KR20110029740A - Fraction of citrus unshiu fruit peel extract and use thereof - Google Patents

Abstract

PURPOSE: A fraction of Satsuma mandarin peel extract is provided to enhance high antioxidation, whitening, UV protection, skin elasticity, and anti-wrinkling. CONSTITUTION: A fraction of Satsuma mandarin peel extract is obtained by dipping Satsuma mandarin peel in water, alcohol of 1-5 carbon atoms, or mixture solvent to obtain extract and by fractioning with n-hexane, methylene chloride, chloroform, or mixture solvent thereof. An antioxidant composition contains the fraction as an active ingredient. The composition is a cosmetic composition.

Description

Fraction of Wenzhou Citrus Peel Extract and its Use {Fraction of Citrus unshiu Fruit Peel Extract and Use Thereof}

The present invention relates to fractions of Wenzhou Mandarin Peel Extract and its use. Specifically, the present invention provides a fraction obtained by fractionating Wenzhou Mandarin Peel Extract with a polar solvent layer, and an antioxidant, skin lightening agent, sunscreen and fractions thereof. It relates to the use as a skin wrinkle improver.

As a person ages, his skin is constantly changing due to skin aging and pigmentation. Factors that determine skin color include, by default, differences in race, region, gender, and age, and partial or total pigmentation, such as tanning due to blemishes, freckles, and sun exposure, as well as the distribution of acne, scars, and keratin. Blood circulation, stress, and health. Among the above factors, the most important factor is pigmentation.

The pigments affecting skin color include melanin, carotene, and hemoglobin, the most important of which is melanin, which is the most important factor affecting melanin biosynthesis. Melanin biosynthesis is a series of processes that begin with the conversion of tyrosine, a type of amino acid from melanocytes of melanocytes to dihydroxy phenylalanine by tyrosinase, to brown (pheomelanin) and black (eumelanin). It is formed of a polymer. This biosynthesis process is carried out in a special type of brown intracellular organelles called melanosomes. Melanosomes, including melanin granules, migrate from the periphery of the nucleus to the end of dendritic processes and then into the cytoplasm by the phagocytosis of keratinocytes. Accumulate around the nucleus of the latinosite. Melanin synthesis and the number of melanosomes and the migration to surrounding keratinocytes are primarily genetically affected, in part by hormones and ultraviolet radiation. In addition, cytokines (cytokine), copper ions, copper, zinc, iron and other metal ions involved in tyrosinase expression, melanin synthesis, and transport, and interferon, prostaglandin, histamine, etc. are known to be involved. 25: 77-127, 1999). Therefore, the skin whitening effect can be expected by inhibiting the synthesis of melanin, which determines the skin color, and the activity of tyrosinase, an enzyme involved therein.

Next, factors that characterize aging with skin color are wrinkle formation and elasticity reduction. Skin aging can be divided into intrinsic, chronological aging and phtoaging [Gilchrest BA: J. Am. Acad. Dermatol., 21, 610-613 (1989). Endogenous aging is a naturally occurring aging phenomenon caused by deterioration of physiological functions of living bodies with increasing age [Braverman IM et al .: J. Invest. Dermatol., 78, 434-443 (1982). Photo-aging means that the skin is repeatedly exposed to light such as ultraviolet rays to change the appearance or function of the skin [Ridder GM et al .: J. Am. Acad. Dermatol., 25, 751-760 (1991). More specifically, photoaging is the damage of skin, tissues, and the like by the damage of lipids, proteins, polysaccharides, and nucleic acids, which are the major constituents of the skin, by ultraviolet rays, resulting in skin aging. Especially, collagen, hyaluronic acid, elastin, proteoglycan, and fibronectin, which are the connective tissues of the skin, are cut, which affects the elasticity of the skin and forms deep wrinkles, a major phenomenon of photoaging. It leads to a situation of induction and a decrease in immune function.

In vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but as aging progresses, the synthesis decreases and promotes degradation, especially at sites chronically exposed to ultraviolet rays. Substrates, enzymes that degrade collagen by ultraviolet rays, etc. Expression of the metalloproteinase (matrixmetalloproteinase, hereinafter referred to as 'MMP') is promoted, thereby reducing skin elasticity and forming wrinkles.

Matrix metalloproteinases (MMPs) are secreted from various cells in the body, including keratinocytes and fibroblasts in human skin. They act as calcium and zinc-dependent endopeptidase and operate at neutral pH. Various extracellular substrates are used as substrates. MMPs are broadly classified into MMP-1, MMP-2, and MMP-9 according to their substrate specificity. MMP-1 is an interstitial enzyme fibroblast type collagenase. Substrates include collagen type I, II, III, Ⅶ, Ⅷ, Ⅹ and gelatin. It is originally a collagen 3/4 or 1/4 length peptide. The cleavage facilitates the action of nonspecific enzymes (gelatinase, etc.). MMP-2 belongs to 72 kD gelatinase (A) and degrades gelatin, collagen types IV, V, VIII, VIII, elastin and fibronectin. MMP-13 also degrades elastin as metalloelastase. Collagen types I and III, elastin and fibronectin, which occupy most of the dermal layer of human skin, impart strength and tension to the skin, and wrinkles, loss of elasticity, and sagging skin when proteins are decomposed by abnormally activated MMP by ultraviolet rays and free radicals. Aging phenomenon is accelerated. In particular, by inhibiting the activity and biosynthesis of MMP-1, which degrades type I collagen, which accounts for the largest proportion of binding proteins, skin binding proteins can be protected to prevent wrinkle formation and elasticity. Therefore, there is a need for the development of a substance capable of regulating or inhibiting MMP expression in which activation is induced in cells by ultraviolet rays. Until now, most of the raw materials used as external skin preparations simply inhibited MMP enzyme activity.

