KR20000034037A - Grape seed extract as tyrosinase inhibitor - Google Patents

Abstract

PURPOSE: A grape seed extract as tyrosinase inhibitor is provided, which shows strong anti-oxidation activity, so can be used as a cosmetic with functions of inhibition of melanin-production, prevention of skin aging, skin protection, and whitening; a medicine for freckles, flecks, spots or hyper pigmentation during pregnancy; an anti-oxidant for food. CONSTITUTION: The process for the preparation of grape seed extract as tyrosinase inhibitor comprises of: washing, drying and pulverizing grape seed; eliminating fat by hexane; extracting with shaking incubator after adding ethanol, methanol or acetone during less than 12hrs; concentrating the extracts under decompression using rotary evaporator at 50°C; and vacuum-drying to gain powdered grape seed extract.

Description

Grape Seed Extract as a Tyrosinase Inhibitor

The present invention relates to grape seed extract having tyrosinase inhibitory activity and its use. More specifically, the present invention relates to grape seed extract which can be used as a whitening cosmetic prepared in hot water or heating conditions using water, ethanol, methanol or acetone, a therapeutic agent for hyperpigmentation and a food browning inhibitor.

Melanin is a biopolymer of phenols that has a complex form of black pigment and protein. Browning occurs when the cut surface of apples, potatoes, and bananas is exposed to air, or it is found on the skin, feathers, skin, hair, and eyes of animals. However, if the melanin is excessively produced, blemishes, freckles, etc. are formed on the skin, skin aging is promoted, skin cancer may also be caused, and in foods, the quality of vegetables, fruits, and fish may also be reduced.

Melanin is reported to be biosynthesized mainly by the action of tyrosinase. Tyrosinase is an enzyme that binds to copper (Cu), which is widely distributed in animals, plants, microorganisms, and humans, and promotes aerobic oxidation in phenolic compounds such as monohydroxy or dihydroxy-phenylalanine (DOPA). In addition, it acts to cause aging or damage of skin by depositing melanintorth on the skin that is severely exposed to ultraviolet rays. In addition, polyphenol oxidase such as tyrosinase also causes browning of food in vegetables or fruits.

Animal melanin, catalyzed by tyrosinase production, is divided into yellow-red-brown pheomelanin and brown-black eumelanin. The synthesis process of melanin is hydroxylated from L-tyrosine to L-3,4-dihydroxy-phenylalanine (DOPA, L-3,4-dihydroxy-phenylalanine) depending on the degree of tyrosinase activity, which is then guided by DOPA. It is oxidized to quinone (dopaquinone), dopaquinone is oxidized to leucochrome, dopachrome is produced by the oxidation of leucochrome, dopachrome is again 5,6-dihydroxyindole (DHI, 5,6-dihydroxyindole), which consists of oxidative polymerization of the dihydroxyindole and finally binding to the protein. In addition to the conversion of dopachrome to DHI in vivo, dopachrome tautomerase acts to convert 5,6-dihydroxyindole carboxylic acid (DHICA) into 5,6-dihydroxyindole carboxylic acid. It is also reported that there is a route.

The biosynthesis metabolism of melanin has recently been intensively studied in connection with skin cancer, and various melanin-producing inhibitors have been developed from this, and skin whitening agents or foods that prevent and treat skin diseases in the pharmaceutical industry and spots and freckles in the cosmetics industry. It is applied as browning inhibitor in industry. Also, in recent years, the demand is rapidly increasing in relation to environmental problems.

Until now, research on melanogenesis inhibitors has been mainly focused on developing tyrosinase inhibitors, and representative tyrosinase inhibitors include ascorbic acid which reduces quinones, which are chelating agents for copper ions of tyrosinase active sites, and quinones ( reducing agents such as ascorbic acid) and bisulfites preparations that denature tyrosinase itself.

