioxidation, and ultraviolet light blocking, containing jujube seed extract as active ingredient - Google Patents

Abstract

The present invention relates to a composition containing a Jujube seed extract. The composition, of the present invention, containing a Jujube seed extract as an active ingredient has a whitening effect by having an effect of inhibiting tyrosinase and has a wrinkle alleviation effect by inhibiting the activity of elastase and collagenase. In addition, the composition has an antioxidative effect by having an excellent scavenging activity for DPPH free radicals, ABTS radical cations, hydrogen peroxide, and superoxide anion radicals and prevents cell death caused by ultraviolet ray irradiation, thereby being useful as an ultraviolet ray blocking agent. Furthermore, the Jujube seed extract of the present invention has an excellent antibacterial activity, is noncytotoxic, and has no skin side effects, and thus can be safely used in cosmetic, pharmaceutical, and food compositions for skin whitening, wrinkle alleviation, antioxidation, and ultraviolet light blocking.

Description

Composition for skin whitening, wrinkle improvement, antioxidant and sunscreen comprising jujube seed extract as an active ingredient

The present invention relates to a composition containing jujube seed extract, and more particularly to cosmetics, pharmaceutical compositions and foods for skin whitening, wrinkle improvement, antioxidant and sunscreen containing jujube seed extract as an active ingredient. It relates to a composition.

Generally, the eyes, hair, and skin color of a person are determined by the concentration and distribution of melanin in the skin, and also by the type of melanin, but are also affected by various environmental and physiological factors such as ultraviolet rays and stress. Melanin is a series of enzymatic enzymes that begins by oxidizing tyrosine, a type of amino acid in melanocytes in the basal layer of the epidermis, into oxidized by merosinase tyrosinase in the melanocytes of melanocytes. Produced through oxidation and non-enzymatic oxidation. The production and synthesis of melanin is a representative influence factor, ultraviolet rays, and other genetic factors, environmental factors, hormones, foods, pharmaceuticals and other chemicals. This melanin absorbs a certain amount of ultraviolet light to protect the skin and maintain body temperature, but when severely irritated, over-produced melanin deposits, causing freckles, senile spots, blemishes, brown or sunspots, sunburn, and wounds. Alternatively, hyperpigmentation and phototoxic reactions may occur after inflammation due to dermatitis.

Recently, Asian women prefer white and clean skin, and this is an important criterion of beauty. Therefore, the development of whitening agents for the treatment of abnormal skin pigmentation and the fulfillment of beauty needs has been actively made. At present, in the development of a whitening agent for skin beauty, a method of reducing the generated melanin pigment by decolorization and a method of inhibiting the activity of tyrosinase, an enzyme that forms melanin pigment, are known. However, since whitening agents using tocopherols and vitamins used to reduce the melanin pigment are known to have a slight decolorizing effect of the melanin pigment, many studies have been conducted on inhibitors that inhibit the production of melanin pigment.

In order to improve skin whitening or hyperpigmentation, conventionally, inhibitory activity against tyrosinase such as hydroquinone, ascorbic acid, kojic acid, glutathione and cysteine Has been used. However, hydroquinone exhibits a predetermined whitening effect, but has a problem of limiting the amount of the compound to a very small amount due to severe skin irritation. Ascorbic acid is easily oxidized, causing problems such as discoloration and discoloration. There is a disadvantage that the manufacturing process of the cosmetic is complicated by the instability in the silver solution. In addition, thiol-based compounds such as glutathione and cysteine not only have a characteristic unpleasant odor, but also have problems with transdermal absorption, and glycosides and derivatives thereof have high polarity, making it difficult to use as a component of a cosmetic composition. In addition, vitamin C has a problem in that it is easily oxidized in the aqueous solution state does not have a lasting effect.

On the other hand, human skin color is determined by the concentration and distribution of melanin (melanin) inside the skin, in addition to genetic factors, it is also affected by environmental or physiological conditions such as solar ultraviolet rays, fatigue, stress. Melanin is synthesized in melanocytes in the epidermal layer of the skin, and the enzyme tyrosinase acts on tyrosine, a type of amino acid, in the melanocytes (melanosome), the dopa (DOPA) and dopaquinone. It is produced by non-enzymatic oxidation. When such synthesis of melanin occurs excessively in the skin, the skin tone is darkened, and the appearance of spots, freckles, and the like. Therefore, by inhibiting the synthesis of melanin pigment by inhibiting the tyrosinase activity in the skin, not only can brighten the skin tone to realize skin whitening, but also hyperpigmentation of skin such as blemishes and freckles caused by ultraviolet rays, hormones and genetic causes Can improve deposition.

Skin is an organ in which aging occurs primarily, and many studies have been conducted in the cosmetic field to delay or prevent it. Normally, skin aging begins about 25 years of age, and skin aging proceeds in earnest around 40 years of age. As a typical phenomenon of skin aging, sebum secretion is reduced, skin is dried, cell regeneration is slowed, and aging keratin is accumulated a lot of skin roughness. In addition, the amount of collagen that supports the epidermis is reduced, and elastin (elastic fiber) is denatured, resulting in wrinkles. In addition, the color of the skin stains, pigmentation symptoms such as blemishes, blotch, etc. appear, the epidermis becomes thinner and the skin protection function is weakened. Other phenomena include an increase in skin problems due to a decrease in the barrier effect associated with a decrease in skin thickness. Research on skin aging is mainly focused on strengthening skin dermal-epidermal junction (DEJ), which protects the skin from UV rays and free radicals, which are the main causes of skin aging, skin wrinkle improvement materials, skin elasticity enhancing materials. In-depth study by classifying related materials.

