KR101726814B1 - Composition comprising Phyllospadix iwatensis Makino extract - Google Patents

Abstract

The present invention relates to a composition containing an extract of Phyllospadix iwatensis Makino as an active ingredient, and more particularly to a pharmacological, food and cosmetic composition containing a shrimp extract or a fraction thereof as an active ingredient, will be. The shrimp horsetail extract of the present invention is safe for skin, has remarkable inhibitory effect on human tyrosinase activity, and thus has excellent melanin production inhibition and skin whitening effect.

Description

Composition comprising Phyllospadix iwatensis Makino extract as an active ingredient

The present invention relates to a composition containing an extract of Phyllospadix iwatensis Makino as an active ingredient, and more particularly to a pharmacological, food and cosmetic composition containing a shrimp extract or a fraction thereof as an active ingredient, will be.

In general, hyperpigmentation of the skin is caused by internal factors that occur in the body, such as hormonal abnormalities, hereditary diseases, and the like, as well as by external factors such as excessive melanin pigmentation and distribution due to excessive ultraviolet irradiation . The proper amount of melanin pigment present on the skin has a positive aspect, such as keeping the skin healthy and absorbing ultraviolet rays (UV), but excessive melanin pigment shows black skin, do. In addition, since the excessive production of melanin causes pigmented skin diseases such as black skin, spots, and freckles, research on whitening ingredients has been started to improve and treat the above diseases.

Melanin, a major factor in pigmentation of the skin, is synthesized through several stages of oxidation in melanosomes, the intracellular organelles of cells called melanocytes, which are present in the epidermis of the skin. It is a phenomenon that an enzyme called tyrosinase acts and a series of oxidation process is followed to overproduce a polymer called melanin and accumulate in the skin. The above tyrosinase is an enzyme that converts tyrosine, a kind of amino acid, into darkening (blackening) of skin which makes DOPA, 3,4-dihydro xyphenylalanine, DOPA-quinone and pheomelanin which are intermediate products of melanin polymer production It is the most important enzyme in the process. As described above, the action of tyrosinase is absolutely necessary in the process of producing melanin. Accordingly, melanin production can be regulated by inhibiting the expression of the enzyme protein or by inhibiting the activity of the enzyme expressed by ultraviolet rays, and a search for functional materials having such an effect has been made.

[Non-Patent Document 1] MIJIN KIM, SANG MI AN, JAE-SOOK KOH, DONG-IL JANG, and YONG CHOOL BOO, Use of non-melanocytic HEK293 cells Stably expressing human tyrosinase for screening of anti- Sci. 2011 Sep-Oct; 62 (5): 515-23.

Accordingly, the inventors of the present invention confirmed that the extract of Phyllospadix iwatensis Makino, a marine plant, inhibited the activity of human tyrosinase and markedly inhibited the production of melanin while studying a natural material having an effect of inhibiting melanin production. Respectively.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating a skin pigmentation disease caused by excessive accumulation of melanin containing an extract of shrimp or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or ameliorating skin pigment diseases caused by excessive accumulation of melanin containing an extract of shrimp or a fraction thereof as an active ingredient.

Another object of the present invention is to provide an extract of Phyllospadix iwatensis Makino or a fraction thereof; And a cosmetically acceptable excipient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating a skin pigmentation disease caused by excessive accumulation of melanin containing an extract of P. shrimp or a fraction thereof as an active ingredient.

In order to accomplish still another object of the present invention, the present invention provides a food composition for preventing or ameliorating a skin pigmentation disease caused by excessive accumulation of melanin containing an extract of P. shrimp or a fraction thereof as an active ingredient.

In order to accomplish still another object of the present invention, the present invention relates to a method for producing an extract of Phyllospadix iwatensis Makino or a fraction thereof; And a cosmetically acceptable excipient.

Hereinafter, the present invention will be described in detail.

The shrimp horsetail extract of the present invention has remarkable inhibitory effect on human tyrosinase activity, and thus has excellent melanin production inhibition and skin whitening effect. A novel use of such a shrimp horse extract is disclosed first in the present invention.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating skin pigmentation diseases caused by excessive accumulation of melanin containing an extract of Phyllospadix iwatensis Makino or a fraction thereof as an active ingredient.

The shrimp ( Phyllospadix iwatensis Makino) is a perennial seaweed of Zosteraceae. It is a perennial seaweed distributed mainly in temperate regions such as Korea, Japan, and China. It grows in deep rocks of depths of 1-8m. The plants crawl to the bottom of the ground and the nodes are short and tight. Leaves are rounded with a length of 20-100cm and a butterfly of 2-4.5㎜. Three veins extend vertically, with a serrate on the upper edge, and a leaflet on the base. The flower blooms in March and is wrapped in a pod at the bottom of the ground. It is about 10cm long. Flower stem is about 3cm long. The female flower as a male-female dioecious is made into one ovary without a small flower stalk, and the male flower has no flower stalk and has one flower-shaped pouch. Fruit is heart-shaped, length and butterfly are about 4mm and both ends are narrow. When you see the buds of flowers from the side, it is called shrimp because it is similar to shrimp.

In the present invention, the shrimp can be used as an extract material in any part constituting the shrimp, including, but not limited to, whole herb of shrimp; Or one or more selected from the group consisting of fruit, leaves, flowers, stems and roots, and preferably using the roots, stems and leaves of shrimp.

The shrimp horsetail extract of the present invention can be extracted by a known natural product extraction method. The kind of the extraction solvent used in the extraction is not particularly limited as long as it is known in the art as a plant component extraction solvent, Water, lower alcohols having 1 to 6 carbon atoms (such as methanol, ethanol, propanol, butanol, pentanol, hexanol, glycerin, propylene glycol and 1,3-butylene glycol) , Acetone, methyl ethyl ketone, etc.), lower alkyl esters having 1 to 4 carbon atoms (such as ethyl acetate and butyl acetate), subcritical fluids, and supercritical fluids, More preferably at least one selected from the group consisting of water, lower alcohols having 1 to 6 carbon atoms, acetone, ethyl acetate, subcritical fluid and supercritical fluid And may be extracted by a tag. Most preferably, one selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms (particularly, methanol, ethanol, propanol, butanol), and mixtures thereof.