As such, tyrosinase inhibitors have been developed and used as various whitening agents, but various problems have been raised at the same time. Indeed, 4-hydroxyanisole and hydroquinone, which are used locally to treat hyperpigmentation such as blemishes, freckles, spots, and hyperpigmentation in pregnancy, have potent melanogenesis inhibitory activities. However, at the same time, it causes side effects such as causing denaturation or lethality of pigment cells and impairing the original function of the cells. In particular, the hydroquinone-based compound was developed and used as a whitening cream that inhibits melanin biosynthesis, but is known to cause skin irritation or skin disease due to cytotoxicity, and is currently licensed only in some countries. Thus, attempts have been made to select tyrosinase inhibitors using natural products that have been previously demonstrated to be stable.

The inventors of the present invention, while researching to develop a tyrosinase inhibitor that inhibits melanin production from natural products, grape seed contains a lot of polyphenol condensed tannin (condensed tannin or proanthocyanidin) and obtained by extracting the grape seed powder with water or an organic solvent The present invention was completed by confirming that grape seed extract effectively inhibits melanin production by inhibiting tyrosinase activity.

It is an object of the present invention to provide grape seed extract and its various uses as tyrosinase inhibitors.

Figure 1 shows the comparison of the tyrosinase inhibitory activity of the grape seed extract of the present invention and the existing hydroquinone according to the concentration,

◆ 70% ethanol extract (30 ° C.); ■: 70% acetone extract;

Δ: hydroquinone; ○: hot water extract;

□: 70% ethanol (90 ° C.); ●: 70% Methanol (90 ℃)

2 is to investigate the degree of inhibition of melanin biosynthesis of the grape seed extract of the present invention using a Streptomyces bikiniensis strain,

Figure 3 is a photograph of the freckles and blemish treatment effect of grape seed extract of the present invention before and after use divided by 16 weeks.

In order to achieve the above object, the present invention provides a grape seed extract having a tyrosinase inhibitory activity extracted using water or an organic solvent and a method for producing the same. At this time, it is preferable to use ethanol, methanol, acetone, or the like as the organic solvent, and the concentration of the organic solvent is preferably in the range of 60 to 90%, and it is preferable to extract the grape seed powder under hydrothermal or heating conditions.

In addition, the present invention provides a cosmetic having a melanin production inhibitory, anti-aging skin, skin protection and whitening function using the grape seed extract as an active ingredient.

The present invention also provides a pharmaceutical composition for the production of melanin inhibitors using the grape seed extract as an active ingredient. It can be used to treat spots, freckles, spots or gestational hyperpigmentation.

In addition, the present invention provides a food additive that suppresses the browning of foods such as fruits, vegetables and fish using the grape seed extract as an active ingredient.

Hereinafter, the present invention will be described in more detail.

In the present invention, in order to prepare a grape seed extract having tyrosinase inhibitory activity, the grape seed powder from which fat is removed is extracted at various temperatures using water and an organic solvent. Preferably, grape seed extract using 60-90% acetone under hydrothermal extraction conditions or 60-90% ethanol or 60-90% methanol under heating conditions, taking into account extraction solvents and drying yield and polyphenol content depending on temperature To prepare a grape seed extract more preferably using 70 to 80% acetone in hot water extraction conditions or 70 to 80% ethanol or 70 to 80% methanol under heating conditions.

The tyrosinase inhibitory activity of the grape seed extract was investigated from absorbance measured by reacting mushroom tyrosinase with L-dihydroxyphenylalanine (L-DOPA) and the like. ), Arbutin, 4-hydroxyanisole, ascorbic acid, ascorbic acid, retinol, hydrocortison showed superior tyrosinase inhibitory activity (see FIG. 1). . In particular, grape seed extract obtained by using 70-80% acetone having a high polyphenol content and grape seed extract obtained by using 70-80% ethanol or methanol under heating conditions exhibit the preferable inhibitory activity.

