JP2000159681A - Extract from grape seed and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an extract from grape seeds which shows inhibitory activity against tyrosinase and is useful as a tyrosinase inhibitor effectively controlling melanin by extracting grape seeds with water and/or an organic solvent. SOLUTION: This extract is obtained by extracting grape seeds with water and/or an organic solvent. As the organic solvent, ethanol, methanol or acetone is favorable. Further, the powder of grape seeds is preferably extracted with an organic solvent of a concentration of 70-80 % under the condition of hot water or heating. This extract is preferably administrated at a dosage of 25-200 mg a day per an adult. This extract may be used as an active ingredient in cosmetics which control melanin formation, prevent skins from aging, protect skins and possess skin-whitening function, an active ingredient in pharmaceutical compositions for treating hyperpigmentation including stain, freckle, spot or superpigmentation in gravidism or an active ingredient in food additives for controlling the browning of foods including fruits, vegetables and perishables.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to tyrosinase (tyro)
ginseng extract having inhibitory activity and uses. More specifically, the present invention relates to the extraction of grape seeds used as whitening cosmetics, therapeutic agents for hyperpigmentation and food browning inhibitors prepared from hot water or heating conditions using water, ethanol, methanol or acetone. About things.

[0001]

2. Description of the Related Art Melanin is a phenolic biopolymer having a complex form of black pigment and protein, and is a browning or animal produced when apple, potato and banana cut surfaces are exposed to air. Hull,
It can be observed from wings, skin, hair, eyes, etc. However, when melanin is overproduced, spots, freckles, etc. are formed on the skin, promoting skin aging and inducing skin cancer, and deteriorating the quality of vegetables, fruits, and fresh foods. There is also action. It has been reported that melanin is biosynthesized mainly by the action of tyrosinase. Tyrosinase is an enzyme linked to copper (Cu) and is widely distributed in animals, plants, microorganisms, humans, etc., and is monohydroxy or dihydroxy-phenylalanine (DOPA).
Promotes aerobic oxidation with phenyl compounds such as melamine toose (melanint)
orth), causing skin aging or damage. Also, in vegetables or fruits, polyphenol oxidase such as tyrosinase (polyphen
ol oxidase) causes browning development of the food. Animal melanin whose production is catalyzed by tyrosinase is classified into yellow-reddish brown pheomelanin and brown-black eumelanin. In the process of synthesizing melanin, L-tyrosine is hydroxylated from L-tyrosine to L-3,4-dihydroxy-phenylalanine (DOPA, L-3,4-dihydroxy-phenylalanine) in accordance with the degree of tyrosinase activity, and furthermore, dopaquinone is obtained from DOPA.
one) and dopaquinone is converted to leucochrome (le
ucochrome), the oxidation of leucochrome produces dopachrome, and dopachrome is further converted to 5,6-dihydroxyindole (DHI, 5,
6-dihydroxyindole), and this dihydroxyindole is polymerized oxidatively and is finally bound to the protein. In addition to the pathway in which dopachrome is converted to DHI, dopachrome tautomerase acts in the body to allow 5,6-dihydroxyindolecarboxylate (DHICA, 5,6-dihyd).
It has also been reported that there is a new pathway for conversion to droxyindole carboxylic acid). The biosynthetic metabolism of melanin has been intensively studied recently in connection with skin cancer, and various melanin production inhibitors have been developed to prevent skin diseases in the pharmaceutical industry, and to prevent spots and freckles in the cosmetics industry. And it is applied as a skin whitening agent to be treated or an anti-browning agent in the food industry. In recent years, the demand for environmental problems has been increasing rapidly. Until now, research on melanogenesis inhibitors has been focused mainly on the development of tyrosinase inhibitors, and typical tyrosinase inhibitors are based on quinones, which are chelating substances for copper ions in the tyrosinase active site. And reducing agents such as ascorbic acid to reduce tyrosinase itself, and bisulfites preparations to modify tyrosinase itself. As described above, tyrosinase inhibitors have been developed as various whitening agents and are currently used, but various problems have been raised at the same time. Indeed, 4-hydroxyanisole and hydroquinone (hydroq) have been used locally for the treatment of hyperpigmentation such as spots, freckles, spots and hyperpigmentation during pregnancy.
uinone) and the like have strong melanin production inhibitory activity, but at the same time, have side effects such as inducing denaturation or killing of pigment cells and impairing the original function of the cells. In particular, compounds of the hydroquinone family have been developed and used in whitening creams that inhibit melanin biosynthesis,
It is known to induce skin irritation or skin disease due to cytotoxicity, and is currently only allowed to be used in some countries. Therefore, an attempt has been made to select a tyrosinase inhibitor using a natural product whose stability has been proved in advance.