As the skin ages, the changes in the dermis appear remarkably, and dermis atrophy after 70 years is a representative aging phenomenon. Changes in the dermis are caused by changes in substances with large molecular weight in the extracellular matrix due to a decrease in the number of fibroblasts and their ability to synthesize. Specific changes include separation of collagen bundles, reduced point polysaccharide synthesis, decreased collagen and elastic fiber counts and diameters, collagen and elastic fiber degradation, and blood vessel dilation. Generally, among the various factors such as skin moisture content, collagen content, and ability to immunize the external environment, the biggest influence on wrinkle formation is collagen, a collagen degrading enzyme that reduces collagen production and collagen content. It is the expression level and activity of genease. Collagenase belongs to the group of Matrix metalloproteinases (MMPs), which play an important role in matrix protein degradation. The collagenase is known to be promoted by ultraviolet rays, growth factors, inflammatory cytokines, phorbol esters to break down collagen type 1, 2, 3, 5, especially in the ultraviolet It is known to play a major role in photoaging. In addition, metalloproteinases (MMPs) may contain type 4 collagen fibers (MMP-2, -3), elastic fibers (MMP-2), or substrate proteins such as fibronectin or laminin (MMP-3). It can be observed that there is a marked increase in expression in aged skin. Therefore, the development of materials that can reduce the activity of metalloproteinases is actively made.

In addition, as aging progresses, the dermal epidermal junction (DEJ) protein, which is responsible for the attachment of the dermis and the epidermis, also decreases in expression, and the epidermal-dermal junction is flattened and the connection is weak. When DEJ is weakened, skin and epidermis cannot communicate smoothly and skin regeneration is not performed smoothly. Recent studies have shown that DEJ is involved not only in wrinkles and skin barriers but also in skin pigmentation due to UV stimulation (Iriyama S, J Dermatol Sci. 64 (3): 223-228, 2011). Laminin-5, collagen-IV, and collagen VII are present at the dermal junction, and materials capable of increasing the expression of these proteins can improve skin wrinkles.

In addition, elastin fibers form crosslinks with collagen and are important skin components in the production of wrinkles involved in skin elasticity. Deficiency and aggregation of elastin fibers and a dramatic increase in the activity of elastase, an elastin degrading enzyme, have been found to be one of the causes of skin wrinkles. Elastase is the only enzyme that can break down elastin and its inhibition is known to fundamentally reduce skin wrinkles.

Human skin is composed of the epidermis, the dermis, and connective tissue including the stratum corneum, of which the dead layer consists of dead cell layers formed through the differentiation of keratinocytes, the basal cells of the epidermis. It protects the human body from the influence of the external environment. There are two major researches on the causes of aging in the skin. One is "internal aging," which is caused by a change in the function of cells, which are components of the skin, with age, and the other is "external aging." As a result, it can be divided into external environment, ie, ultraviolet light, pollution, and stress.

When skin cells are subjected to oxidative stress by reactive oxygen species, which occur in a constantly occurring biochemical reaction to supply the energy needed in vivo, the constituents of lipids, proteins, Carbohydrates and DNA oxidative damage and changes in enzyme activity cause various diseases such as skin cancer, and promotes aging.

In particular, with the prolonged summer, the domestic sunscreen products market grew steadily from 330 billion won in 2010 to 420 billion won in 2014. According to the research data of the data monitor, the size of the domestic sunscreen market recorded KRW 34 billion in 2010, KRW 357 billion in 2011, KRW 377.7 billion in 2012, and KRW 398 billion in 2013, and the average annual growth rate was 6.34%. As the risk of photoaging is known, sunscreens are becoming a necessity for the four seasons, but they are still strong in the summer season and are sold as representative seasonal products.

About 90% of the ultraviolet light that reaches the earth's surface is UV A and less than 10% is UV B. The wavelength of the light is inversely proportional to the energy, so the absolute amount of UVB at least has a strong energy, which can affect the cell's genes. The minimum amount of light that a particular wavelength of light causes erythema on the skin is called the minimal erythema dose (MED). In Asians, the minimum amount of erythema of the skin is 50-70 J / cm2 for UV A and 50-70 mJ / cm2 for UV B, and 1 / 1,000 of UV A can cause erythema. The minimum amount of UV B erythema in hairless mice used in skin barrier studies varies from report to report, but is approximately 20-80 mJ / cm ² .

Ultraviolet rays can cause skin cancer by altering DNA, and are unwelcome in the skin enough to suppress skin immune responses by reducing the number of cells responsible for skin immunity. Due to the increased awareness of UV rays, sunscreen and absorption related cosmetics have been developed in various formulations and types.

Therefore, in the cosmetics field, research and development of functional cosmetics having skin whitening and wrinkle improvement, antioxidant and sunscreen effects are being actively conducted, and in particular, researches using natural materials to avoid toxicity or irritation to the skin continue. have.

On the other hand, the jujube seed is a seed part in the flesh of the jujube and is a jujube processing by-product generated after processing the jujube pulp. Jujube seed by-products produced during jujube processing account for about 30% of the weight of jujube. Jujube is often added to sweeten like licorice in oriental medicine, and has its own pharmacological effect to improve the symptoms of gastric cramps, insomnia, indigestion, intestinal hemorrhage, blue blood, and hypersensitivity.

At present, the leaves and fruits of the jujube are edible, but the seeds are limitedly edible. Jujube seed has high nutritional value due to its high content of protein, minerals and fiber, and has been used as a herbal medicine called Sanjoin in Chinese medicine. Despite these excellent nutritional and pharmacological values, most jujube seed resources are not industrially utilized and are discarded.

Jujube seeds contain fatty oils consisting mainly of unsaturated fatty acids of oleic acid and linoleic acid, as well as saponins, eblin and lactone. As medicinal ingredients, betulin, betulicacid and the like are known, and as medicinal ingredients of jujube, various sterols, alkaloids, vitamins, saponins, serotonin, fatty acids, polyphenols, flavonoids and the like are known. In particular, the cyclic-AMP component is about 1,000 times higher than the general plant, and other nutrients such as sugars and organic acids are known (Ministry of food and drug safety, 2013).

However, there have been no reports about the skin whitening, wrinkle improvement, antioxidant and sunscreen effects of jujube seeds.

Accordingly, the present inventors have continued to develop new natural substances having skin whitening, anti-wrinkle, anti-oxidant and sunscreen effects and less human side effects, and jujube seed extract has excellent skin whitening effect without cytotoxicity, The present invention has been completed by discovering the fact that the anti-wrinkle effect as well as the anti-wrinkle effect and antioxidant effect are exhibited.

Therefore, the technical problem to be solved by the present invention is to provide a material having excellent human safety while having skin whitening, wrinkle improvement, antioxidant and sunscreen effect.