In addition, the melanin biosynthesis inhibitory activity of the grape seed extract is investigated using Streptomyces bikiniensis strain, a microorganism that biosynthesizes melanin (see FIG. 2). As a result of measuring the size of the melanin inhibition ring (Inhibition zone, mm), the grape seed extract produced a relatively larger and cleaner inhibitory ring than kojic acid, hydroquinone, abutin, 4-hydroanisole.

Therefore, the grape seed extract may be used for the prevention and treatment of melanoma, freckles, spots or hyperpigmentation as a melanin inhibitor that inhibits tyrosinase activity. In addition, the grape seed extract may also be used as a cosmetic having a melanin production inhibitory, anti-aging skin, skin protection or whitening function can be added to the softening longevity, nourishing cosmetics, nutrition cream, massage cream, essence, pack, etc. ). In addition, the grape seed extract may be used as a food additive to increase the elongation by inhibiting the browning of foods such as fruits, vegetables and fish.

The grape seed extract is an extract of an edible fruit that has already been confirmed to be non-toxic, and the extract prepared through the above process may be prepared by a known method in the pharmaceutical field to be used as a therapeutic agent, itself or in pharmaceuticals. It can be mixed and used in the form of ointments, powders, granules, tablets, capsules or injections by mixing with an acceptable carrier, excipient (forming agent), diluent and the like. They can also be used parenterally or orally or by application to the skin.

The dosage of the active ingredient according to the present invention is appropriately selected depending on the absorbency, inactivation rate, rate of excretion, the age, sex and condition of the patient, the severity of the disease to be treated, etc. 5 to 300 mg should be used, but 25 to 200 mg should be used. Such formulated formulations may be applied to the skin several times at regular time intervals, or by using specialized dosing regimens at the discretion of the expert who monitors or observes the administration of the agent as needed and as desired by the individual.

Hereinafter, the present invention will be described in detail by Examples, Preparation Examples and Experimental Examples.

However, the following Examples, Preparation Examples and Experimental Examples illustrate the present invention, but the content of the present invention is not limited by these contents.

<Example 1> Preparation of grape seed extract

Wash the grape seed with water, dry it, crush it with a pulverizer to remove fat with hexane, and extract 100 g of ethanol, methanol or acetone with different solvent and water into 100 g of the grape seed powder. Depending on the temperature was extracted with a shake incubator (30 ℃) or a reflux extractor (60 ~ 95 ℃) attached to a temperature control tank. Next, the extract was concentrated under reduced pressure at 50 ° C. using a rotary evaporator and dried under vacuum to obtain a grape seed extract (G.S.E.) in powder form. In addition, the weight of the grape seed before extraction and the weight of the dried grape seed powder after extraction according to the extraction solvent and the extraction temperature were measured, respectively, and the drying yield was calculated according to the comparison, and the polyphenol content of the grape seed was determined by Folin-Denis. Obtained by the law. Considering the dry yield and polyphenol content according to the extraction solvent and the extraction temperature as a whole, the extract using 70-80% acetone in hot water extraction conditions and the extract using 70-80% ethanol or methanol under heating conditions are most effective. Confirmed.

Drying Yield and Polyphenol Content of Grape Seed Extract (G.S.E) According to Extraction Solvent and Extraction Temperature Extraction solvent Extraction temperature (℃) Dry yield (%) Polyphenol Content of Grape Seed Extract (%) water 80 to 95 7.7 14.5 10% ethanol 30 2.1 5.1 40% ethanol 30 2.9 10.1 70, 80% Ethanol 30 2.8 19.2 10% ethanol 60 to 80 3.5 15.0 40% ethanol 60 to 80 7.4 27.4 70, 80% Ethanol 60 to 80 7.7 38.2 99% ethanol 60 to 75 1.5 50.0 10% acetone 30 2.3 4.0 40% acetone 30 4.1 19.0 70, 80% Acetone 30 3.7 40.3 10% methanol 60 to 80 3.0 14.1 40% methanol 60 to 80 7.1 25.2 70, 80% methanol 60 to 80 7.5 35.4