[0002]

One of the objects of the present invention is to provide grape seed extracts as tyrosinase inhibitors and their various uses.

[0003]

Means for Solving the Problems So far, the present inventors have been studying to develop a tyrosinase inhibitor which suppresses melanin production from natural products, and found that condensed tannin which is a polyphenol is added to grape seeds. Or pr
oanthocyanidin), to confirm that grape seed extract obtained by extracting grape seed powder with water or organic solvent inhibits tyrosinase activity and effectively suppresses melanin production. Thus, the present invention has been completed. Accordingly, the present invention provides an extract of grape seeds having tyrosinase inhibitory activity extracted using water or an organic solvent, and a method for producing the same. At this time, it is desirable to use ethanol, methanol or acetone as the organic solvent, and it is preferable to extract the grape seed powder under hot water or heating conditions with the concentration of the organic solvent in the range of 60 to 90%. . Further, the present invention suppresses melanin production using the extract of the grape seed as an active ingredient, prevents skin aging,
Provided is a cosmetic having a skin protection and whitening function. The present invention also provides a pharmaceutical composition for a melanin production inhibitor comprising an extract of the grape seed as an active ingredient. This can be caused by spots, freckles, spots or gestational hyperpigmentation (hyperpig
mentation). The present invention also provides a food additive which suppresses browning of foods such as fruits, vegetables and fresh foods using the extract of the grape seed as an active ingredient.

[0004]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. According to the present invention, fat-free grape seed powder is extracted at various temperatures using water and an organic solvent to prepare a grape seed extract having tyrosinase inhibitory activity. Preferably, when the drying yield and the polyphenol content depending on the extraction solvent and the temperature are taken into consideration, the hot water extraction condition is 60 to 9%.
A grape seed extract is prepared using 0% acetone or 60-90% ethanol or 60-90% methanol under heating conditions, and more preferably under hot water extraction conditions.
Use 0-80% acetone or 70-80% under heating conditions.
A grape seed extract is prepared using% ethanol or 70-80% methanol. The tyrosinase inhibitory activity of the grape seed extract may be determined by adding mushroom tyrosinase or the like.
As a result of investigating the absorbance measured by reacting with dihydroxyphenylalanine (L-DOPA) or the like, the extract of the grape seeds was found to contain the existing kojic acid, hydroquinone, arbutin, and 4-hydroxy. Anisole (4-hydroxyanisole), ascorbic acid (ascorbic acid), retinol (retinol),
It was shown that tyrosinase inhibitory activity was superior to hydrocortison (see FIG. 1). In particular, grape seed extract obtained using 70-80% acetone having a high polyphenol content exhibits the desired inhibitory activity. The melanin biosynthesis inhibitory activity of the grape seed extract is also investigated using Streptomyces bikiniensis, which is a microorganism that biosynthesizes melanin (see FIG. 2). At this time, as a result of measuring the size of the melanin formation inhibition ring (Inhibition zone, mm), the grape seed extract produced a relatively large and beautiful inhibition ring than kojic acid, hydroquinone, arbutin and 4-hydroanisole. Was.

Accordingly, the extract of the grape seed is used as an inhibitor of melanin production that inhibits tyrosinase activity, such as spots, freckles, spots or hyperpigmentation during pregnancy.
mentation) and the like. Also, the grape seed extract may inhibit melanin production,
It is also used as a cosmetic having a skin aging prevention, skin protection or whitening function, and can be added to a softening lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, a pack, etc. (see FIG. 3). In addition, the grape seed extract can be used as a food additive that suppresses browning of foods such as fruits, vegetables, and freshness, and increases freshness.

[0006] The grape seed extract is an edible fruit extract which has been confirmed to be non-toxic, and the extract produced through the above process is used in the pharmaceutical field to be used as a therapeutic drug. Ointments, powders, granules, tablets, capsules can be manufactured by a known method, or mixed with a pharmaceutically acceptable carrier, a forming agent, a diluent, or the like. It is manufactured and used in various forms such as drugs and injections. In addition, they can be used parenterally or orally or applied to the skin.