In order to solve the above technical problem, the present invention provides a composition for skin whitening, anti-wrinkle, antioxidant and sunscreen, comprising jujube seed extract as an active ingredient.

Preferably, the composition comprising the jujube seed extract in the present invention may be a cosmetic composition, a pharmaceutical composition or a food composition.

The term "extract" of the present invention refers to a formulation prepared by squeezing the herbal medicine with a suitable leach solution and evaporating the leach solution, but not limited thereto, but the extract obtained by the extraction treatment, the dilution or concentrate of the extract, the extract It may be a dried product obtained by drying, these adjustment products, or a refined product.

Jujube seed extract of the present invention can be extracted using a conventional extraction solvent known in the art, the extracted liquid can be used in liquid form or concentrated and / or dried. At this time, the extraction solvent is, for example, (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) the lower alcohol and water A mixed solvent, (d) acetone, (e) ethyl acetate, (f) chloroform or (g) 1,3-butylene glycol can be obtained as an extraction solvent. Preferably, the extraction is performed using methanol, ethanol or butane. Depending on the extraction solvent, the extraction degree and loss degree of the active ingredient of the extract may vary, so select an appropriate extraction solvent. The extraction method is not particularly limited, and examples thereof include cold needle extraction, ultrasonic extraction, reflux cooling extraction, and the like.

In addition, the jujube seed extract may be obtained through a conventional purification process in addition to the extraction by the extraction solvent. Obtained by various additional purification methods, such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). Jujube seed extract can also be obtained through the fraction.

According to an embodiment of the present invention, the jujube seed extract is prepared by pulverizing the jujube seed using a solvent selected from the group consisting of an extraction solvent, such as water, anhydrous or hydrous lower alcohol having 1 to 3 carbon atoms, and a mixed solvent thereof. It can be extracted, the amount of the extraction solvent may be 2 to 20 weight times, preferably 5 to 15 weight times the dry weight of the herbal medicine.

In one specific embodiment of the present invention, the jujube seed extract is recovered after being deposited for 20 to 30 hours, preferably 22 to 26 hours, using 20 to 95% aqueous ethanol solution, preferably 50 to 70% aqueous ethanol solution. Then, filtered through filter paper and concentrated to obtain a jujube seed extract.

The composition of the present invention may include the jujube seed extract in 0.005 to 50% by weight, more preferably 0.01 to 30% by weight, most preferably 0.1 to 10% by weight based on the total weight of the composition. At this time, when the content of jujube seed extract is less than 0.005% by weight, the skin whitening effect and the wrinkle improvement effect, which are the objective effects of the present invention, cannot be obtained, and when the content exceeds 50% by weight, the effect is not proportional to the increase of the content. It may be inefficient and there is a problem in that formulation stability is not secured.

The composition containing the jujube seed extract of the present invention exhibits skin whitening, anti-wrinkle, anti-oxidant and sunscreen effects, and has little cytotoxicity as a natural substance.

According to one embodiment of the present invention, it provides a cosmetic composition comprising the jujube seed extract as an active ingredient.

Cosmetic compositions according to one embodiment of the present invention include ingredients commonly added to cosmetic compositions in addition to jujube seed extract as active ingredients, such as conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and Carriers may be further added.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a nourishing cream, astringent lotion, a flexible lotion, a lotion, an essence, a nourishing gel or a massage cream.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components. Can be.

Toss, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component when the formulation of the present invention is a powder or a spray, and in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Castle cellulose, aluminum metahydroxy, bentonite, agar or tracant gum can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The pharmaceutical composition according to one embodiment of the present invention comprises a pharmaceutically acceptable carrier in addition to the jujube seed extract. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. 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 may be administered orally or parenterally, and is preferably applied by topical application by parenteral administration, more preferably by application.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. The dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg / kg on an adult basis. In addition, in the case of external preparations, it is recommended to continue the application for 1 month to 5 times a day in an amount of 1.0 to 3.0 ml on an adult basis. However, the dosage does not limit the scope of the present invention.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form or in multidose form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which can be easily carried out by those skilled in the art. It may be prepared by incorporation into a container. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or an aqueous medium, or may be in the form of an exir, powder, powder, granule, tablet or capsule, and may further include a dispersing or stabilizing agent.

In addition, the food composition according to one embodiment of the present invention contains not only jujube seed extract as an active ingredient, but also components commonly added in food preparation, such as proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. It may further comprise.

Examples of such carbohydrates are monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

For example, when the food composition of the present invention is prepared with a drink, in addition to the jujube seed extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract, etc. may be further included. Can be.

In one specific embodiment of the present invention, the present invention provides a method and its use for obtaining a skin whitening and wrinkle improvement effect of a subject by administering to the subject a composition comprising a jujube seed extract as an active ingredient.

In one specific embodiment of the present invention, the present invention provides a method and use thereof for obtaining an antioxidant and sunscreen effect of a subject by administering to the subject a composition comprising a jujube seed extract as an active ingredient.

In one specific embodiment of the present invention, the present invention is to improve the skin condition of the individual by administering a composition comprising jujube seed extract as an active ingredient to the subject to enhance the skin whitening, wrinkle improvement, antioxidant and sunscreen effect of the subject It provides a method and its use.

The composition to be administered to the subject of the present invention has the effect of improving the skin condition of the subject or a disease, disease or abnormality caused by them due to skin whitening, wrinkle improvement, antioxidant and sunscreen effects as described above.

In the present invention, the term "individual" refers to monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, chimpanzees, including humans with skin conditions whose symptoms can be improved by administering the composition of the invention. It means a mammal such as.

In the present invention, the term "improvement" means (a) suppression of (a) development, (b) alleviation, and (c) removal of pigmentation, skin aging, wrinkle formation, skin disease or inflammatory disease caused by ultraviolet rays in a subject. do.

On the other hand, jujube seed extract of the present invention is a natural substance, harmless to the human body, has little toxicity and side effects, so can be used safely even in long-term use, in particular can be safely applied to cosmetics, pharmaceutical and food compositions as described above .