The shrimp horse extract may include a pretreatment step to increase the extraction efficiency. For example, the shrimp horse may be dried and pulverized by a grinder.

The extraction temperature of the extract of the present invention is not particularly limited, and may be, for example, from 0 ° C to 150 ° C, preferably from 10 ° C to 80 ° C. The extraction time of the extract of the present invention is not particularly limited as long as it can be appropriately set and changed by a person skilled in the art according to the extraction temperature. For example, it may be 10 minutes to 10 days, preferably 1 hour to 72 hours, Can be from 3 hours to 60 hours.

The extract of the present invention can be extracted by a known natural substance extraction method. For example, it can be extracted by cold extraction, normal temperature extraction, hot water extraction, reflux cooling extraction, heat extraction, high pressure extraction, ultrasonic extraction, subcritical extraction, supercritical extraction, It can be repeatedly extracted seven times or seven times.

In order to facilitate the processing, storage and the like in the production of the shrimp horsetail extract of the present invention, a filtration process, a concentration and purification process, a drying process, a freezing process, and a pulverization process may be arbitrarily added.

The filtration process may be performed by a known filtration method. For example, filtration using a filter net or a microfilter, centrifugal separation and separation funnel may be used.

The concentration process may be performed by a conventional concentration process. For example, the concentration process may be performed using precipitation concentration, evaporation concentration, reduced pressure concentration, ultrafiltration, reverse osmosis, and centrifugation.

The drying may be performed by a known drying method, but not limited thereto, for example, freeze drying, spray drying or hot air drying.

The pulverization process may be performed by a known pulverization method and is not limited thereto. For example, it may be pulverized by a process such as dextrin inclusion.

In the present invention, the fractions can be obtained by fractionating or purifying a substance having an activity of interest in the present invention from the shrimp extract (especially, crude extract) using a specific solvent ). For example, fractions obtained by further fractionating the shrimp extract with a polar or nonpolar solvent, fractions obtained by passing through an ultrafiltration membrane having a constant molecular weight cut-off value, various chromatographies (separation by size, charge, hydrophobicity or affinity For example, by fractionation by means of a variety of additional purification methods, including, but not limited to,

In the present invention, the fraction may include all of the non-polar solvent fraction of the shrimp extract or the polar solvent fraction of the shrimp extract. Preferably, the fraction in the present invention may be a polar solvent fraction of a shrimp extract, and the type of said polar solvent is well known in the art.

Most preferably, the polar solvent fraction of the shrimp horse extract is obtained by sequentially fractionating the shrimp extract with water and a non-polar solvent, and then fractionating the water fraction with a polar solvent of butanol or ethyl acetate. At this time, the nonpolar solvent may be at least one solvent selected from the group consisting of n-hexane, dichloromethane and ethyl ether.

The fraction may be optionally further added with a filtration process, a concentration and purification process, a drying process, a freezing process, and a pulverization process for the sake of ease of treatment, storage, etc., as described above.

The skin pigmentation disease is caused by excessive accumulation of melanin and is not particularly limited as long as it is known in the art. For example, there are hereditary dysplasia (hereditary dyschromatosis), reticular dysplasia Pigmentation caused by pigmentation, spots, freckles, black spots, nevi, drugs (eg, drugs selected from minocycline, bleomycin, vaginal plate or zidovudine), post inflammatory pigmentation, It has been shown to be effective in the treatment of chronic skin diseases such as epidermal melanocytic lesions, caffeine au lait macules, dermal melanocytic lesions, Mongolian spot, surplus Lentigines, Melanoma, Lentigo maligna melanoma, Superficial spreading melanoma, Acral lentiginous melanoma, The present invention relates to a method for the treatment of nodular melanoma, pigment basal cell carcinoma, dermatofibromas, dermoid cysts, pigmented keloids and keratoacanthomas ), And the like. Specifically, the nevus is composed of a flattened nest, pigmented moths, Becker's nevus, Nevus spilus, Nevus of Ota, acquired bilateral otalbia sheep (Nevus of Ito), Blue nevus, Melanocytic nevus, Junctional nevus, Compound nevus, and the like. Intradermal nevus, Halo nevus, Congenital nevocytic nevus, Spitz nevus, and Dysplastic nevus, among others.

Preferably, the skin pigment diseases caused by the excessive accumulation of melanin according to the present invention are selected from the group consisting of hereditary dystrophy, dysentery, , Pigmentation by nevus, drugs (e.g., drugs selected from minocycline, bleomycin, vesicol, or zidovudine), post-inflammatory pigmentation, and hyperpigmentation resulting from dermatitis.

 The pharmaceutical composition of the present invention may contain the shrimp extract or its fraction alone, or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. The carrier for parenteral administration may also contain water, suitable oils, saline, aqueous glucose and glycols, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI >

The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the route of administration as described above.

In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

 In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.

The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses . The pharmaceutical composition of the present invention may vary in the content of the active ingredient depending on the degree of the disease. When administered parenterally, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per 1 kg of body weight per day on the basis of the shrimp horse extract or its fraction, and when administered orally, May be administered in one to several divided doses so as to be administered in an amount of preferably 0.01 to 100 mg, more preferably 0.1 to 50 mg per kg of body weight per day on the basis of the extract or fraction thereof. However, the dose of the shrimp hull extract or its fraction may be varied depending on various factors such as a patient's age, body weight, health status, sex, severity of disease, diet and excretion rate as well as route and frequency of administration of the pharmaceutical composition, In view of this point, one of ordinary skill in the art will be able to determine the appropriate effective dose of the composition of the present invention, since an effective dose will be determined. The pharmaceutical composition according to the present invention is not particularly limited to the formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The shrimp extract or its fractions of the present invention may be provided as a food composition for preventing or improving skin pigment diseases caused by excessive accumulation of melanin by containing it as an effective ingredient. The food composition according to the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.