In order to prevent aging due to UV rays, the most common method is to apply a product containing sunscreen directly to the skin, but in 1988, retinoic acid was reported to be effective in reducing roughness and fine wrinkles of aged skin (KS Weiss et al. , JAMA, 259, 527-532, 1988), research is being actively conducted to develop substances that inhibit or improve skin aging worldwide. Among them, derivatives of vitamins such as vitamins A, C and E and AHA (alpha-hydroxy acid) are representative aging skin improvement substances known to date (Hermitte, Cosmetics & Toiletries, 107, 63-67, 1992, DR Rosenthal et al. , J. Invest.Dermatol., 95, 510-515, 1990, TDDitre et al, J. Invest.Dermatol, 34, 187-195, 1996). However, they are very unstable and have a problem in use, or a problem that is somewhat insufficient to show a visible effect.

Recently, in order to reduce skin irritation caused by various chemicals, a lot of attention has been focused on functional cosmetics having functions such as wrinkle relaxation and whitening obtained from natural extracts. Natural materials have less side effects on the skin, and as the consumer's response to the external skin preparations using the natural materials has recently increased, the development value of the anti-aging cosmetic composition is further increased. For example, Japanese Patent Application Laid-open No. Hei 9-672662 describes seaweed extracts having a hyaluroronidase inhibitory effect. Japanese Patent Application Laid-open No. Hei 10-85905 describes the skin texture improvement effect of Fucoidan extracted from Wakame. Japanese Patent Application Laid-Open No. 2-245087 describes the antioxidant effect of Sargassum genus extract. Japanese Patent Application Laid-Open No. 3-294384 describes an antioxidant composition extracted from the genus Hijikia. Japanese Patent Laid-Open No. 7-10769 describes a extract of Phaeophyceae having an astringent effect. U.S. Patent No. 5,508,033 describes an anti-radical active cosmetic including citrus fruits and blood, and Japanese Patent Application Publication No. 2008-056643 discloses citrus peels having functions such as antioxidant activity, cell metabolism activity, and skin aging prevention ( Sargassum fulvellum) extract-containing cosmetics are disclosed, and Japanese Patent Laid-Open No. 1998-330280 discloses a citrus peel extract-containing cosmetics that inhibit collagen production promotion, wrinkles or reduced elasticity. Korean Patent No. 810134 includes the steps of washing and desalting citrus peel or 톳; Primary enzymatic digestion consisting of endo-β1,4-glucanase, Alginic acid lyase, and exocellulose; And a series of steps of digesting with a secondary enzyme consisting of Arabanase, cellulase and β1,4-glucanase, and enzymatic digests of citrus peels or mussels and methods for preparing the same are disclosed. It is disclosed that it can be used as a cosmetic or the like. Korean Unexamined Patent Publication No. 2009-27387 discloses an extract of citrus peel which can be used as a cosmetic for improving skin wrinkles and elasticity.

Therefore, the present inventors have studied the possibility of application to a whitening and anti-aging cosmetic composition in various natural products, and as a result of selecting an extract of Wenzhou mandarin peel and preparing an extract and fractionating it with various solvents, antioxidant activity, melanin production inhibitory activity, etc. This high fraction could be obtained.

The technical problem of the present invention is to provide a fraction of Wenzhou mandarin skin extract having high antioxidant activity, whitening activity, sunscreen activity, skin elasticity improving activity and / or skin wrinkle improvement activity.

Another technical problem of the present invention is to provide a composition for skin antioxidant comprising the fraction.

Another technical problem of the present invention is to provide a composition for sun protection comprising the fraction.

Another technical problem of the present invention is to provide a composition for improving skin wrinkles comprising the fraction.

The inventors extracted the Citrus unshiu peel with 80% ethanol to obtain lyophilized powder (hereinafter referred to as "80% ethanol extract of Wenzhou mandarin peel"), and the powder was mixed with water, methanol and ethanol (high polar solvent Layer) and a mixture of hexane, dichloromethane, and chloroform (low polar solvent layer), the fraction of the high polar solvent layer of Wenzhou Mandarin Peel, the low polarity of 80% ethanol extract of the Wenzhou Mandarin Peel or Wenzhou Mandarin Peel. Compared with the fraction of the solvent layer, it was confirmed that it has a significantly higher antioxidant activity, whitening activity, sunscreen activity, skin elasticity improving activity and skin wrinkle improving activity.

In addition, according to the inventors of the present invention, the fraction of the high polar solvent layer of the Wenzhou citrus rind contained a very high content of the glutinous assets compared to the fraction of the low polar solvent layer of the Wenzhou citrus rind. Activity, whitening improving activity, etc. (Luigi Sartor et al., Biochemical Pharmacology Vol 64, Issue 2, 15 July 2002, Pages 229-237; Sundaram Jagan et al., Molecular and Cellular Biochemistry (2008) 319 : 51-59, October 21, 2008).

The present invention is provided based on these experimental results.

In one aspect, the present invention can be regarded as a fraction of the solvent layer of the polar solvent of Wenzhou mandarin skin extract having antioxidant activity, whitening activity, UV protection activity, and / or skin wrinkle improvement activity.