As such, the composition comprising the jujube seed extract of the present invention as an active ingredient has a tyrosinase inhibitory effect and not only have a whitening effect, but also have an anti-wrinkle effect by inhibiting elastase and collagenase activity. In addition, the DPPH free radical, ABST radical cation, hydrogen peroxide and superoxide anion radical scavenging activity is excellent and has an antioxidant effect, the cell is not killed by irradiation with ultraviolet light can be usefully used as a sunscreen agent. In addition, the jujube seed extract of the present invention is excellent in antibacterial activity, there is no cytotoxicity and skin side effects can be used safely in cosmetics, pharmaceutical and food compositions.

1 is a graph showing the tyrosinase activity inhibitory effect of jujube seed extract.

2 is a graph showing the inhibitory effect of DOPA oxidation inhibitory activity of jujube seed extract.

Figure 3 is a graph showing the elastase inhibitory activity according to the concentration of ethanol as the extract solvent of jujube seed extract.

Figure 4 is a graph showing the collagenase inhibitory activity according to the concentration of ethanol, the extract solvent of jujube seed extract.

5 is a graph showing the results of DPPH electron donating ability according to the concentration of ethanol as an extract solvent of jujube seed extract.

Figure 6 is a graph showing the ABTS radical scavenging ability according to the concentration of ethanol, the extraction solvent of jujube seed extract.

7 is a graph showing the hydrogen peroxide (H ₂ O ₂ ) scavenging ability according to the concentration of ethanol as an extract solvent of jujube seed extract.

8 is a graph showing the superoxide anion radical scavenging ability according to the concentration of ethanol which is an extract solvent of jujube seed extract.

9 and 10 are photographs showing the antimicrobial activity of the jujube seed extract against microorganisms.

Figure 11 shows the cell viability of jujube seed extract.

12 is a graph showing the protective effect of jujube seed extract against UVB-induced apoptosis in HaCaT cells.

Figure 13 is a graph showing the effect on the expression of MMP-2 and MMP-9 of jujube seed extract.

14 is a graph showing the effect of jujube seed extract on UV-irradiated Pro-Collagen Type 1 and MMP-2 and MMP-9 expression.

Figure 15 is a graph showing the measurement of the pH change of the sunscreen formulation according to the jujube seed extract content.

Figure 16 is a graph showing the viscosity change of the sunscreen formulation according to the jujube seed extract content.

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example 1 Preparation of Jujube Seed Extract

Jujube seed extract used in this experiment was used to crush the one provided from Mae Nam Nongsan (Cheongdo, Gyeongbuk). 1000 ml of 20%, 40%, 60% 80%, 95% ethanol was immersed in 1 kg of sample for 24 hours, and then collected three times. Filtered with 2 filter paper, the filtrate was concentrated using a vacuum rotary concentrator, lyophilized, and used in the experiment while storing at -20 ℃.

Test Example 1 Measurement of Whitening Effect

(1) Tyrosinase Inhibitory Activity

Tyrosinase inhibition activity was measured using the dopachrome method. The reaction zone was reacted for 15 minutes at 37 ° C by adding 10 ul of mushroom tyrosinase (2,000U / mL) to a mixture of 10 ul of substrate solution dissolved in 11.5 mM L-tyrosine in 110 ul of 0.1 M phosphate buffer (pH 6.5) and 10 ul of sample solution. It measured at 490 nm. DOPA oxidation inhibition activity was measured by the absorbance decrease rate of the sample solution added and no added solution.

Tyrosinase (monophenol, dihydroxy-L-phenylalanin: oxygen oxidoreductase, EC 1.14.18.1) is a copper-containing enzyme in L-tyrosine, the initial stage of melanin synthesis, in L-3,4-dihydroxyphenylalanine (DOPA), in DOPA. It acts on the transition to L-dopaquinone. Ultraviolet mitosis of melanocytes occurs followed by melanocyte activation. In activated melanocytes, tyrosinase synthesis is promoted and melanin is produced and transported out of the epidermis, causing pigmentation such as blemishes and freckles. Therefore, tyrosine activity inhibitors can effectively inhibit the synthesis of melanin polymer in the skin. Tyrosinase activity inhibition experiments have been recognized as useful evaluation methods in the development of skin whitening agents. Therefore, tyrosinase plays an important role in melanin biosynthesis process, so tyrosinase inhibitors can be used as substances that can control melanin pigmentation of skin. In addition, tyrosinase inhibitors such as 4-hydroxyanisole and hydroquinone have been used to treat hyper-pigmentation in humans, and have been reported to be effective in inhibiting the proliferation of melanoma cells.

1 is a graph showing the tyrosinase activity inhibitory effect of jujube seed extract. Here, J20 is jujube seed extract extracted with 20% ethanol, J40 is jujube seed extract extracted with 40% ethanol, J60 is jujube seed extract extracted with 60% ethanol, J80 is jujube seed extract extracted with 80% ethanol , J95 is a jujube seed extract extracted with 95% ethanol, and arbutin was used as a positive control.

As shown in Fig. 1, as a result of measuring the whitening effect of jujube seed extract, when Tysonise was used as a substrate, an effect of about 15% was measured even at a concentration of 1,000 ug / mL, and the rest of the extracts showed a slight inhibitory activity of 22.8% to 31.7%. It was confirmed.

(2) measurement of DOPA oxidation inhibitory activity

DOPA oxidation inhibition activity was measured according to the method of Yagi et al. The reaction zone was added with 0.2 mL of mushroom tyrosinase (110U / mL) in a mixture of 0.2 mL of substrate solution dissolved in 10 mM L-DOPA in 0.5 mL of 1/15 M sodium phosphate buffer (pH 6.8) and 0.1 mL of sample solution. DOPA chrome produced in the reaction solution was measured at 475 nm. DOPA oxidation inhibition activity was measured by the absorbance decrease rate of the sample solution added and no added solution.

2 is a graph showing the inhibitory effect of DOPA oxidation inhibitory activity of jujube seed extract. Here, J20 is jujube seed extract extracted with 20% ethanol, J40 is jujube seed extract extracted with 40% ethanol, J60 is jujube seed extract extracted with 60% ethanol, J80 is jujube seed extract extracted with 80% ethanol , J95 is a jujube seed extract extracted with 95% ethanol, and arbutin was used as a positive control.