For example, as a health food, the shrimp horsetail extract or fraction itself of the present invention may be prepared in the form of tea, juice, and drink and then taken for drinking, granulated, encapsulated and powdered. In addition, the shrimp extract or fraction of the present invention can be prepared in the form of a composition by mixing it with a known substance or active ingredient known to have a whitening effect.

Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled, jam, maalmalade, etc.), fish, meat and processed foods such as ham, Etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, , Retort food, frozen food, various kinds of seasonings (for example, soybean paste, soy sauce, sauce, etc.) by adding the shrimp horse extract or fraction of the present invention.

The preferred content of the shrimp extract or fraction of the present invention in the food composition of the present invention is not limited thereto, but may be, for example, 0.01 to 80% by weight of the finally prepared food, 0.01 to 50% by weight.

In addition, in order to use the shrimp extract or fraction of the present invention in the form of a food additive, it may be used in the form of powder or concentrate.

The present invention relates to a shrimp ( Phyllospadix iwatensis Makino) extract or a fraction thereof; And a cosmetically acceptable excipient. The cosmetic composition of the present invention is not limited to a specific use, but may be preferably used for skin whitening.

The 'whitening' is a function to prevent excessive deposition of melanin pigment in the skin or to remove or reduce the melanin pigment deposited in the skin. In the present invention, the effect of preventing or improving skin pigment diseases caused by excessive accumulation of melanin May be inclusive. The skin pigmentation diseases caused by excessive accumulation of melanin are as described above.

The cosmetic composition according to the present invention may further comprise cosmetically and / or dermatologically acceptable excipients as an active ingredient in the shrimp extract of the present invention or a fraction thereof, so that it can be manufactured in any form suitable for topical application . ≪ / RTI > For example, but not by way of limitation, the cosmetic composition according to the present invention may be in the form of a solution, a gel, a solid, an emulsion, a suspension, a microemulsion, a microcapsule, a microgranule, an ionic and / Lotion, powder, spray or stick. Specific examples of the classification according to the purpose of use include basic cosmetic compositions (cleansers, packs, body oils such as lotions, essences, creams, essences, cleansing foams and cleansing water), color cosmetic compositions (foundation, lipstick, mascara , Make-up base), hair product composition (shampoo, conditioner, hair conditioner, hair gel) and soap.

The content of the shrimp extract or its fraction contained in the cosmetic composition of the present invention is not limited thereto, but is preferably 0.01 to 50% by weight, more preferably 0.1 to 30% by weight based on the total weight of the whole composition.

The cosmetic composition of the present invention includes dermatologically (or cosmetically, cosmetically) acceptable excipients which act as diluents, dispersants or carriers for the active ingredient. The excipient may be selected from materials conventionally used in skin care products such as water, liquid or solid softener (skin softening agent), silicone oil, emulsifier, solvent, wetting agent, thickener, powder, propellant, skin permeation enhancer, colorant, . ≪ / RTI >

Specifically, the skin softening agent is used for softening, soothing, coating, smoothing, or moisturizing the skin. The emollient may be any kind known in the art including, but not limited to, a petroleum base, a fatty acid ester, an alkyl ethoxylate, a fatty acid ester ethoxylate, a fatty alcohol, a polysiloxane, or a mixture thereof Lt; / RTI > In addition to these, it is also possible to use other fatty acid esters such as propylene glycol, butylene glycol, glycerin, triethylene glycol, wax, wax, fatty acid, fatty alcohol ether, glyceride, acetoglyceride, ethoxylated glycerides, other fatty acid esters of polyhydroxy alcohol, And the like. The emulsifier serves to mix the aqueous phase of the cosmetic composition with the water phase. The emulsifier may optionally contain one or more kinds of nonionic, anionic or cationic surfactants. The kind of the emulsifier can be determined according to whether the emulsion is in a water dispersion type or an oil-water dispersion type. Examples of suitable emulsifying agents include, but are not limited to, sorbitan trioleate, sorbitan tristearate, glycerol monooleate, glycerol monostearate, glycerol monolaurate, sorbitan sesquioleate, sorbitan monooleate, Sorbitan monostearate, polyoxyethylene stearyl ether, polyoxyethylene sorbitol beeswax derivatives, PEG 200 dilaurate, PEG 200 monostearate, PEG 400 diolate, sorbitan monopalmitate, polyoxyethylene monostearate and Polyoxyethylene sorbitan monostearate, and the like. The thickener includes, but is not limited to, crosslinked carboxypolymethylene polymer, ethylcellulose, polyethylene glycol, tragacanth gum, karaya gum, xanthan gum, bentonite, hydroxyethylcellulose and hydroxypropylcellulose. As the solvent, purified water, alcohol, or a mixture of purified water and alcohol may be used. Other examples of common cosmetic additives that may optionally be used include, but are not limited to, preservatives such as para-hydroxybenzoate esters, antioxidants such as butylhydroxytoluene, ascorbic acid and its derivatives and tocopherol and its derivatives, glycerol, Wetting agents such as sorbitol, 2-pyrrolidone-5-carboxylate, dibutyl phthalate, gelatin, polyethylene glycol, pH buffers such as acetate, phosphate, citrate, triethanolamine and carbonate, waxes such as waxes, paraffin Thickeners, activity enhancers, coloring agents and perfumes may be used.