In the present specification, "wenzhou mandarin rind extract" refers to the extraction subject as an extracting subject, and the extraction subject as water, a C1-C5 alcohol or a mixed solvent thereof. It means an extract obtained by immersing in a solvent, and includes a concentrated liquid extract or solid extract from which the extraction solvent is removed. Regardless of the extraction method, it is to be understood that the extraction method may be applied to any method such as cold soaking, refluxing, warming, and ultrasonic wave, as long as the extract is immersed in the extraction solvent in the wet mandarin peel. Nevertheless, the "wenzhou mandarin rind extract" preferably means an extract obtained by extracting the Wenzhou mandarin rind which is the extraction target with a mixed solvent of water and ethanol, in particular 80% ethanol.

In addition, in the present specification, the "fraction fraction of the strong solvent layer of the Wenzhou mandarin peel extract" is a polar polarity selected from the group consisting of water, C1-5 alcohol and a mixed solvent thereof as defined above Means a fraction of a polar solvent layer obtained when the solvent is fractionated with a weak polar solvent selected from the group consisting of n-hexane, methylene chloride, chloroform and a mixed solvent thereof, and the fractionated solvent is removed from the fraction. It is meant to include liquid fractions or solid fractions. Preferably, the extract obtained by extracting the Wenzhou mandarin peel, which is the extraction target, with a mixed solvent of water and ethanol, in particular 80% ethanol, is divided into a mixed solvent composed of water, methanol and ethanol, and a mixed solvent composed of methylene chloride, chloroform and n-hexane. Refers to the fraction of the mixed solvent layer composed of water, methanol and ethanol obtained. Most preferably, the extract obtained by extracting with 80% ethanol and removing the extracting solvent is a mixture of water, methanol and ethanol in a volume ratio of 3: 4: 3 with methylene chloride, chloroform and n-hexane 1: 2. It means a fraction of the mixed solvent layer of water, methanol and ethanol obtained when fractionated with a mixed solvent composed of a volume ratio of 7 :.

In addition, in the present specification, "antioxidant activity" means the removal activity of active oxygen species such as singlet oxygen, superoxide anion, hydroxyl free group, hydrogen peroxide, etc., preferably In the following experimental example, the antioxidant activity was examined through superoxide scavenging activity and DPPH radical scavenging activity, which means superoxide scavenging activity and / or DPPH radical scavenging activity.

Also herein, "whitening activity" is defined as melanin production inhibitory activity.

In another aspect, the present invention relates to an antioxidant composition comprising a fraction of the solvent layer of the strong polar solvent extract of Wenzhou mandarin peel of the present invention as an active ingredient.

As used herein, the term "active ingredient" alone refers to a component that can exhibit the desired activity or exhibit itself with a carrier which is inactive.

Also herein, "antioxidant" means amelioration, treatment of pathological symptoms of a disease caused by reactive oxygen species and inhibition / delay of the development of such pathological symptoms. Diseases caused by reactive oxygen species include arteriosclerosis, stroke, myocardial infarction, blemishes, freckles, fine lines, cancer, leukemia, collagen disease (a disease that causes abnormalities in connective tissues of the body such as skin, joints, and blood vessels), Hypogonadism, stroke, myocardial infarction, diabetic vascular disorders, hyperlipidemia, acute inflammation, rheumatoid disease, alcoholic hepatitis, etc. in men (Harrison et al, Am J Cardiol, 2003. 2; 91 (3A): 7A-11A; Behrend et al, Biochem Soc Trans, 12 Dec. 2003; 31: 1441-1444; Agarwal et al, Fertil Steril, 2003. 4; 79 (4): 829-843; (Orr, WC and Sohal, RS , Science, 1994, 263, 1128-1130; Sohal, RS et al., J. Biol. Chem., 1995, 270, 15671-15674. Preferably, the antioxidant composition of the present invention is mainly applied as a topical skin composition. Given that, "antioxidant" refers to the treatment, treatment and improvement of diseases associated with the skin caused by reactive oxygen species, ie blemishes, freckles and wrinkles. It means on the onset of inhibition / delay.

In the antioxidant composition of the present invention, the fraction of the solvent layer having a strong polarity of Wenzhou mandarin peel extract as an active ingredient may be included in any amount (effective amount) as long as it can exhibit antioxidant activity, the usual effective amount is the whole composition It will be determined in the range of 0.001% to 99.900% by weight based on weight. As used herein, "effective amount" refers to an amount of an active ingredient that can induce improvement, treatment, or inhibition / delay of expression of symptoms caused by reactive oxygen species. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.

In another aspect, the present invention can be understood as a skin lightening composition comprising a fraction of the solvent layer of the strong polar solvent extract of Wenzhou mandarin peel of the present invention as an active ingredient.

As used herein, "skin whitening" is understood as a result of melanin production being inhibited. Melanin is produced by tyrosinase. Tyrosine is oxidized to dopa (DOPA, 3,4-dihydro xyphenylalanine), and the dopa is oxidized again to dopaquinone, followed by a non-enzymatic oxidation reaction. Is made through. This melanin protects the skin organs under the dermis by blocking UV rays, and at the same time serves to protect the damage of proteins and genes in the skin by free radicals generated in the skin, but when melanin is produced more than necessary Induces hyperpigmentation, such as freckles and blotch, and leads to skin aging. Thus "skin whitening" can be understood as an improvement in symptoms resulting from an increase in melanin, blemishes, freckles, blotch and / or skin aging.

In the skin whitening agent composition of the present invention, the terms described in connection with the antioxidant composition of the present invention are applied mutatis mutandis to the effective amount of the active ingredient.

In another aspect, the present invention can be understood as a sunscreen composition comprising a fraction of the solvent layer of the strong polar solvent extract of Wenzhou citrus peel of the present invention as an active ingredient.