As shown in FIG. 2, when DOPA was used as a substrate, J60 showed an inhibitory activity of 52.4% at a concentration of 500 ug / mL, and it was confirmed that DOPA inhibited the transfer to L-dopaquinone.

Test Example 2 Wrinkle Improvement Effect Measurement

(1) Determination of Elastase Inhibitory Activity

Elastase inhibitory activity was measured as follows. The extract was prepared at a constant concentration, 0.5 mL each was taken in a test tube, 0.5 mL of porcine pancreas elastase (2.5 U / mL) solution dissolved in 50 mM tris-HCl buffer (pH 8.6) was added, followed by 50 mM tris-HCl buffer (pH). Substrate N-succinyl- (L-Ala) 3-p-nitroanilide (0.5 mg / mL) dissolved in 8.6) was added and reacted for 20 minutes, and the amount of p-nitroanilide produced from the substrate was measured at 405 nm. Elastase inhibitory activity was expressed by the absorbance reduction rate of the sample solution added and the group added.

Matrix -metalloproteinases (MMPs) induced by UV light and free radicals in the dermal layer of skin are closely related to skin aging, especially wrinkle formation. Main components of MMPs include collagenase, gelatinase, and elastase, and elasticity and moisture are decreased due to internal and external stress such as ultraviolet rays, and the elastin network structure is increased due to overexpressed elastase. When broken, the skin sags and wrinkles, so the reduction of the elastase activity is very important in reducing the elasticity of the skin and wrinkles. Elastase, which hydrolyzes elastin, is an enzyme associated with skin wrinkles and the effect of jujube seed extract on elastase activity was observed.

Figure 3 is a graph showing the elastase inhibitory activity according to the concentration of ethanol as the extract solvent of jujube seed extract. Here, J20 is jujube seed extract extracted with 20% ethanol, J40 is jujube seed extract extracted with 40% ethanol, J60 is jujube seed extract extracted with 60% ethanol, J80 is jujube seed extract extracted with 80% ethanol , J95 is a jujube seed extract extracted with 95% ethanol, and ursolic acid was used as a positive control.

As shown in Figure 3, ursolic acid showed a concentration-dependent inhibition of elastase, whereas jujube seed extract showed a slight inhibition at 60% ethanol, but other extracts in the experimental concentration showed little inhibition.

(2) Measurement of collagenase inhibitory activity

Collagenase inhibitory activity was measured as follows. In other words, 4 mM CaCl ₂ was added to 0.1 M tris-HCl buffer (pH 7.5) and 0.25 mL of substrate solution in which 4-phenylazobenzyl oxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (0.3 mg / mL) was dissolved. 0.15 mL of collagenase (0.2 mg / mL) was added to a 0.1 mL mixture of the sample solution, and the mixture was allowed to stand at room temperature for 20 minutes. Then, 0.5 mL of 6% citric acid was added to stop the reaction. Then, 1.5 mL of ethyl acetate was added thereto. Absorbance was measured at 320 nm. Collagenase inhibitory activity was expressed by the decrease in absorbance of the sample solution added and non-added.

Collagenase shows the elasticity of connective tissue, constitutes more than 90% of the dermis, has wrinkles and moisturizing function of the skin, and decreases biosynthesis with age. There are many types of enzymes that break down collagen, the most well known is collagenase.

Figure 4 is a graph showing the collagenase inhibitory activity according to the concentration of ethanol, the extract solvent of jujube seed extract. Here, J20 is jujube seed extract extracted with 20% ethanol, J40 is jujube seed extract extracted with 40% ethanol, J60 is jujube seed extract extracted with 60% ethanol, J80 is jujube seed extract extracted with 80% ethanol , J95 is the jujube seed extract extracted with 95% ethanol, EGCG is Epigallocatechin gallate as a positive control.

As shown in Figure 4, the result of measuring the collagenase inhibitory activity of jujube seed extract showed the highest inhibitory activity of 30.7% of J60 at a concentration of 1,000ug / ml.

Test Example 3 Antioxidant Effect Measurement

(1) Measurement of DPPH radical scavenging ability

Electron donating ability (EDA) was measured according to Blois's method (1958). 1 mL of 0.2 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) was added to 2 mL of each sample solution, stirred for 30 minutes, and the absorbance was measured at 517 nm. The electron donating ability was expressed as the absorbance decrease rate of the sample solution added group and the group without addition.

DPPH radical scavenging ability is a kind of measuring electron donating ability. The higher the reducing power, the more powerful the antioxidant. Based on the reduction of DPPH, it is possible to estimate the reducing power and antioxidant power of the measured substance. DPPH electron donating ability to inhibit oxidation by donating electrons of free radicals in the chain reaction of lipid peroxidation was measured by adjusting and adding the jujube seed extract to a concentration of 10-1,000 ug / ml.

The results are shown in FIG. 5 is a graph showing the results of DPPH electron donating ability according to the concentration of ethanol as an extract solvent of jujube seed extract. Here, J20 is jujube seed extract extracted with 20% ethanol, J40 is jujube seed extract extracted with 40% ethanol, J60 is jujube seed extract extracted with 60% ethanol, J80 is jujube seed extract extracted with 80% ethanol , J95 is a jujube seed extract extracted with 95% ethanol, BHA is a butylated hydroxyanisole as a positive control.

As shown in Figure 5, it was confirmed that the jujube seed extract using 60% ethanol shows a high DPPH radical scavenging ability compared with other extracts.

(2) Measurement of ABTS radical cat ion scavenging ability

ABTS radical cation scavenging ability becomes distinctive index blue green when ABTS + radical is formed by reaction of 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) with potassium persulfate. The addition of hydrogen donating antioxidants and chain breaking antioxidants is a measure of decolorization to light green as added.

Antioxidant activity using ABTS radical was measured by ABTS + cation decolorization assay method (Roberta, et al., 1999). 7 mM 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) and 2.4 mM potassium persulfate were mixed for 24 hours in a dark room at room temperature to form ABTS +, diluted with ethanol, and sampled in 0.1 mL ABTS +. 0.1 mL was added and allowed to stand for 1 minute, and then the absorbance was measured at 732 nm.