As described above, the cosmetic composition of the present invention may further contain a perfume, a coloring agent, a bactericide, an antioxidant, an antiseptic, a moisturizing agent, and the like, and may contain a thickening agent, an inorganic salt, a synthetic polymer material and the like for the purpose of improving physical properties. For example, when a cleanser and a soap are prepared using the cosmetic composition of the present invention, the shrimp extract or its fraction can be easily added to a common cleanser and a soap base. In the case of producing a cream, the shrimp horse extract or its fraction may be added to a cream base of a typical underwater type (O / W). A synthetic or natural material such as a flavor, a chelating agent, a coloring matter, an antioxidant, an antiseptic, and a protein, a mineral, and a vitamin for the purpose of improving a physical property may be further added.

In addition, the cosmetic composition according to the present invention may contain, in addition to the shrimp extract or its fractions, other ingredients capable of elevating or assisting the main effect of the present invention. For example, arbutin, niacinamide, ascorbic acid derivatives, resveratrol derivatives, and para-coumarinic acid derivatives, which are conventionally known to have whitening action.

In the present invention, the excipient can usually constitute the cosmetic composition of the present invention as a remaining amount other than the above-mentioned active ingredient, if no other cosmetic auxiliary agent (auxiliary cosmetic material) is used.

The shrimp horsetail extract of the present invention is safe for skin, has remarkable inhibitory effect on human tyrosinase activity, and thus has excellent melanin production inhibition and skin whitening effect.

FIG. 1 shows the results of measuring the change of human tyrosinase activity according to the treatment concentration (0.025 to 0.1%) in the shrimp horse extract treatment of the present invention.
FIG. 2 shows the results of measuring the inhibitory activity of human tyrosinase against the non-polar solvent fraction or the polar solvent fraction obtained during the production of the shrimp active fraction of the present invention.

Hereinafter, the present invention will be described in detail.

However, the following Preparation Examples and Examples are illustrative of the present invention, and the content of the present invention is not limited to the following Production Examples and Examples.

≪ Preparation Example 1 &

Preparation of shrimp horse extract

The roots, stems and leaves of the marine plant shrimp ( Phyllospadix iwatensis Makino) were washed with purified water more than 3 times and completely dried at 50 ° C for 24 hours or more using a hot air drier and then pulverized. An aqueous 80% (v / v) ethanol solution was added at a ratio of about 10 times the weight of the shrimp ground powder, and the mixture was stirred at room temperature for 48 hours to prepare an extract. This was concentrated under reduced pressure at 50 캜 or below and lyophilized to prepare a shrimp extract powder. At this time, the yield was 15.5%.

<Manufacturing Comparative Examples 1 to 49>

 Manufacture of marine plant extracts

The roots, stems and leaves of various marine plants described in Table 1 below were treated according to the method of Preparation Example 1 to prepare extracts (powders) of each marine plants.

Experimental Example 1. Measurement of human tyrosinase inhibitory action

(1) Acquisition of human tyrosinase

Normal human melanocytes in the laboratory are not only difficult to perform accurate experiments because the melanin production is decreased and the survival rate is lowered when incubated for a long time and the cost for culturing is higher than that of normal cells. In order to overcome this disadvantage, human embryonic kidney cells (HEK293-TYR), which permanently express human tyrosinase by permanent injection of human tyrosinase gene into human embryonic kidney cells (HEK293 cell) Was used for the experiment.

Briefly, human embryonic kidney cells (HEK 293 cells) were purchased from the American Type Culture Collection (USA) and were purchased from MIJIN KIM et al., 2011 HEK293-TYR cells were prepared as described. The cultured HEK293-TYR cells were suspended in a cell lysis solvent (10 mM Tris-Cl, pH 7.4, 120 mM NaCl, 25 mM KCl, 2.0 mM EGTA, 1.0 mM EDTA, and 0.5% Triton X- To obtain a cell lysate. The cell lysate was then centrifuged at 13,000 x g for 15 minutes, and the supernatant was used as a human thylosinase cell extract.

(2) Comparison of human tyrosinase inhibitory activity of 50 marine plants

Preparation Example 1 and Preparation Comparative Example 1-49 To measure the effect of the marine plant extract on human tyrosinase, 0.1 M phosphate buffer (pH 6.8) was added to a 96-well microplate, The reaction was carried out at 37 ° C for 120 minutes so that the reaction solution containing 200 μg / mL of human thylosinase cell extract, 0.5 mM L-tyrosine, 1 μM L-dopa (DOPA) and 0.1% of each marine plant extract (powder) . Using a microplate reader, the absorbance of the reaction product DOPA chrome was measured at an absorption wavelength of 490 nm and the inhibition rate was calculated to determine the ability of each marine plant extract to inhibit melanin formation and skin whitening ability Respectively. As a positive control, arbutin, a representative material commonly used for skin whitening effect, was used. The inhibitory activity of tyrosinase activity of each of the marine plant extracts of Comparative Examples 1 to 49 and shrimp extract obtained according to Preparation Example 1 was calculated and shown in Table 1 below. As a negative control, an aqueous 80% (v / v) ethanol solution was used instead of the plant extract.

The effects of the marine plant extracts on mushroom tyrosinase (Sigma-Aldrich) were evaluated in the same manner as in Preparation Example 1 and Comparative Preparation Example 1-49, and are shown in Table 1 as well.