In the present specification, "ultraviolet ray blocking" may improve symptoms caused or aggravated by ultraviolet rays (UVA and / or UVB), such as skin cancer, wrinkles, promoting melanin synthesis, erythema, freckles, and swelling. Defined as efficacy. Therefore, for example, the sunscreen composition of the present invention is regarded as meaning including a composition for preventing skin cancer caused by ultraviolet rays or a composition for reducing skin cancer exacerbated by ultraviolet rays.

Also in the sunscreen composition of this invention, what was demonstrated in connection with the antioxidant composition of this invention is applied mutatis mutandis as regards the effective amount of the effective ingredient.

In another aspect, it can be seen as a skin wrinkle improver composition comprising the fraction of the solvent layer of the strong polar solvent extract of Wenzhou citrus peel of the present invention as an active ingredient.

Skin wrinkles are usually caused by a decrease in collagen or elastin, which maintains skin elasticity by various harmful substances such as ultraviolet rays and free radicals. Fibroblasts are involved in the production of collagen or elastin. Ingrid Emerit et al. Free radical and aging, 1992; Arther K. Balin et al. Aging and the skin, 1998; Hermitte, Cosmetics amp, Toiletries, 107, 63-67, 1992; DR Rosenthal et al , J. Invest.Dermatol., 95, 510-515, 1990; TDDitre et al, J. Invest.Dermatol, 34, 187-195, 1996).

As described above, the fraction of the solvent layer having a strong polarity of the Wenzhou mandarin peel extract of the present invention has the effect of blocking and removing ultraviolet rays, active oxygen, etc., which cause skin wrinkles, and the following experimental example is 80 It showed no cytotoxicity against fibroblasts involved in the production of collagen or elastin at concentrations 5 times higher than% ethanol extract and 20 times higher than fractions of low polar solvent layer of Wenzhou citrus rind. Giving.

In particular, the experimental example below shows that the fraction of the solvent layer having a strong polarity of the Wenzhou mandarin peel extract of the present invention reduced the skin wrinkles and improved skin elasticity through the clinical experiment.

Based on the foregoing, in the present specification, the term "skin wrinkle improvement" is defined as meaning including skin wrinkle reduction, inhibition, prevention as well as an increase in skin elasticity.

Also in the antiwrinkle composition of this invention, what was demonstrated in connection with the antioxidant composition of this invention is applied mutatis mutandis with respect to the effective amount of the effective ingredient.

The antioxidant composition, skin lightening composition, sunscreen composition and skin wrinkle improving composition (hereinafter “composition” of the present invention) of the present invention may be regarded as a cosmetic composition in specific embodiments.

When the composition of the present invention is conceived as a cosmetic composition, the cosmetic composition can be prepared in various forms, for example, emulsions, lotions, creams (oil-in-water, water-in-oil, multiphase), solutions, suspensions (anhydrous and Aqueous), anhydrous products (oil and glycol based), gels, masks, packs or powders and the like.

The composition of the present invention may include an acceptable carrier in the cosmetic preparation in addition to the fraction of the solvent layer of the strong polar extract of Wenzhou mandarin peel as an active ingredient.

Herein, "acceptable carriers in cosmetic preparations" are compounds or compositions already known and used that may be included in cosmetic preparations, or compounds or compositions which will be developed in the future, which have no toxicity, instability or irritation more than the body can adapt to in contact with the skin. Say that.

The carrier may be included in the cosmetic composition for skin whitening of the present invention at about 1% by weight to about 99.99% by weight, preferably about 50% to about 99% by weight of the composition.

However, since the ratio depends on the form of the above-described preparation of the cosmetic of the present invention and its specific application site (face or hand) or its preferred application amount, the ratio is in any aspect the scope of the present invention. It should not be understood as limiting.

Meanwhile, examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, sunscreen agents, colorants, fragrances, and the like.

Alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, sunscreens, colorants, compounds / compositions that can be used as fragrances, etc., which can be used as the carrier are already known in the art. As a result, those skilled in the art can select and use appropriate materials / compositions.

Meanwhile, the composition of the present invention may be regarded as a pharmaceutical composition in another specific embodiment.

The pharmaceutical composition of the present invention is usually administered directly to the skin in a topical formulation.

In addition, the pharmaceutical composition of the present invention may be formulated using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants and surfactants commonly used when formulated, cream, lotion, ointment (semi-solid) External preparations), microroemulsions, gels, pastes, transdermal preparations (TTS) (such as patches, bandages, etc.) and the like.

The daily dosage of the pharmaceutical composition of the present invention is usually 0.001 ~ 150 mg / kg body weight range, it can be administered once or divided into several times. However, since the actual dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, and the severity of the patient, the dosage is in any aspect the scope of the present invention. It should not be understood as limiting.

The composition of the present invention may be regarded as a soap composition in another specific embodiment.

When the composition of the present invention is understood as a soap composition, the soap composition of the present invention may be prepared by including a fraction of the solvent layer having a strong polarity of Wenzhou Mandarin Skin Extract, which is an active ingredient on the soap base, and as an additive a skin moisturizer, It may be prepared including an emulsifier, water softener, and the like.

As the soap base material, vegetable oils such as palm oil, palm oil, soybean oil, perm oil, olive oil, palm kernels, or animal oils such as tallow, pork, sunny and fish oils can be used, and the skin moisturizing agent is glycerin, erythritol and polyethylene glycol. , Propylene glycol, butylene glycol, pentylene glycol, hexyl glycol, isopropyl myristate, silicone derivatives, aloe vera, sorbitol and the like can be used, and as the emulsifier natural oil, wax fatty alcohol, hydrocarbons, natural plant extracts Etc. may be used, and as the water softener, tetrasodium idieti may be used.