<Example 2> Tyrosinase Inhibition Activity of Grape Seed Extract

In order to investigate the tyrosinase inhibitory activity of the powdered grape seed extract prepared in Example 1, the degree of inhibition was measured using mushroom tyrosinase (mushroom tyrosinase, Sigma, E.C. 1.14.18.1). At this time, tyrosinase was dissolved in 1/15 M phosphate buffer (phosphate buffer, pH 6.8) at a concentration of 1560 units / mL and stored in a freezer. As a substrate, L-3,4-dihydroxyphenylalanine (L-3,4-dihydroxyphenylalanine, L-DOPA) was dissolved in 1 / 15M phosphate buffer in a shading bottle, and the solution was prepared immediately before use. .

Tyrosinase inhibitory activity was pre-cubated at 25 ° C. for 5 minutes by adding 0.0625 mL of tyrosinase (78 units / mL) and 1.4615 mL of 1 / 15M phosphate buffer, followed by absorbing 0.476 mL of L-DOPA for 10 minutes at 475 nm. Was measured. That is, a sample group containing both a tyrosinase enzyme and a solvent extract or fraction of grape seed (C), a control group having an equivalent amount of 1 / 15M phosphate buffer instead of the sample (A), an enzyme control having a 1 / 15M phosphate buffer instead of the enzyme ( D) And the experiment was performed by dividing the test group (B) with a 1 / 15M phosphate buffer instead of enzymes and samples. Each group was repeated three times (triplicate), and the enzyme inhibition rate (%) was calculated from the following formula. In this case, kojic acid, hydroquinone, arbutin, 4-hydroxyanisole, ascorbic acid, retinol, hydrocortisone (hydrocortison) was used.

As a result, the grape seed extract using 70-80% acetone having a high polyphenol content and the grape seed extract using 70-80% ethanol and methanol under heating conditions showed higher inhibitory activity than the control group (see FIG. 1).

A: absorbance at 475 nm of control without sample;

B: absorbance at 475 nm of sample and control without enzyme;

C: absorbance at 475 nm of the experimental group containing the sample and the enzyme;

D: absorbance at 475 nm of the control without enzyme;

Inhibition of Tyrosinase Activity of Grape Seed Extract (G.S.E.) (%) Extraction solvent Extraction temperature (℃) Concentration (μg / mL) Inhibition (%) water 80 to 95 50 30.0 10% ethanol 30 50 7.0 40% ethanol 30 50 28.3 70, 80% Ethanol 30 50 29.4 10% ethanol 60 to 80 50 15.1 40% ethanol 60 to 80 50 30.8 70, 80% Ethanol 60 to 80 50 58.1 99% ethanol 60 to 80 50 61.1 10% acetone 30 50 7.1 40% acetone 30 50 31.8 70, 80% Acetone 30 50 52 10% methanol 60 to 80 50 15.1 40% methanol 60 to 80 50 25.2 70, 80% methanol 60 to 80 50 56.2

% Inhibition of tyrosinase activity in the control group Control Concentration (μg / mL) Inhibition (%) Kojisan 50 51.0 Hydroquinone 50 5.0 Abutin 50 7.0 4-hydroanisole 50 3.0 Ascorbic acid 50 20.0 Retinol 50 4.9 Hydrocortisone 50 3.7

<Example 3> Melanin biosynthesis inhibitory activity of grape seed extract

In order to investigate the melanin biosynthesis inhibitory activity of the powdered grape seed extract prepared in Example 1, melanin biosynthesis microorganism Streptomyces bikiniensis B-1049 (Streptomyces bikiniensis NRRL B-1049) was used. Specifically, the Streptomyces bikini strain contains V-8 juice (Cambell Soup Co., USA) 200mL, glucose (2g), yeast extract (2g), CaCO ₃ 1g, 2g Bakto agar and 800mL distilled water Inoculated into Papavizas' VDYA (15) slope medium at pH 7.2 and incubated at 28 ° C. for 2 weeks, then 2 mL of distilled water was added to the surface of the slope culture medium to form spores formed in Bacillus mycelia. The spore suspension was scraped off and the spore suspension was stored frozen in sterile test tubes.