Inhibition rate of tyrosinase activity (%)

= [(ODblank-ODblank con.) - (ODexp.-ODcon.)] / (ODblank-ODblank con.) X 100

ODexp. : Measured value of sample when containing tyrosinase

ODcon. : Measured value of sample not containing tyrosinase

ODblank: Measurement value of barium when containing thiocyanate

ODblank con. : Measurement value of blanching test not containing tyrosinase

Comparison of inhibitory activity of human tyrosinase activity of shrimp horses and other marine plant extracts NO. And name
Country name
Academic name
human
Inhibition rate of tyrosinase activity
(%) mushroom
Inhibition rate of tyrosinase activity
(%) Positive control group Arbutin 35.6 10.2 Manufacturing example One Leeches Shrimp Phyllospadix iwatensis Makino 39.7 -50.43 Manufacturing Comparative Example One Bora leaves and Pressed pink leaf Acrosorium yendoi Yamada 11.2 -55.43 Manufacturing Comparative Example 2 Kelp Yakisiridaki seaweed Agarum cribrosum -113.5 -67.28 Manufacturing Comparative Example 3 Coral horse Wide gap Amphiroa anceps (Lamarck) Decaisne -2.0 -20.43 Manufacturing Comparative Example 4 With a hook Sea ring pool Asparagopsis taxiformis ( Delila ) Trevisan -1.7 -77.72 Manufacturing Comparative Example 5 With a hook Chaff Bonnemaisonia hamifera Hariot -15.4 -13.59 Manufacturing Comparative Example 6 Red rashes Crested red leaf Callophylis japonica Okamura in DeToni & Okamura -10.8 -26.96 Manufacturing Comparative Example 7 Jinuari Wrinkled red leaf Callophyllis crispata Okamura -5.4 -5.11 Manufacturing Comparative Example 8 Jinuari Black-eyed Carpopeltis affinis (Harvey) Okamura -17.7 -12.07 Manufacturing Comparative Example 9 Chain grass True Chain Pool Champia parvula (C. Agardh) Harvey -9.2 -21.41 Manufacturing Comparative Example 10 Stone scarf Stone bracelet Chondracanthus tenellus (Harvey) Homm . in Homm. -26.0 -72.5 Manufacturing Comparative Example 11 Red-nosed Seosil Chondria crassicaulis Harvey -14.5 -10.43 Manufacturing Comparative Example 12 Stone scarf Jin Duc Chondrus ocellatus Holmes -25.2 -16.3 Manufacturing Comparative Example 13 And Brown vs. Mare Cladophora wrightiana Harvey -9.4 -31.74 Manufacturing Comparative Example 14 Hearing ear Codium fragile (Suringar) Hariot -9.7 -15.54 Manufacturing Comparative Example 15 Kori Mae Flame Term Colpomenia sinusa (Mertens ex Roth) Derbes et Solier in Castagne -18.5 -22.07 Manufacturing Comparative Example 16 Kelp Swordfish Costaria costata (C. Agardh) Saunders -16.0 -39.78 Manufacturing Comparative Example 17 Mt. Tobacco leaves Desmarestia tabacoides Okamura -11.2 -43.26 Manufacturing Comparative Example 18 The net base horse and True net Dictyoperis dichotoma (Hudson) Lamouroux -12.3 -37.93 Manufacturing Comparative Example 19 The net base horse and Gag water base horse Dictyota okamurae (Dawson) H? Tning, Schnetter, et Prud'homme van Reine -2.2 -96.85 Manufacturing Comparative Example 20 Dictio Duck fan Distromium decumbens -79.1 96.52 Manufacturing Comparative Example 21 Kelp Moth Ecklonia cava Kjellman in Kjellman & Petersen -120.2 108.04 Manufacturing Comparative Example 22 Kelp rhubarb Eisenia bicyclis (Kjellman) Setchell -66.6 -5.43 Manufacturing Comparative Example 23 With scissors A scissors ruler Galaxaura falcata Kjellman -12.6 -32.39 Manufacturing Comparative Example 24 Myrtle Mugwort Gelidium amansii (Lam.) Lamouroux -6.0 -29.24 Manufacturing Comparative Example 25 Full grass Baby grass Gloiopeltis compianata (Harvey) Yamada -12.3 -22.61 Manufacturing Comparative Example 26 Cicada Croaker Gracilaria verrucosa (Hudson) Papenfuss -9.8 -24.78 Manufacturing Comparative Example 27 Jinuari Wrinkled Grateloupata crispata (Okamura) Lee -11.8 -29.13 Manufacturing Comparative Example 28 Jinuari True betting Grateloupia elliptica Holmes -10.5 -14.89 Manufacturing Comparative Example 29 Kori Mae Net basket Hydroclathrus clathratus (C. Agardh) Howe -11.8 -16.2 Manufacturing Comparative Example 30 Fruit tree branch Participation Hypnea charoides -12.3 -31.63 Manufacturing Comparative Example 31 Loss tile Ishige okamurae Yendo -33.7 30.54 Manufacturing Comparative Example 32 Red-nosed Black standing room Laurencia intermedia Lamouroux -12.6 -13.26 Manufacturing Comparative Example 33 A blurred horse Rock bank Lethesia difformis (Linnaeus) Areschoug -14.5 -21.85 Manufacturing Comparative Example 34 Madigail Baby Lomentaria hakodatensis Yendo -13.4 -20.65 Manufacturing Comparative Example 35 Bora leaves and Silk mesh Martensia denticulata Harvey -19.2 -105.11 Manufacturing Comparative Example 36 With mothballs An alien dog Myagropsis myagroides (Martens ex Turner) Fensholt -15.7 -33.91 Manufacturing Comparative Example 37 With a rocky whisker Rock beard Myelophycus simplex (Harvey) Papenfuss -16.5 -19.13 Manufacturing Comparative Example 38 The net base horse and Horse leather netting Pachydictyon coriaceum (Holmes) Okamura -19.8 -39.46 Manufacturing Comparative Example 39 The net base horse and Suburban Padina arborescens Holmes -37.8 -75.65 Manufacturing Comparative Example 40 Kori Mae Starfish Petalonia binghamiae (j.Agardh) Vinogradova -17.2 -31.85 Manufacturing Comparative Example 41 Curly science A true curl Plocamium telfairiae (Harvey) Harvey -8.8 -12.93 Manufacturing Comparative Example 42 Jinuari Redcurrant Prionitis cornea (Okamura) Dawson -17.5 -23.7 Manufacturing Comparative Example 43 Myrtle Dogwood Pterocladia capillacea (Gmelin) Bornet -34.9 -42.17 Manufacturing Comparative Example 44 With mothballs Top Sargassum fusiformis (Harvey) Okamura -10.3 -22.5 Manufacturing Comparative Example 45 Kori Mae Seaweed room Scytosiphon gracilis Kogame -17.1 -34.46 Manufacturing Comparative Example 46 Green thread Abstract room Ulothrix flacca (Dillwyn) Thuret in LeJolis 0.3 -17.39 Manufacturing Comparative Example 47 A cauldron Creeper Ulva fasciata Delile -5.7 -46.74 Manufacturing Comparative Example 48 Seaweed and Seaweed Undaria pinnatifida (Harvey) Suringar -9.1 -37.17 Manufacturing Comparative Example 49 Leeches and Leech Zostera Marina L. 1.2 -38.26