The soap composition of the present invention may further include an antimicrobial agent, a dispersant, a foam inhibitor, a solvent, a scale inhibitor, a corrosion inhibitor, a perfume, a pigment, a metal ion blocking agent, an antioxidant, a preservative, and the like as an additive.

In the soap composition of the present invention, the soap base or additives may be included in an amount generally used in the art, and the soap base is generally 99.9 to 50 wt% based on the total content of the soap composition. It may be added, the additive may be added in 1% to 20% by weight.

As described above, the present invention can provide a fraction of Wenzhou Mandarin Skin Extract, which has high antioxidant activity, whitening activity, sunscreen activity, skin elasticity improving activity and / or skin wrinkle improving activity, and has a high molar content.

The fraction of Wenzhou mandarin peel extract of the present invention is formulated as an external preparation for skin, especially cosmetics, and can be used for antioxidants, sunscreens, skin whitening agents, skin wrinkle improvers and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are only illustrative of the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

EXAMPLES Preparation of Wenzhou Mandarin Skin Extract and Its Fractions

Example 1 Preparation of Wenzhou Mandarin Skin Extract

The dried and dried citrus (Wenju citrus) peel was refluxed three times for 5 hours with an aqueous 80% (V / V) ethanol solution, chilled, and filtered through Whatman # 5 filter paper. The filtered extract was concentrated under reduced pressure and freeze-dried at 50 ℃ or less to prepare a tangerine peel extract.

Example 2 Preparation of Fraction of Solvent Layer with Strong Polarity of Wenzhou Citrus Peel Extract

10 g of the lyophilisate extract of the citrus peel extract obtained in Example 1 was evenly dispersed and dissolved in 1 L of a highly polar mixed solvent containing purified water, methanol and ethanol at a volume ratio of 3: 4: 3, and then hexane, dichloromethane, and chloroform were 7: The mixture was stirred with 1 L of the low polar mixed solvent mixed at a volume ratio of 2: 1. After stirring the mixed liquid for 8 hours in a sealed state at room temperature, the low polar mixed solvent layer and the high polar mixed solvent layer were carefully separated. The recovered high polar mixed solvent layer was filtered through Whatman # 5 filter paper, and then concentrated under reduced pressure and freeze-dried at 50 ° C. or lower to prepare a high content citrus peel.

Example 3 Preparation of Fraction of Solvent Layer with Low Polarity of Wenzhou Citrus Peel Extract

The low-polar mixed solvent layer separated in Example 2 was filtered through Whatman # 5 filter paper, and concentrated under reduced pressure and freeze-dried at 30 ° C. or lower to prepare an extract of a citrus peel.

Experimental Experiments of Wenzhou Mandarin Skin Extracts and Their Fractions

Experimental Example 1 Cytotoxicity Test on Fibroblasts

The cytotoxicity against fibroblasts of the citrus peel extract and its fractions obtained in Examples 1 to 3 was evaluated.

Fibroblasts (fibroblasts) were placed in a 24-well test plate 1 x 10 ⁵ and attached for 24 hours. Each well was washed once with PBS (Phosphate buffered saline), and each sample was treated with 1 ml of cell culture medium (10% FBS added to DMEM) and diluted to an appropriate concentration for 24 hours. Incubated for After 24 hours, the medium was removed, and 500 μl of cell culture medium and MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) solution (2.5 mg / ml) were added to each well. 60 μl was added thereto and then incubated in a 37 ° C. CO ₂ incubator for 2 hours. The medium was removed, and 500 μl of isopropanol-HCl (0.04N) was added thereto. After shaking for 5 minutes, the cells were lysed and 100 μl of the supernatant was transferred to a 96-well test plate, followed by measurement of 565 nm absorbance in a microplate reader. Cell survival rate (%) was measured by the following formula, and the concentration of the sample that did not affect the survival of the cells was determined and shown in Table 1 below.

Cell survival rate (%) = [(St-Bo) / (Bt-Bo)] X 100

Bo: 565 nm absorbance of wells that only reacted with cell culture media

Bt: 565 nm absorbance of wells that developed color reaction without treatment

St: 565 nm absorbance of wells treated and developed

Sample concentration does not affect cell viability Sample Name 100% cell survival concentration Extract of Example 1 0.1% Extract of Example 2 0.5% Extract of Example 3 0.025%

As can be seen in Table 1, the 100% cell viability of the citrus peel extract of Example 1 was found to have a cytotoxicity similar to that of conventional plant extracts. In addition, the fraction of the solvent layer of weak polarity of Example 3 was confirmed that the cytotoxicity was significantly increased to 100% cell survival concentration of 0.025%. On the contrary, the fraction of the solvent layer having the strong polarity of Example 2 was found to have a 100% cell viability concentration of 0.5%, which significantly improved cytotoxicity, and thus relatively low incidence of skin irritation and was safe.

Experimental Example 2 Experiment of Measuring Antioxidant Effect Using NBT Method

In order to confirm the antioxidant effect of the citrus peel extract and its fractions obtained in Examples 1 to 3 as a comparative sample, BHT (Butylated hydroxytoluene), which is well known as an antioxidant under laboratory conditions, was used as a comparison sample and antioxidant was performed using NBT (Nitro Blue Tetrazolium) method. Activity was measured.