In addition, glucose 1.5%, L-tyrosine 0.05%, L-asparagine 0.1%, K ₂ HPO ₄ 0.05%, MgSO ₄ · 7H ₂ O 0.05%, NaCl 0.05%, FeSO ₄ · 7H ₂ O 0.001%, bacto agar Bacto-yeast extract was added to the tyrosine agar (ISP No. 7) medium composition containing 2% by 0.2% to make a solid medium, and sterilized and dispensed at 40 mL per phytidish (90 mm id). . 0.4 mL of the above S. bikiniensis spore suspension was added to each agar plate thus prepared, and uniformly coated with a glass hockey bar. Next, the surface of the medium was dried and placed on an 8 mm diameter paper disc (Toyo Roshi Kasha Ltd., Japan) soaked with 50 μL of the sample and incubated at 28 ° C. for 48 hours to inhibit the melanogenesis inhibition zone (mm). ) Is generated. The size of the melanogenesis inhibitory ring was measured on the back of the medium, and koji acid, hydroquinone, arbutin, and 4-hydroanisole were used as controls.

As a result, the inhibitory ring was relatively large and clear in hot water extracts, 70-80% acetone grape seed extracts, and 70-80% ethanol or methanol grape seed extracts under heating conditions (see FIG. 2).

Inhibition of Melanin Biosynthesis of Grape Seed Extract in Streptomyces Bikinssis Strains Extraction solvent Extraction temperature (℃) Concentration (%) Inhibitory ring (mm) water 80 to 95 8 13.0 10% ethanol 30 8 9.0 40% ethanol 30 8 10.0 70, 80% Ethanol 30 8 11.0 10% ethanol 60 to 80 8 9.0 40% ethanol 60 to 80 8 12.0 70, 80% Ethanol 60 to 80 8 14.5 99% ethanol 60 to 75 8 12.0 10% acetone 30 8 8.5 40% acetone 30 8 12.5 70, 80% Acetone 30 8 13.5 10% methanol 60 to 80 8 9.5 40% methanol 60 to 80 8 12.0 70, 80% methanol 60 to 80 8 13.0

Inhibition of Melanin Biosynthesis of Streptomyces Bikinensis in the Control Group Control Concentration (%) Inhibitory ring (mm) Kojisan 8 12 Hydroquinone 1.7 15 Abutin 8 0 4-hydroanisole 8 14

<Production example 1>

An example of the prescription of a flexible lotion among cosmetics containing grape seed extract is as follows. The grape seed extract used 75% ethanol extract of the heating conditions obtained in Example 1.

Parts by weight

Grape Seed Extract 0.01

Glycerin 3.0

Butylene Glycol 2.0

Propylene Glycol 2.0

Carboxy Vinyl Polymer 0.1

Ethanol 10.0

Triethanolamine 0.1

Preservative traces

A small amount of pigment

A small amount of spices

Purified Water Balance

Total 100.0

<Production example 2>

An example of nutritional lotion prescription among cosmetics containing grape seed extract is as follows. The grape seed extract is the same as the preparation example.

Parts by weight

Grape Seed Extract 0.01

Beeswax 4.0

Polysorbate 60 1.5

Solbitan Sesquioleate 0.5

Liquid Paraffin 5.0

Squalane 5.0

Caprylic / Capric Triglycerides 5.0

Glycerin 3.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Carboxy Vinyl Polymer 0.1

Triethanolamine 0.2

Preservative traces

A small amount of pigment

A small amount of spices

Purified Water Balance

Total 100.0

<Production example 3>

An example of nutritional cream prescription among cosmetics containing grape seed extract is as follows. The grape seed extract is the same as the preparation example.