As a result, as shown in Table 1, the shrimp extract of Preparation Example 1 was superior to the marine plant extracts of Preparation Example 1-49 in inhibiting human tyrosinase. The extracts of Comparative Examples 1-49 largely lacked the inhibitory action of human tyrosinase, or rather promoted the activity of the enzyme, and the weak human thyrosinase inhibitory action was only observed in the extracts of pale yellow leaves and seaweed. Duck ferns, mung bean, and lobster strongly inhibited the mushroom tyrosinase but increased the activity of human tyrosinase. These results demonstrate that many of the existing studies have demonstrated melanogenesis inhibitory and whitening efficacy of certain substances based on the inhibitory effect of mushroom tyrosinase, but this attestation proves the precise biochemical efficacy of the substance on humans Suggesting that it is inappropriate to do so. The shrimp extract of Preparation Example 1 according to the present invention showed an excellent inhibitory effect on tyrosinase inhibitory activity at a slightly higher level than that of the positive control (arbutin), and it is expected that the extract would be more effective when purified there was.

(3) Identification of human tyrosinase inhibitory activity of shrimp horse extract

The shrimp extract of Preparation Example 1 was treated at various concentrations of 0.025 to 0.1% in the above human tyrosinase cell extract to confirm its effect. To determine the effect of human tyrosinase, a 96-well microplate was coated with 0.1 M phosphate buffer (pH 6.8), human tyrosinase cell extract 200 μg / mL, 0.5 mM L- Tyrosine, 1 μM L-dopa (DOPA) and the shrimp extract of Preparation Example 1 at various concentrations ranging from 0.025 to 0.1% were reacted at 37 ° C. for 120 minutes so as to be 200 μL. The degree of enzyme activity was evaluated by measuring the absorbance of the reaction product DOPA chrome at an absorption wavelength of 490 nm using a microplate reader. As a negative control, an aqueous 80% (v / v) ethanol solution was used instead of the plant extract.

As a result, as shown in FIG. 1, it was confirmed that the shrimp extract of the present invention inhibited human tyrosinase in a concentration-dependent manner.

&Lt; Production Examples 2 to 12 >

Preparation of shrimp horse extract

The roots, stems and leaves of the shrimp were washed with purified water more than 3 times and then completely dried at 50 ° C for 24 hours or more using a hot air drier and then pulverized. Each of the extraction solvents shown in Table 2 below was added at about 10 times as much as the weight of the shrimp ground product, and the mixture was stirred at room temperature for 48 hours to prepare an extract. The extract was concentrated under reduced pressure at 50 캜 or below and lyophilized to prepare an extract powder.

Experimental Example 2. Measurement of human thyrosinase inhibitory action of shrimp extract according to extraction solvent

The inhibition rates of human tyrosinase activity according to the method of Experimental Example 1 were measured and calculated for the shrimp extracts of Preparation Examples 2 to 12 as shown in Table 2 below.

Experimental Results As shown in Table 2, extraction efficiency and inhibition of human tyrosinase were excellent when water, ethanol, methanol, and acetone were used alone or in combination as an extraction solvent.

&Lt; Preparation Examples 13 to 18 &

Preparation of shrimp horse fractions

The shrimp hull extract of Preparation Example 1 was diluted with about 10 times the volume of water and was dispersed with the same volume of nonpolar solvent described in Table 3 to extract and remove the impurities. The water layer was further divided into the same volume To obtain a polar solvent fraction of the shrimp extract, by extracting the active ingredient. The concentrate was concentrated under reduced pressure at 50 ° C or below and lyophilized to prepare a shrimp fraction powder. (The term 'shrimp fraction' refers to the 'polar fraction of the polar solvent in the shrimp' unless otherwise stated).

Experimental Example 3. Determination of inhibitory effect of human tyrosinase on shrimp fractions by solvent

(1) First, the non-polar or polar solvent fraction obtained in each step in the shrimp horse shrimp production was evaluated for its ability to inhibit human tyrosinase activity. Typically, non-polar or polar solvent fractions obtained in the preparation of the fraction of Preparation Example 16 were used in this experiment. Briefly, the shrimp hull extract of Preparation Example 1 was diluted in water of about 10 times volume and sequentially dispersed with dichloromethane and butanol, and each was concentrated under reduced pressure and lyophilized to prepare a dichloromethane fraction, a butanol fraction and a water fraction . According to the method of Experimental Example 1, the three fractions were added to 0.05% , And human tyrosinase activity was measured.

As a result, as shown in Fig. 2, human tyrosinase activity was greatly reduced by the butanol fraction. These results suggest that the polar solvent fraction of shrimp contains a high concentration of whitening active ingredient as compared with the non-polar solvent fraction.

(2) Human thyrosinase activity was measured for the shrimp polar solvent fraction of Production Examples 13 to 18 according to the method of Test Example 1 above.

 Experimental Results As shown in Table 3, it was found that while the extract was purified, the impurities were removed and the yield was decreased while the inhibitory effect of human tyrosinase was improved.