In order to measure the antioxidant effect, the active oxygen produced by xanthine and xanthine oxidase was measured by NBT method, and the effect of removing the active oxygen by the test substance, that is, the effect of scavenging the active oxygen, was evaluated. Free radicals are produced from xanthine by xanthine oxidase. The active oxygen scavenging rate can be measured by measuring the blue color produced by reaction of this active oxygen with Nitro Blue Tetrazolium (NBT) at a wavelength of 560 nm.

As a measuring method, 0.05M Na2CO3 (2.4ml), 3mM xanthine solution (0.1ml), 3mM EDTA solution (0.1ml), BSA solution (0.1ml), 0.72mM NBT solution (0.1ml) were added thereto, and each sample solution was added thereto. 0.1 ml is added and left at 25 ° C for 10 minutes. Xanthine oxidase solution (0.1 ml) is added and stirred quickly and incubation at 25 ° C. for 20 minutes is started. Then 6 mM CuCl ₂ solution (0.1 ml) was added to stop the reaction and the absorbance St at 560 nm was measured. In the blank test, absorbance Bt was measured by using distilled water instead of the sample solution as described above. In addition, the absorbance (So) when only the sample solution was added without using the enzyme and the absorbance (Bo) when both the enzyme and the sample solution were not added were measured by the same method as described above using the same amount of distilled water.

The antioxidant effect was calculated by the following formula, the results are shown in Table 2 below.

% Inhibition = [1- (St-So) / (Bt-Bo)] X 100

St: absorbance at 560 nm after enzyme reaction of sample solution

Bt: absorbance at 560 nm after enzymatic reaction of blank test solution

So: absorbance at 560 nm before reaction without enzyme in sample solution

Bo: absorbance at 560 nm before reaction in the absence of enzyme in blank test solution

Antioxidative effect Sample Name content Antioxidative effect(%) Extract of Example 1 0.5 wt% 42.1% Extract of Example 1 0.1 wt% 16.2% Fraction of Example 2 0.1 wt% 92.7% Fraction of Example 2 0.02% by weight 43.6% Fractions of Example 3 0.5 wt% 48.3% Fractions of Example 3 0.1 wt% 21.2% BHT 0.1 wt% 89.3%

As can be seen in Table 2, the fractions of the Wenzhou mandarin peel extract of Example 1 and the solvent layer of weak polarity of Example 3 was found to have a weak and similar antioxidant power than BHT. However, it was confirmed that the fraction of the solvent layer having a strong polarity of Example 2 has an antioxidant power better than BHT. That is, the fraction of the solvent layer of the strong polarity of Example 2 can be seen that the antioxidant activity is significantly improved.

Experimental Example 3 Free Radical Scavenging Activity Measurement Experiment

In order to measure the free radical scavenging activity of the citrus peel extract and its fractions obtained in Examples 1 to 3, BHT was compared with the laboratory conditions and free radical scavenging activity was measured using the DPPH method.

The DPPH method measures free radical scavenging activity by reducing force using a free group called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical). The free radical scavenging rate is measured at a wavelength of 560 nm by comparing the degree to which the DPPH is reduced by the test substance to reduce the absorbance.

Samples of 0.1%, 0.01%, and 0.001% concentrations were prepared for DPPH free radical scavenging activity measurement. Samples of the above concentrations were placed in 96-well plates, respectively, and DPPH prepared in 100 μM methanol solution was added thereto so that the total volume of the solution was 200 μl. After leaving it at 37 ° C. for 30 minutes, absorbance was measured at 560 nm. The free radical scavenging activity (%) was calculated by the following equation and is shown in Table 3 below.

Free radical scavenging activity (%) = 100- (B / A) X100

A: absorbance of control wells not treated citrus peel extract of the present invention

B: Absorbance of Experimental Wells Treated with Citrus Peel Extracts of the Present Invention

Free radical scavenging activity measurement Sample Name content Antioxidative effect(%) Extract of Example 1 0.5 wt% 19.3% Extract of Example 1 0.1 wt% 9.7% Fraction of Example 2 0.1 wt% 84.6% Fraction of Example 2 0.02% by weight 22.2% Fractions of Example 3 0.5 wt% 22.3% Fractions of Example 3 0.1 wt% 12.1% BHT 0.1 wt% 81.0%

As can be seen in Table 3, the fraction of the Wenzhou mandarin peel extract of Example 1 and the solvent layer of weak polarity of Example 3 was found to have a weaker and similar antioxidant power than BHT. However, it was confirmed that the fraction of the solvent layer having a strong polarity of Example 2 has an antioxidant power better than BHT. That is, the fraction of the solvent layer of the strong polarity of Example 2 can be seen that the antioxidant activity is significantly improved.

Experimental Example 4 Measurement of Melanin Inhibition Effect

In order to confirm the whitening effect of the Wenzhou citrus peel extract and its fractions obtained in Examples 1 to 3 was confirmed the degree of inhibition of melanin production for B16F1 melanocytes.

B16F1 melanocytes used in this example are cell strains derived from mice, and cells that secrete a black pigment called melanin. Samples were treated during the artificial culture of these cells to compare and evaluate the degree of reduction of melanin pigment. The B16F1 melanocytes used in this Experimental Example were used after being sold from ATCC (American Type Culture Collection, Accession No .: 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were dispensed in 6 well plates at a concentration of 2 × 10 ⁶ per well, cells were attached, and the samples were incubated for 72 hours by treating the samples at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, and the cells were counted and centrifuged to recover the cells. Quantification of intracellular melanin was carried out with a slight modification of the method of Rotan: Cancer Res. , 40: 3345-3350, 1980. The cell pellet was washed once with PBS, and then 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt the cells. Melt the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), measure the absorbance of melanin at 405 nm with a microplate reader, and then quantify the melanin in the sample. Percent inhibition of production was measured.