Figure 6 is a graph showing the ABTS radical scavenging ability according to the concentration of ethanol, the extraction solvent of jujube seed extract. Here, J20 is jujube seed extract extracted with 20% ethanol, J40 is jujube seed extract extracted with 40% ethanol, J60 is jujube seed extract extracted with 60% ethanol, J80 is jujube seed extract extracted with 80% ethanol , J95 is a jujube seed extract extracted with 95% ethanol, BHA is a butylated hydroxyanisole as a positive control.

As shown in Figure 6, jujube seed ABTS radical cation scavenging ability was measured 44.56% activity at a concentration of 100 ug / ml, it was excellent effect of 99.38% at 500 ug / ml. This result is similar to that of the control group BHA, and thus, it is judged that the possibility of industrialization as a natural antioxidant is very high as the DPPH effect.

(3) Determination of hydrogen peroxide scavenging ability

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

Hydrogen peroxide inhibitory activity measurement is one of the most useful methods of measuring the ability of antioxidants to reduce the content of prooxidants such as hydrogen peroxide. Hydrogen peroxide is a weak oxidant and is known to directly reduce the activity of some enzymes by oxidizing the thiol group, an essential group of enzymes. Hydrogen peroxide is also important because it is a reactive, non-radical substance that can pass directly through the biofilm. Hydrogen peroxide is converted into highly reactive singlet oxygen and hydroxyl radicals through various reactions through biofilms to induce lipid peroxidation or toxicity in cells.

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

As shown in FIG. 7, the inhibitory activity of the jujube seed extract was increased depending on the concentration of hydrogen peroxide. Among them, 60% ethanol extract showed similar activity as the positive control.

(4) Superoxide anion radical scavenging ability measurement

Superoxide anion radical scavenging ability was measured by NBT reduction method according to Fridovich's method. To 1 ml of each sample solution and 0.4 ml of 0.1 M potassium phosphate buffer (pH 7.5), add 1 ml of xanthine (0.4 mM) and NBT (0.24 mM), and 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. Then, the amount of superoxide anion radical generated in the reaction solution was measured at 560 nm.

Since superoxide anion radicals act as precursors of other harmful free radicals, the scavenging activity of these substances is known to be an effective method for screening the antioxidant activity of samples.

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

As shown in Figure 8, the jujube seed extract showed about 20% superoxide anion radical scavenging activity at all concentrations.

Test Example 4 Antibacterial Activity

(1) Target strain

Candida in a liquid medium for the culture of the albicans (KCTC 7965) it was used as the YM broth (YMB), Escherichia Nutrient broth (NB) was used for the liquid medium of coli (KCTC 1039) and Staphylococcus epidermidis (KCTC 1917), tryptic soy broth (TSB) was used for the liquid medium of Staphylococcus aureus (KCTC 1621), and Propionibacterium acnes (KCTC) was used. 3314) used GAM and solid medium was used by adding agar to the liquid medium. Strains were cultured by growth temperature in a BOD incubator.

(2) Disk Diffusion Method for Susceptibility Testing

Antibacterial activity was measured using a paper disc method. In other words, each strain cultured on a plate medium with 1 platinum, incubated for 18-24 hours in 10 mL of liquid medium, dissolved in 0.85% saline and mixed well, and then adjusted to 0.5 McFarland turbidity (spectrophotometer, 530 nm), and the concentration of the strain was about 1-5X10. ⁶ CFU / mL. The sterilized swabs were evenly spread on MH-GMB medium, and the disc and antibiotic (control) discs prepared with jujube seed extract in concentration-dependent manner were loaded. 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 FIGS. 5 and 6.

9 and 10 are photographs showing the antimicrobial activity of the jujube seed extract against microorganisms. Here, J20 is jujube seed extract extracted with 20% ethanol, J40 is jujube seed extract extracted with 40% ethanol, J60 is jujube seed extract extracted with 60% ethanol, J80 is jujube seed extract extracted with 80% ethanol , J95 is a jujube seed extract extracted with 95% ethanol. As a result, the jujube seed extract was confirmed that there is no antimicrobial activity against gram positive, negative, fungi.

Test Example 5 Regeneration of Cells

(1) Cell culture

CCRF S-180 II (Mouse skin fibroblast cell line, KCLB No. 10008) was distributed from Korean Cell Line Bank (Seoul, Korea). Cell culture medium was used 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. Subculture was used 0.05% trypsin, 0.53 mM EDTA.

(2) Cell viability evaluation by MTT assay

Toxicity of jujube cells was analyzed by MTT assay method. This method measures the conversion of mitochondrial dehydrogenases into formazan by dispensing CCRF S-180 Ⅱ (Mouse skin fibroblast cells) of 1X 10 ⁴ cells / well in 96 well plates and extracting 60% ethanol extract of jujube seeds by concentration (62.5). ~ 1000 ug / ml) was incubated for 24 hours. 20 ul of MTT solution per well was added and reacted for 4 hours at 37 ° C. 5% CO ₂ , and then the absorbance change was measured at 490 nm using a microplate reader to express cell viability as a percentage.

Based on in vitro experiments, the cell survival rate of CCRF S-180 II (Mouse fibroblast cells) was measured at a concentration of 50-200 ug / ml using J60, the most inhibitory effect.

Figure 11 shows the cell viability of jujube seed extract. As shown here, the cell viability was 97.9% at the concentration of 200 ug / ml.

(3) Measurement of the inhibitory effect of J60 on apoptosis on UVB

When UV rays are irradiated to the skin cells, the UV rays directly or indirectly damage the cell structure by directly inducing DNA structure changes or oxidative reactions.

UVB was irradiated to the cells to kill the cells, and using the sample to determine the extent of inhibition of cell death, UVB was examined and treated with MTT reagent to confirm the degree of cell death.

Seeding HaCaT cells (Human Keratynocyte Cells) in 96 well plates for 24 hours, incubating with serum free medium, and irradiating UVB (250mJ / cm ² ) to keratinocytes incubated in 96 well plates for 24 hours After treatment with J60 (60% ethanol extracts of Jujube seed) at various concentrations, incubate for 24 hours, treatment with Thiozolyl Blue Tetra-zolium Bromide (MTT) reagent, remove supernatant from plate after 4 hours, and remove Ethanol: DMSO = A 1: 1 solution was treated in each well and the absorbance was observed at 540 nm.