Experimental Example 4 Measurement of Melanogenesis Inhibitory Effect in Cells

The inhibitory effect on the production of melanin of the shrimp horses of Preparation Examples 1 to 10 and the shrimp fractions of Preparation Examples 13 to 18 was measured in human epidermal melanocytes. The human epidermal melanocytes were purchased from Cascade Biologics (Portland, OR, USA) and cultured in Medium 254 supplemented with human melanocyte growth supplements (Cascade Biologics) and antibiotics.

First, the cell viability was evaluated by a known trypan blue exclusion assay. Briefly, each of the shrimp horsetail extracts or fractions was treated with cells at a concentration of 0.1% and cultured at 37 DEG C for 48 hours. Cells were trypsinized and then harvested by centrifugation at 1,200 rpm for 3 minutes. The cells were collectively suspended in the culture broth and mixed at a ratio of 1: 1 with 0.1% trypan blue solution (Sigma-Aldrich). The number of stained dead cells and non-stained living cells was counted microscopically using a hemocytometer.

To determine the effect of shrimp extracts and fractions on cellular pigmentation, the test substances were pretreated with 0.1% concentration of melanocytes for 60 minutes, followed by stimulation with 1.0 mM L-tyrosine for 48 hours . This process was repeated 3 times with medium exchange. Melanin was extracted from the cells by treatment with 0.1 M NaOH at 60 ° C for 60 minutes, and the absorbance at 490 nm was quantified and corrected for the amount of protein. The amount of protein was assessed by Bio-Rad DC analysis.

Experimental Results As shown in Table 4, the shrimp extracts of Preparative Examples 1 to 10 inhibited cell melanin production with almost the same or superior activity as arbutin, a known whitening substance, and showed almost no cytotoxicity. In addition, the shrimp fractions (polar solvent fractions) of Production Examples 13 to 18 exhibited superior cell melanogenesis inhibitory action and showed almost no cytotoxicity.

Examples 1 to 4 and Comparative Examples 1 and 2 Preparation of a whitening cosmetic comprising shrimp extract or its fractions

Each active ingredient was added to a cream base as described in Table 5 below to prepare each cosmetic containing the ingredients in Table 6 below.

Active ingredient in cosmetics for testing NO. Active ingredient Formulation ratio (%) Example 1 Shrimp horse extract Preparation Example 6 2 Example 2 Shrimp horse extract Preparation Example 6 5 Example 3 Shrimp horse fraction 18 0.5 Example 4 Shrimp horse fraction 18 2 Comparative Example 1 (positive control) Arbutin 2 Comparative Example 2 (negative control) - -

Cream Formulation Ingredients ingredient content(%) Active ingredient Table 5 Allantoin 0.1 glycerin 4 1,3-butylene glycol 3 Carbomer 0.15 Dipropylene glycol 2 Clofenine 0.25 Propylparaben 0.1 Full payment 2 Glyceryl stearate One Glyceryl stearate / phage 100 stearate 1.5 Sorbitan sesquioleate 0.8 Polysorbate 60 1.6 Cetearyl alcohol 2 Olive oil 8 Tocopheryl acetate 0.1 Dimethicone One Sodium polyacrylate / hydrogeneate 0.4 Triethanolamine 0.15 Purified water Balance Total (total) 100

Experimental Example 5. Human skin skin test

In order to examine the irritation of the cosmetic products manufactured according to the conventional methods in Examples 1-4 and Comparative Example 1-2 to human skin, a human skin primary stimulation test was conducted. Unlike medicines, which are used for a certain period of time for the treatment of certain diseases, cosmetics are products that are normally used by normal healthy people for a long period of time. Therefore, the human skin primary stimulation test is essential for predicting skin irritation under actual use conditions.

A suitable amount (20 μl) of each cosmetic material was dropped into the IQ chamber for the foaming test for 30 healthy adult males and females, and the chamber was removed for 48 hours. At 30 minutes and 24 hours after the removal, changes in skin condition were visually observed. Evaluation criteria and scores of skin reactions are shown in Table 7 below.

score Evaluation standard 0 No reaction One Weak erythema, 2 Moderate erythema 3 Marked erythema, accompanied by edema 4 Apparent erythema, edema and small scrotal accompanied

The reaction was firstly determined at 30 minutes after removal of the patch, at second after 24 hours, and the response was calculated according to the following equation. The average degree of reactivity of the first and second determinations showed the degree of stimulation to the human skin, and the results are shown in Table 8 below.

Response score = [(response score × number of respondents) / maximum response score (4) × total number of testers (n)] × 100

Number of respondents 48 hours
(Primary judgment) 72 hours
(Secondary judgment) Reactivity 1+ 2+ 3+ 4+ 1+ 2+ 3+ 4+ 48
time 72
time Average Example 1 0 - - - - - - - - 0.0 0.0 0.0 Example 2 One One - - - - - - - 0.8 0.0 0.4 Example 3 0 - - - - - - - - 0.0 0.0 0.0 Example 4 One One - - - - - - - 0.8 0.0 0.4 Comparative Example 1
Positive contrast 2 2 - - - 2 - - - 1.6 1.6 1.6 Comparative Example 2
Negative contrast 0 - - - - - - - - 0.0 0.0 0.0

As shown in Table 9, the cosmetic compositions of Examples 1 to 4 according to the present invention showed significantly less skin irritation than the cosmetic composition containing 2% arbutin (Comparative Example 1) used as a positive control in terms of primary skin irritation on human skin.

Experimental Example 6. Skin whitening effect test on human skin

The cosmetic products prepared according to the conventional methods in Examples 1-4 and Comparative Example 1-2 were tested for whitening effect on human skin, and the results are shown in Tables 9 and 10 below. Specifically, in order to test skin whitening effect on human skin, opaque tape having four holes of 1.5 cm x 1.5 cm was attached to the inner part of both sides of the testicles in 20 healthy male adults, (UV) of about twice the erythema level (MED) to induce skin pigmentation. Each of the cosmetic compositions of the examples according to the present invention and the cosmetic preparations of the comparative examples were applied to the pigment deposits in the morning and evening twice a day for 8 weeks. The evaluation of melanin index and skin color was performed using Mexameter and Spectrophotometer before and after 8 weeks of use.