Parts by weight

Grape Seed Extract 0.005

Beeswax 10.0

Polysorbate 60 1.5

Solbitan Sesquioleate 0.5

Liquid Paraffin 10.0

Squalane 5.0

Caprylic / Capric Triglycerides 5.0

Glycerin 5.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Triethanolamine 0.2

Preservative traces

A small amount of pigment

A small amount of spices

Purified Water Balance

Total 100.0

<Production example 4>

A massage cream prescription example of a cosmetic containing grape seed extract is as follows. The grape seed extract is the same as the preparation example.

Parts by weight

Grape Seed Extract 0.005

Beeswax 10.0

Polysorbate 60 0.8

Solbitan Sesquioleate 0.5

Liquid Paraffin 40.0

Squalane 5.0

Caprylic / Capric Triglycerides 4.0

Glycerin 5.0

Butylene Glycol 3.0

Propylene Glycol 3.0

Triethanolamine 0.2

Preservative traces

A small amount of pigment

A small amount of spices

Purified Water Balance

Total 100.0

<Production example 5>

A prescription example of a pack of cosmetics containing grape seed extract is as follows. The grape seed extract is the same as the preparation example.

Parts by weight

Grape Seed Extract 0.005

Polyvinyl Alcohol 13.0

Sodium Carboxymethylcellulose 0.2

Allantoin 0.1

Nonylphenyl Ether 0.3

Preservative traces

A small amount of pigment

A small amount of spices

Purified Water Balance

Total 100.0

<Experiment 1> Suppression of pigmentation of skin

A site of 2 × 2 cm ² was set in the lower arm of both arms of 20 healthy men and women. Put the aluminum foil on the arm so that the ultraviolet ray is irradiated only on the test target area, and at the same time, each of the two FL20S BLB lamps and the FL20S E-30 lamps manufactured by Toshiba, Japan, at the same time, 10 cm at the same time, 0.8 × 10 ⁷ erg / cm ³ / circuit 1 day One survey was conducted three times consecutively. The area to be tested was well washed with an aqueous 70% isopropyl alcohol solution before irradiation. After irradiation, the formulations of Preparation Examples 1 to 5 containing grape seed extract were applied three times a day. After 3 weeks of application, the degree of pigmentation was visually determined, and how much the experimental group suppressed the pigmentation compared to the control group (base only) was evaluated in three stages of remarkable effect, effectiveness, and invalidity. As shown in Table 6, the positive effect was shown to be higher than the effective one, showing the effect of inhibiting pigmentation excellently.

Experimental group Significant Effect (People) Effective (persons) Void (persons) Preparation Example 1 2 11 7 Preparation Example 2 2 12 6 Preparation Example 3 3 13 4 Preparation Example 4 2 13 5 Preparation Example 5 One 13 6

As described above, the grape seed extract of the present invention is prepared from natural products, has no side effects, and inhibits tyrosinase activity, thereby inhibiting melanin production. It is useful as a therapeutic agent for hyperpigmentation such as spots or gestational hyperpigmentation, and as a food additive for suppressing browning of food.

Claims (6)

Grape seed extract having tyrosinase inhibitory activity extracted using water or an organic solvent. [Claim 2] The grape seed extract according to claim 1, wherein ethanol, methanol or acetone is used as the organic solvent, and grape seed powder is prepared by extracting grape seed powder under hydrothermal or heating conditions with an organic solvent in a concentration range of 60 to 90%. Claim 1, a cosmetic having melanin production, skin aging, skin protection and whitening function as an active ingredient grape seed extract. Pharmaceutical composition for the production of melanin using the grape seed extract of claim 1 as an active ingredient. The pharmaceutical composition according to claim 4, which is used for the treatment of hyperpigmentation including blemishes, freckles, spots or gestational hyperpigmentation. A food additive for inhibiting browning of foods including fruits, vegetables and fish using the grape seed extract of claim 1 as an active ingredient.