12 is a graph showing the protective effect of jujube seed extract against UVB-induced apoptosis in HaCaT cells. As shown here, the addition of the jujube seed extract after irradiation with ultraviolet rays shows the result of increasing the survival of the cells, which can be said that the jujube seed extract is effective in suppressing the aging phenomenon.

(4) Gelatin Zymography

Gelatin zymography was performed to investigate the effect of jujube seed extract on MMP-2 and MMP-9 activity using the properties of MMP-2 and MMP-9, which degrades gelatin. Gelatin Zymography was used to investigate the effect of jujube seed on the expression level of MMP-2 and MMP-9. Medium and sample quantified on 10% SDS-polyacrylamide gel (30% acrylamide / 0.8% bis-acrylamide, 1.5M Tris-HCl (pH8.8), 10% SDS, 10% APS, TEMED, 0.2% Gelatin, DW) The buffer was mixed 1: 1 and electrophoresed. To remove SDS, shake for 30 minutes in renaturing buffer (Triton X-1002% in DW), add to Developing buffer (50mM Tris-HCl, 0.2M NaCl, 5mM CaCl ₂ , 1% TritonX-100), and then at 37 ° C. The reaction was carried out for 12 hours. After staining for 1 hour in 0.5% coomassie blue, a staining solution, destaining solution (Methanol: Acetic acid: DW = 5: 1: 4) for 1 hour and then confirmed a transparent band.

Figure 13 is a graph showing the effect on the expression of MMP-2 and MMP-9 of jujube seed extract. As shown here, it can be seen that the expression level of MMP-2 and MMP-9 increased in the section treated with UVB (50mJ / cm ² ). Thus, when treated with 60% extract of jujube seed EtOH by concentration, 50 ug / ml treatment did not show a change in expression, but the concentration-dependent MMP-2 and MMP- in the section treated with 100 ug / ml, 200 ug / ml It was confirmed that the expression level of 9 decreases.

(5) Western blot

To investigate the effect of UVB on human keratinocytes, we investigated the effects of UVB (50mJ / cm ² ) on HaCaT cells on the activation of kinase related to the expression of collagen precursors Pro-Collagen Type 1 and MMPs. In order to see the protein expression was measured using Western blot method.

After irradiating UVB 50mJ / cm ² , the jujube seed extract was treated by concentration, cells were collected, washed with PBS, dissolved in RIPA buffer (M.biotek) for 1 hour, and then centrifuged at 12,000rpm and 4 ° C for 10 minutes. Was recovered. 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 isolated protein was transferred to nitrocellulose membrane (Whatman, UK) for 1 hour at 100V, blocked with 0.1% BSA solution, and the membrane was immersed in Antibody solution for 18 hours at 4 ℃. Washed three times for 10 min with TBST (10 mM Tris-HCl, pH 7.5), 150 mM NaCl, 0.2% Tween 20). The washed membrane was reacted for 2 hours to combine with the chemically fluorescent horse radish peroxidase (HRP), and then used a chemiluminescence imaging device (ATTO).

14 is a graph showing the effect of jujube seed extract on UV-irradiated Pro-Collagen Type 1 and MMP-2 and MMP-9 expression. As shown here, as a result of confirming the expression level of Pro-Collagen Type 1, it was confirmed that the UVB irradiation decreased, and the positive control group and J60 increased in a concentration dependent manner. In the case of MMP-2 and MMP-9, it was confirmed that MMP-9 suppressed the visible expression level.

Formulation Example 1 Development of Sunscreen Formulation According to Jujube Seed Extract Content

A sunscreen formulation was prepared according to the content of jujube seed extract (hereinafter referred to as "HC-Phytoju") as shown in Table 1 below.

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-Hexandiol	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)

<Test Example 6> Stability Evaluation Method

(1) pH measurement

pH measurement was performed using a pH meter of Ohaus (Starter 3100F). Before the 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 and left for 10 minutes or more. The detector was washed well with water and wiped lightly before use.

Figure 15 is a graph showing the measurement of the pH change of the sunscreen formulation according to the jujube seed extract content. As shown here, the pH of the sunscreen stored at 25 ° C. for 12 weeks on days 0, 1, 3, 5, 7, 14, 28, 60 and 90 was measured, and the prescription HC-A, HC-B and HC- In the C formulation, slight differences in pH values were observed depending on the content of the jujube seed extract during the storage period.

(2) Measurement of viscosity change over time

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

Figure 16 is a graph showing the viscosity change of the sunscreen formulation according to the jujube seed extract content. As shown here, the viscosity of the sunscreen stored at 25 ° C. for 12 weeks on days 0, 1, 3, 5, 7, 14, 28, 60, and 90 was measured. As a result, prescription HC-A, HC-B and HC- C All formulations showed a difference in viscosity to 26,000-17,500 cps for 3 months, and especially in the case of HC-B and HC-C formulations, a decrease in viscosity was observed during the storage period.

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

The sunscreen is placed in a transparent container and stored at 0 ° C., 25 ° C. and 45 ° C. for 12 weeks, respectively, and evaluated for stability at temperatures up to 0, 1, 3, 5, 7, 15, 30, 60, and 90 days. Temperature stability was evaluated to investigate chemical and physical changes caused by spontaneous deterioration of sunscreen over time. At high temperature (45 ℃), the effect was observed on rancidity, discoloration, discoloration, flotation, sedimentation, separation, etc., and at the low temperature, physical and chemical changes such as coagulation, precipitation, and separation were observed.

 The sunscreen prepared for each prescription was placed in a transparent container and stored for 12 weeks at 0 ° C, 25 ° C and 45 ° C, respectively, and evaluated for stability by temperature until the 1st, 3, 5, 7, 14, 28, 60 and 90 days. 2 (0 ° C), Table 3 (25 ° C) and Table 4 (45 ° C) show the stability results over time, respectively.

SampleDay	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

SampleDay	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

SampleDay	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, HC-C) stored at 0 ℃ was observed to be stable for 28 days without the phenomenon of creaming or aggregation, but stored at 25 ℃ in Table 3 In the case of sunscreen, B was 28 days, C was 14 days, and it was observed to be unstable due to the creaming and agglomeration. As shown in Table 4, B was 14 days and C was 7 days after the sunscreen stored at 45 ℃. Creaming and flocculation occurred, resulting in instability.