The melanin indexes measured using a mexameter are shown in Table 9 below. The lower the value of the melanin index, the greater the whitening effect.

Melanin Index (mean ± standard error) Before use after use Example 1 231.56 ± 3.16 208.51 + - 7.55 Example 2 229.45 ± 6.65 191.26 + - 5.57 Example 3 228.10 ± 5.26 207.33 + - 5.25 Example 4 231.10 + - 8.14 189.23 + - 5.68 Comparative Example 1
Positive contrast 230.74 + - 8.52 210.57 + - 2.54 Comparative Example 2
Negative contrast 229.85 ± 6.01 216.21 + - 6.45

As shown in Table 9, it can be seen that the cosmetic compositions of Examples 1 to 4 have an excellent inhibitory effect on the melanin index as compared with Comparative Example 1-2. The cosmetic containing 2% of the extract of Preparation Example 6 (Example 1) showed a whitening effect equal to or higher than that of the cosmetic containing 2% arbutin (Comparative Example 1), and the cosmetic containing 5% of the extract of Preparation Example 6 ) Was much better than cosmetic composition containing 2% arbutin (Comparative Example 1). The cosmetic containing 0.5% of the fraction of Production Example 18 (Example 3) showed a whitening effect equal to or higher than that of the cosmetic containing 2% arbutin (Comparative Example 1), and the cosmetic containing 2% of the fraction of Production Example 18 ) Was much better than cosmetic composition containing 2% arbutin (Comparative Example 1).

The effect on skin color was calculated according to the ITA ° (Individual Typological Angle) value reflecting the degree of whiteness of skin using L *, a *, b * values measured by a spectrophotometer, The value calculation formula is as follows. The larger the ITA value, the greater the whitening effect.

ITA ° = arctg [(L * - 50) / b *] × (180 / π).

L *: Brightness factor; brightness

b *: color factor; Blue-Yellow

ITA ° value (mean ± standard error) Before use after use Example 1 19.96 ± 1.40 27.33 + - 1.31 Example 2 19.54 ± 2.00 31.84 ± 1.22 Example 3 20.61 + - 0.95 27.11 + - 0.94 Example 4 19.88 ± 0.86 32.24 + - 1.14 Comparative Example 1
Positive contrast 19.31 + - 1.07 26.87 ± 0.68 Comparative Example 2
Negative contrast 19.45 ± 0.99 24.12 ± 1.27

As shown in Table 10, it was confirmed that the cosmetic compositions of Examples 1 to 4 had better skin whitening effect than Comparative Example 1-2 in skin color. The cosmetic containing 2% of the extract of Preparation Example 6 (Example 1) showed a whitening effect equal to or higher than that of the cosmetic containing 2% arbutin (Comparative Example 1), and the cosmetic containing 5% of the extract of Preparation Example 6 ) Was much better than cosmetic composition containing 2% arbutin (Comparative Example 1). The cosmetic containing 0.5% of the fraction of Production Example 18 (Example 3) showed a whitening effect equal to or higher than that of the cosmetic containing 2% arbutin (Comparative Example 1), and the cosmetic containing 2% of the fraction of Production Example 18 ) Was much better than cosmetic composition containing 2% arbutin (Comparative Example 1).

As described above, the present invention relates to a composition containing an extract of Phyllospadix iwatensis Makino as an active ingredient, and more particularly to a pharmacological, food and cosmetic composition containing a shrimp extract or a fraction thereof as an active ingredient, And their use.

Since the shrimp horse extract of the present invention is safe for skin and has a remarkable inhibitory effect on human tyrosinase activity and is thus excellent in the effect of inhibiting melanin production, it is possible to provide a composition for skin whitening and a preventive and ameliorating disease caused by excessive melanin accumulation Or as a therapeutic composition.

Claims (9)

A pharmaceutical composition for preventing or treating a skin pigmentation disease caused by an excessive accumulation of melanin containing an extract of Phyllospadix iwatensis Makino or a fraction thereof as an active ingredient.
A food composition for preventing or ameliorating a skin pigmentation disease caused by an excessive accumulation of melanin containing an extract of Phyllospadix iwatensis Makino or a fraction thereof as an active ingredient.
Phyllospadix iwatensis Makino extract or fraction thereof; And a dermatologically acceptable excipient.
delete 3. A method according to claim 1 or 2, wherein the skin pigmentation disorder due to melanin overaccumulation Genetic polymorphic dysplasia (dystrophic dystrophy), reticular dystrophy, pigmentation, freckles, black spot, nevus, drug-induced pigmentation, post-inflammatory pigmentation and dermatitis &Lt; / RTI &gt; wherein the composition is selected from the group consisting of hyperpigmentation.
The shrimp hull extract according to any one of claims 1 to 3, wherein the shrimp extract is selected from the group consisting of water, a lower alcohol having 1 to 6 carbon atoms, a lower ketone having 1 to 4 carbon atoms, a lower ester having 1 to 4 carbon atoms, Wherein the composition is an extract from at least one solvent selected from the group consisting of water-soluble solvents and critical fluid.
4. A composition according to any one of claims 1 to 3, wherein the fraction is a polar solvent fraction of a shrimp extract.
The method according to claim 7, wherein the polar solvent fraction of the shrimp extract
Wherein the shrimp horse extract is sequentially fractionated with water and a non-polar solvent, and the water fraction is fractionated again with a polar solvent of butanol or ethyl acetate.
The composition of claim 8, wherein the non-polar solvent is at least one solvent selected from the group consisting of n-hexane, dichloromethane, and ethyl ether.

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