[0007] The dose of the active ingredient according to the present invention is determined by the degree of water absorption, inactivation rate, excretion rate, age of the patient,
It is appropriately selected according to gender and condition, the severity of the disease to be treated, etc., but generally 5-30 days a day for adults.
Although about 0 mg is used, it is desirable to use about 25-200 mg. Formulations formulated in this way can be administered several times at regular intervals, using specialized medications according to expert judgment and individual requirements to monitor and observe drug administration as needed. And can be applied to the skin.

Hereinafter, the present invention will be described in detail with reference to Examples, Production Examples and Experimental Examples. However, the following Examples, Production Examples, and Experimental Examples illustrate the present invention, and the contents of the present invention are not limited by these contents. In the following,% indicates weight% in principle.

Example 1 Production of Grape Seed Extract Grape seeds were washed with water, dried, crushed with a crusher to remove fat with hexane, and dried to obtain 100 g of grape seed powder. And 0.5 to 3 L of ethanol, methanol or acetone, each having a different temperature from water with an extraction solvent, is added to the mixture for 12 hours or less, and according to each extraction temperature, a shaking incubator (30 ° C.) or temperature control is used. Extraction was performed with a reflux extractor (60 to 95 ° C) equipped with a water tank. Next, the extract was concentrated under reduced pressure at 50 ° C. using a rotary evaporator and dried again under vacuum to obtain a grape seed extract (GSE) in a powder state. Obtained. Also, according to the extraction solvent and the extraction temperature, the weight of the grape seed before extraction and the weight of the grape seed powder dried after the extraction were measured, and the drying yield was calculated based on the measured weight. The polyphenol content of the species was determined by the Follin-Dennis method. Table 1 shows the relationship between the drying yield of the grape seed extract (GSE) and the polyphenol content according to the extraction solvent and the extraction temperature.
Shown in When the drying yield and polyphenol content according to the extraction solvent and the extraction temperature are taken into consideration as a whole, an extract using 70-80% acetone under hot water extraction conditions and 70-80% ethanol or methanol under heating conditions are used. It was confirmed that the extract used was the most effective.

[Table 1]

Example 2 Investigation of Tyrosinase Inhibitory Activity of Grape Seed Extract In order to investigate the tyrosinase inhibitory activity of the powdery grape seed extract prepared in Example 1, mushroom tyrosinase (mushroom) was used. tyrosinase, Sigma, EC 1.1.
The degree of inhibition was measured using 14.18.1). At this time, tyrosinase was dissolved in a 1/15 M phosphate buffer solution (phosphate buffer).
ffer, pH6. 8) 1560 units / mL. It was thawed at a concentration and stored frozen in a freezer, and thawed on ice for use as an enzyme source at the time of use. As a substrate, L-3,4-dihydroxyphenylalanine (L-3,4-dihydroxyphenylalaniline) is used.
ne, L-DOPA) was dissolved in a 1/15 M phosphate buffer solution in a light-shielding bottle and used, and this solution was dispensed immediately before use. The tyrosinase inhibitory activity was 0.1% tyrosinase (78 units / mL).
Add 0625 mL and 1.4615 mL of 1/15 M phosphate buffer solution together, and pre-incubate at 25 ° C for 5 minutes (preincubation).
Then, 0.476 mL of the L-DOPA was added thereto, and 47
The absorbance was measured at 5 nm for 10 minutes. That is, a sample group (C) containing the tyrosinase enzyme and all the solvent extracts or fractions of grape seeds, a control group (A) containing the same amount of 1/15 M phosphate buffer instead of the sample, 1/15 instead
The experiment was divided into an enzyme control group (D) containing M phosphate buffer solution and a blank test group (B) containing 1 / 15M phosphate buffer solution instead of enzyme and sample. Each group was repeated three times (triplicate), and the enzyme inhibition rate (%) was calculated from the following formula. At this time, kojic acid (kojic acid) and hydroquinone (hydroquinon
e), arbutin, 4-hydroxyanisole, ascorbate (ascorb)
ic acid), retinol and hydrocortison. Table 2 shows the results of the relationship between the degree of inhibition (%) of the grape seed extract (GSE) on tyrosinase activity, and Table 3 shows the degree of inhibition (%) on the tyrosinase activity of the control group. Show. The degree of tyrosinase inhibition (%) is determined by Equation 1. As a result, the grape seed extract using 70-80% acetone having high polyphenol content and the grape seed extract using 70-80% ethanol and methanol under heating conditions had higher inhibitory activities than the control group. (See FIG. 1).