The melanin production inhibition rate (%) of B16F1 melanocytes was calculated by the following equation, and the melanin production inhibition rate of the sample as a concentration value (IC ₅₀ value) of 50% inhibiting melanin production is shown in Table 4 below. It was.

% Inhibition = [(A-B) / A] × 100

A: Melanin amount in wells without sample

B: Melanin amount in the wells to which the sample was added

Melanin production inhibition rate Sample Name Melanin synthesis inhibitory effect (IC ₅₀ ) Extract of Example 1 0.55% Fraction of Example 2 0.23% Fractions of Example 3 0.81% Arbutin 0.46% Soluble Licorice Extract 0.04%

As a result of testing the melanin production inhibitory effect of B16F1 melanocytes, as shown in Table 4, the IC ₅₀ value of the fraction of the solvent layer of the strong polarity of Example 2 was 0.23%, which is weaker than the conventionally known whitening agent useful licorice extract. However, it showed a better effect than arbutin. However, the IC ₅₀ value of the extract of Example 1 was 0.55% and the IC ₅₀ value of the fraction of Example 3 was 0.81%, indicating a lower melanin production inhibitory effect than arbutin.

That is, it can be seen that the extract of citrus peel of the glutinous asset of Example 2 was significantly improved in the whitening activity.

Experimental Example 5 UV Protection Effect

The UV protection effect of the citrus peel extract and its fractions obtained in Examples 1 to 3 was evaluated by measuring the MMP-1 concentration by enzyme immunoassay (ELISA) after UV irradiation and sample addition.

Human dermal fibroblasts were irradiated with UVA at an energy of 6.3 J / cm 2 using a UV chamber. UV irradiation dose and incubation time established conditions to maximize MMP expression in fibroblasts through preliminary experiments. Negative controls were wrapped in tinfoil to maintain the same time in the UVA environment. UVA emission was measured using a UV radiometer. The cells during the UVA irradiation were intact in the previously dispensed medium and irradiated with UVA and then exchanged with the medium to which the sample was added. The primary antibody (MMP-1 (Ab-5) monoclonal antibody) was treated and reacted at 37 ° C. for 60 minutes. After reacting the secondary antibody anti mouse IgG (whole mouse, alkaline phosphatase conjugated) for about 60 minutes, the alkaline phosphatase substrate solution (1 mg / ml ρ-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes. The reaction was carried out for a minute and the absorbance was measured at 405 nm with a microplate reader, and is shown in Table 5 below. As a control, one without addition of a sample was used.

Sample Name content MMP-1 expression inhibition rate Control group - - Extract of Example 1 0.1 wt% 52.6% Extract of Example 1 0.02 wt% 15.7% Fraction of Example 2 0.1 wt% 64.8% Fraction of Example 2 0.02 wt% 22.3% Fractions of Example 3 0.02 wt% 12.9% Retinol 0.005% by weight 38%

As can be seen in Table 5, the fraction of the citrus peel extract of Example 1 and the solvent layer of Example 2 having a strong polarity compared to the control group did not process the sample for MMP-1, which is induced during ultraviolet irradiation, is 0.1 wt%. Inhibition rate of more than 50% in the concentration was relatively low compared to the retinol used as a control, but showed a very good result as a natural extract. However, the fraction of the solvent layer of weak polarity of Example 3 was able to experiment at a concentration of 0.025% by weight or less due to high solubility and cytotoxicity, it was confirmed that the inhibitory activity is also very low.

<Experiment 6> Comparison test of molar asset content

The molar content of the Wenzhou Mandarin Peel Extract obtained in Examples 1 and 2 and the fraction of its polar solvent layer was analyzed by the following method using HPLC.

The analysis equipment used Waters Alliance 2695, reverse phase C-18 column (Xterra), and guard column, and methanol and purified water were separated by concentration gradient conditions as a mobile phase solvent. In addition, the attraction of the peak was improved by adding a pH 4.75 acetate buffer. The development conditions are a flow rate of 0.8 mL per minute, the initial mobile phase conditions are 75% purified water, 20% methanol and 5% acetic acid buffer, and developed under the same conditions for the first 5 minutes, 15% purified water, the final mobile phase solvent composition, 80% methanol To give a concentration gradient of 5% acetic acid buffer, 60 minutes was separated. Flavonoid content was analyzed by the area of the peak measured by fixing the absorbance at 280 nm using a Waters 2695 PDA (photodiode array) detector.

First, FIG. 1 shows a result of measuring a mixture of standard materials targeting a plurality of flavonoids. As a result, the informal assets were detected with a delay of about 7.8 minutes. As a result of confirming the citrus peel extract and its fractions of Examples 1 and 2 on the basis of these results, as shown in Figure 2, the Wenzhou mandarin peel extract of Example 1 is that the content of various flavonoids including a glutinous asset is very low level On the other hand, as can be seen from the results of FIG. 3, the strong polar solvent layer of Example 2 was found to contain a high content of a glutinous asset near the delay time of about 7.8 minutes.

That is, the fraction of the solvent layer of the strong polarity of Example 2 can be seen that the content of the molar asset significantly compared to the citrus peel extract of Example 1.

Experimental Example 7 Skin Elasticity Improvement Effect and Skin Wrinkle Improvement Effect Experiment

A cosmetic composition containing a fraction of the extract of the high content Wenzhou citrus rind obtained in Example 2 was prepared (cosmetic composition of Preparation) to evaluate skin elasticity improvement effect and wrinkle improvement effect in humans. At this time, for the comparative experiments, a cosmetic composition (the cosmetic composition of Comparative Example) that does not contain the fraction was used as a comparative example.