(4) Stability test according to temperature cycle (Cycle chamber)

Put the sunscreen in a transparent container and store it for 8 hours at -5 ℃, 25 ℃, 50 ℃, and repeat it 14 times with 1 dah / 1 cycle for 2 weeks on 0, 1, 3, 5, 7, 14 days. The stability with temperature cycling during the process was evaluated. The stability test method according to the temperature cycle (Cycle chamber) is carried out to observe the change of physical properties according to the storage conditions of low and high temperature of cosmetics in Korea with four distinct seasons, and also the change of physical properties in emulsification and solubilization according to temperature change. To observe. Most of the products are hydrolysis and other phenomena occur quickly at high temperatures, and at low temperatures, volume expansion, interfacial membrane damage due to solubility difference, and precipitation occur. The stability test by the cycle chamber can check this phenomenon in a short period of time. The results are shown in Table 5 below.

SampleDay	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, physical properties such as creaming, agglomeration, and separation were shown in sunscreen HC-B and HC-C, and HC-A was stable without phase separation, discoloration, and discoloration in all 14 cycles. It could be confirmed as.

Test Example 7 Evaluation of Effectiveness (SPF, PA)

This test is based on the `` Regulations on the Functional Cosmetics Examination '' (Food and Drug Safety Notice No. 2015-14) on the `` Functional Cosmetics that Help to Burn Skin Finely or Protect Skin from UV Light '' as defined in Article 9 of the Cosmetic Act. It was carried out according to 'Method and Criteria for Measuring UV Protection Effect'.

(1) Test site

The test was conducted on the subject and the like. The test site was selected to avoid skin damage, excessive hairs or areas of particular color tone and was clean and dry.

(2) Test substance application method

A standard sample and test sample application area on the subject's back, except for the spinal region, were applied to the test site in an amount of 2.0 mg / cm 2 (70.0 mg total). Micro pipettes were used to apply the sample in the correct amount. Samples were evenly applied to the surface of the skin several times and then uniformly applied using a finger thimble or spatula.

Section of ultraviolet irradiation site

Six square irradiation sites having an area of 0.8 cm × 0.8 cm were partitioned with a product application area of 35 cm 2.

(3) Equipment used

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

The instrument is equipped with a 300watt Xenon arc lamp, which has a continuous emission spectrum similar to sunlight and does not exhibit a specific peak, as the light source is irradiated with a dichroic mirror and aiming lens. After passing through a collimating lens, the liquid is emitted in a square size of 0.8 cm X 0.8 cm through six liquid light guides. Special filters are used to remove wavelengths in the ultraviolet region below 290 nm, which are particularly harmful to humans.

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

Light intensity meter in units of ㎼ / cm ² , measured by combining the SUV probe at the end of each light guide, and confirm that the measured value is maintained before and after UV irradiation.

(C) standard sample

Standard samples with a high UV protection index should be prepared and used in accordance with the "Methods and Standards for Measuring UV Protection Effects" of the Regulations on the Evaluation of Functional Cosmetics (KFDA No. 2015-14).

(4) SPF measurement result

Sunscreen index evaluation test of 12 subjects, including 2 preliminary subjects, was conducted at the Korean Dermatological Research Institute, a cosmetic clinical research institute, and the sunscreen index averaged 50.5 ± 2.3. Could be confirmed. In order to ensure the reliability of the test, standard samples were prepared and tested at the same time according to the test method suggested by the Ministry of Food and Drug Safety, and the UV protection index of 15.3 ± 1.3 was confirmed. ] Was within the range of 15.5 ± 3.0. The 95% confidence intervals of the UV protection index of each of the standard sample and the test sample measured through this test were confirmed to be within ± 20% of the measured value, thereby verifying the reliability of the measured value. In conclusion, the product is considered to have ultraviolet power equivalent to SPF 50.5 ± 2.3.

(5) PA measurement result

In the dermatology research institute, a cosmetics clinical research institute, a sunscreen conducted by this study was tested on a total of 12 subjects including two preliminary subjects. I could check the index. In order to ensure the reliability of the test, the standard samples were prepared and tested at the same time according to the test method suggested by the Ministry of Food and Drug Safety, and the UVA blocking index of 4.04 ± 0.41 was confirmed. Was within the range of 3.75 ± 1.01. The standard error measured by this test and the standardization error of the UVA blocking index of each test sample were confirmed to be within ± 10% of the measured value, thereby verifying the reliability of the measured value. In conclusion, the product is considered to have UVA blocking ability equivalent to PFA 8.77 ± 0.71 and PA +++.

As such, the composition comprising the jujube seed extract of the present invention as an active ingredient has a tyrosinase inhibitory effect and not only have a whitening effect, but also have an anti-wrinkle effect by inhibiting elastase and collagenase activity. In addition, the DPPH free radical, ABST radical cation, hydrogen peroxide and superoxide anion radical scavenging activity is excellent and has an antioxidant effect, the cell is not killed by irradiation with ultraviolet light can be usefully used as a sunscreen agent. In addition, the jujube seed extract of the present invention is excellent in antibacterial activity, there is no cytotoxicity and skin side effects can be used safely in cosmetics, pharmaceutical and food compositions.

Claims (5)

1. Cosmetic composition for skin whitening, anti-wrinkle, antioxidant and sun protection comprising jujube seed extract as an active ingredient.
2. A pharmaceutical composition for skin whitening, anti-wrinkle, antioxidant and sunscreen comprising jujube seed extract as an active ingredient.
3. Food composition for skin whitening, anti-wrinkle, antioxidant and sunscreen comprising jujube seed extract as an active ingredient.
4. The method according to any one of claims 1 to 3,

   The jujube seed extract is pulverized jujube seed after 20 to 30 hours by immersion using 20 to 95% ethanol aqueous solution and then recovered, and then filtered by filter paper and concentrated to obtain a composition.
5. The method according to any one of claims 1 to 3,

   The jujube seed extract is characterized in that it comprises an amount of 0.005 to 50% by weight based on the total weight of the composition.

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