(Equation 1) A: Absorbance from 475 nm of control group without sample; B: Absorbance from 475 nm of control group without sample and enzyme; C: Absorbance from 475 nm of experimental group containing sample and enzyme; D: Control without enzyme Absorbance of the group from 475 nm;

[Table 2]

[Table 3]

<Example 3> Investigation of melanin biosynthesis inhibitory activity of grape seed extract The melanin biosynthesis inhibitory activity of the powdery grape seed extract prepared in Example 1 was investigated. Streptomyces bikiniensis B, a biosynthetic microorganism
-1049 (Streptomyces bikiniensis NRRL B-1
049) was used. Specifically, the Streptomyces bikiniensis strain was used in V-8 juice (Cambell Soup C).
o., USA) 200 mL of glucose, 2 g of glucose, 2 g of yeast extract, 1 g of CaCO ₃ , 2 g of Bacto agar and 800 mL of distilled water at a pH 7.2 Papavizas
VDYA (Papavizas'VDYA (15))
After culturing this at 28 ° C. for 2 weeks, 2 mL of distilled water was added to the surface of the slant medium, and spores formed in the mycelia were scraped out with a loop to produce a spore milk solution. The liquid was stored frozen in a sterile test tube. Further, glucose 1.5%, L - 0.05% tyrosine, L - asparagus Jin _{0.1%, K 2 HPO 4 0.05} %, MgSO 4 · 7H 2 O 0.0
5%, 0.05% NaCl, FeSO 4 · 7H 2 O 0.001
%, 2% Bacto-aga (ISP No.
7) Bacto-yeast ext
ract) was added to 0.2% to prepare a solid medium, sterilized, and then separated into 40 mL per Petrizara (90 mm id). 0.4 mL per plate for the agar plate thus produced
Said S. Bikiniensis spore milk solution was added, and the mixture was uniformly applied with a glass hockey bar. Next, the surface of the culture medium was dried, and a 50-μL sample was wetted with an 8 mm diameter paper disc (paper disc, Toyo Roshi Kas).
ha Ltd. , Japan) and cultured at 28 ° C. for 48 hours to form a melanin production inhibition ring (inhibition zone, mm). The size of the melanin production-inhibiting ring was measured from the back of the medium, and at this time, kojic acid, hydroquinone, arbutin, and 4-hydroanisole were used as control groups. Table 4 shows the relationship between the degree of inhibition of melanin biosynthesis of extracts of grape seeds from Streptomyces bikiniensis strains, and Table 5 shows the relationship between the degree of inhibition of melanin biosynthesis of Streptomyces bikiniensis in the control group.
Are shown below. As a result, hot water extract, 70-80%
Grape seed extract of acetone, 70% under heating conditions
Grape seed extracts of 〜80% ethanol or methanol showed relatively large and clear inhibitory rings (see FIG. 2).

[Table 4]

[Table 5]

<Production Example 1> Among cosmetics containing an extract of grape seeds, an example of formulating a soft lotion is as follows. Here, the grape seed extract was used under the heating condition of 75 obtained from Example 1.
% Ethanol extract was used. Parts by weight parts by weight grape seed extract 0.01, glycerin 3.0, butylene glycol 2.0 propylene glycol 2.0 carboxyvinyl polymer 0.1 ethanol 10.0 triethanolamine 0.1 preservative trace pigment trace Perfume trace amount Purified water Residual liquid Total 100.0

<Production Example 2> Among cosmetics containing an extract of grape seeds, an example of a nutritional lotion formulation is as follows. Here, the grape seed extract is the same as in the above preparation example. Parts by weight Parts by weight Grape seed extract 0.01 Beeswax 4.0 Polysorbate 60 1.5 Sorbitan sesquiolate 0.5 Liquid paraffin 5.0 Squalane 5.0 Caprylic acid / capryl triglyceride 5.0 Glycerin 3.0 Butylene Glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Triethanolamine 0.2 Preservatives Trace dyes Trace fragrance Traces purified water Residual liquid Total 100.0