Cosmetic compositions of Preparation Examples and Comparative Examples are in the form of a cream, the composition is shown in Table 6 below.

First, the phase b) recorded in Table 6 is heated and stored at 70 ° C. A) phase is added thereto, followed by pre-emulsification, which is then uniformly emulsified with a homomixer, and then gradually cooled to prepare a cosmetic composition in the form of a cream. Twenty test subjects (20-35 year old female) were coated with the cream of the preparation on the right side of the face and the cream of the comparative example on the left side of the face, twice a day for 2 consecutive months. After the experiment was completed, the skin elasticity improvement effect was measured using a skin elasticity measuring instrument (cutometer SEM 575, C + K Electronic Co., Germany) before and after using the product for 2 months. The experimental results are described in Table 7 below as the ΔR7 value of the cutometer SEM 575, where the R7 value represents the viscoelasticity of the skin.

Component Composition and Component Content (wt%) of Cosmetic Composition Production Example  Comparative example



end Stearyl alcohol 8 8 Stearic acid 2 2 Stearic cholesterol 2 2 Squalane 4 4 2-octyldodecyl alcohol 6 6 Polyoxyethylene (25 mol addition) alcohol ester 3 3 Glyceryl monostearic acid ester 2 2
I Fraction of Example 2 One - Propylene glycol 5 5 Quantity Quantity Quantity

Skin elasticity improvement effect Experimental products Skin elasticity effect (△ R7) Production Example 0.29 Comparative example 0.14  * n = 20, p <0.05

The results of Table 7 show that the cream containing a fraction of the high content Wenzhou mandarin peel skin extract of Example 2 has a high skin elasticity improving effect.

On the other hand, the experiment of the wrinkle improvement effect was also performed with the cosmetic composition of the preparation example and the cosmetic composition of the comparative example.

For 20 experimenters (20-35 year old female), the cream of the preparation was applied to the right side of the face and the cream of the comparative example was applied to the left side of the face twice a day for 2 consecutive months. After completion of the experiment, skin wrinkle improvement effect is made of silicone replica to make skin wrinkle improvement effect before and after using the product for 2 months. SV60, C + K Electronic Co., Germany).

The results are shown in Table 8 below as the average of subtracting the parameter value two months ago from each parameter value two months later. This value means that the negative number, the higher the wrinkle improvement effect.

Experimental product R1 R2 R3 Production Example -0.29 -0.24 -0.13 Comparative example -0.14 -0.11 -0.05 R1: difference between the highest and lowest of the wrinkle contour
R2: Average of R1 values after randomly dividing the wrinkle contour by 5 spaces
R3: Highest value of R1 divided by 5

The results in Table 8 show that the skin wrinkle improvement effect of the cream containing the fraction of Example 2 was very high compared to the cream without it.

1 shows the results of HPLC analysis of a mixture of flavonoid standards.

Figure 2 is a HPLC analysis of the Wenzhou mandarin peel extract (extract of Example 1).

FIG. 3 shows the results of HPLC analysis of a fraction of the Wenzhou Mandarin Peel Extract (fraction of Example 2).

Claims (11)

(a) has antioxidant activity, whitening activity, sunscreen activity and skin wrinkle improvement activity, (b) a polar solvent selected from the group consisting of water, alcohols having 1 to 5 carbon atoms and mixed solvents thereof, by dipping the Wenzhou mandarin peel into water, alcohols having 1 to 5 carbon atoms or mixed solvents thereof; n-hexane, methylene chloride, chloroform and the like obtained in the polar solvent when fractionated with a weak polar solvent selected from the group consisting of Fraction of Wenzhou Mandarin Skin Extract. (a) has antioxidant activity, whitening activity, sunscreen activity and skin wrinkle improvement activity, (b) When the extract obtained by dipping Wenzhou mandarin peel in a mixed solvent of water and ethanol was partitioned into a mixed solvent of water, methanol and ethanol, and a mixed solvent of n-hexane, methylene chloride and chloroform, Obtained in the mixed solvent layer Fraction of Wenzhou Mandarin Skin Extract. (a) has antioxidant activity, whitening activity, sunscreen activity and skin wrinkle improvement activity, (b) Wenzhou Mandarin Peel Extract Powder obtained by dipping Wenzhou Mandarin Peel in 80% Ethanol and then removing the extraction solvent was fractionated into a mixed solvent of water, methanol and ethanol, and a mixed solvent of n-hexane, methylene chloride and chloroform. When obtained in a mixed solvent layer of water, methanol and ethanol Fraction of Wenzhou Mandarin Skin Extract. An antioxidant composition comprising the fraction of the Wenzhou mandarin peel extract of any one of claims 1 to 3 as an active ingredient. The method of claim 4, wherein The composition is an antioxidant composition, characterized in that the cosmetic composition. Skin whitening agent composition comprising a fraction of the Wenzhou mandarin peel extract of any one of claims 1 to 3 as an active ingredient. The method of claim 6, The composition is a skin lightening composition, characterized in that the cosmetic composition. A sunscreen composition comprising the fraction of the Wenzhou mandarin peel extract of any one of claims 1 to 3 as an active ingredient. The method of claim 8, The composition is a skin lightening composition, characterized in that the cosmetic composition. A skin wrinkle improver composition comprising a fraction of the Wenzhou mandarin peel extract of any one of claims 1 to 3 as an active ingredient. The method of claim 10, The composition is a skin wrinkle improver composition, characterized in that the cosmetic composition.

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