<Production Example 3> Formulation examples of a nutritional cream among cosmetics containing an extract of grape seeds are as follows. Here, the grape seed extract is the same as in the above preparation example. Parts by weight grape seed extract 0.005 beeswax 10.0 polysorbate 60 1.5 sorbitan sesquiolate 0.5 liquid paraffin 10.0 squalane 5.0 caprylic acid / caprylic triglyceride 5.0 glycerin 5.0 butylene Glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservative trace amount pigment trace amount fragrance trace amount purified water residual liquid total 100.0

<Production Example 4> Among cosmetics containing grape seed extract, an example of a massage cream formulation is as follows. Here, the grape seed extract is the same as in the above preparation example. Parts by weight grape seed extract 0.005 beeswax 10.0 polysorbate 60 0.8 sorbitan sesquiolate 0.5 liquid paraffin 40.0 squalane 5.0 caprylic acid / caprylic triglyceride 4.0 glycerin 5.0 butylene Glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservative trace amount pigment trace amount fragrance trace amount purified water residual liquid total 100.0

<Production Example 5> Among cosmetics containing an extract of grape seeds, a formulation example of a pack is as follows. Here, the grape seed extract is the same as in the above 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 preservatives trace pigment trace fragrance trace purified water residual liquid Total 100. 0

<Experimental Example 1> Inhibition of skin pigmentation 2 × 2 in the lower arm of both arms of 20 healthy male and female subjects
A site of cm ² was set. Put an aluminum foil on your arm so that only the part to be tested is irradiated with ultraviolet rays, and place it at a distance of 10 cm.
Two −30 lamps were simultaneously irradiated at a rate of 0.8 × 10 ⁷ erg / cm ³ / time, once a day, three times continuously. Prior to irradiation, the target site was thoroughly washed with a 70% aqueous isopropyl alcohol solution. After irradiation, the formulations of Production Examples 1 to 5 containing the extract of grape seeds were applied to the ultraviolet irradiation site three times a day. 3
After weekly application, the degree of pigmentation was determined by the naked eye, and how the pigmentation was suppressed compared to the experimental group (only the base was applied) was evaluated in three levels of remarkable effect, effectiveness, and ineffectiveness. As shown in Table 6, the effect of suppressing the pigmentation was shown by showing a positive effect higher than the effective effect.

[Table 6]

[0018]

As can be seen from the above, the grape seed extract of the present invention has no side effects since it is manufactured from natural products, and inhibits tyrosinase activity and suppresses melanin production, thus preventing skin aging. It is useful for cosmetics having skin protection and whitening functions, for treating hyperpigmentation such as spots, freckles, spots or hyperpigmentation during pregnancy, and as food additives for suppressing browning of foods.

[Brief description of the drawings]

FIG. 1 is a graph showing the tyrosinase inhibitory activities of grape seed extract of the present invention and existing hydroquinone according to concentration, and the symbols in the figure mean the following. ◆: 70% ethanol extract (30 ° C.); ■: 70% acetone extract; Δ: hydroquinone; ●: hot water extract; □: 70% ethanol (90 ° C.); ○: 70% methanol (90 ° C.) ;

FIG. 2 shows the degree of inhibition of melanin biosynthesis of the grape seed extract of the present invention.
(ptomyces bikinienis) A photograph showing the results of irradiation using a strain.

FIG. 3 is a photograph showing the therapeutic effect of the grape seed extract of the present invention on freckles and spots before and after use (after 16 weeks).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 7/00 A61K 7/00 W U 7/48 7/48 31/00 617 31/00 617 C12N 9 / 99 C12N 9/99 (71) Applicant 596065337 108-2,5-Ka, Yangpyung-Dong, Youngdeungpo-ku, Seoul, Korea. (72) Inventor Lee, Sun-Jae, Republic of Korea, Gyeonggi-do 422-240, # 7-205 (72) Inventor Jean, Jae-Kwon South Korea, Seoul 138-190, Songpaak, Sokchon-dong 229 −8, Jaewon Villa # 102 (72) Inventors Park, Chillon, Republic of Korea, SO Le 143-224, Kuwanji down - click, Junkoku 4- Don 81-10 (72) inventor Lee, Hyun - Amblyseius Korea, Seoul 139-210, Nowon - click, gas production 10 Don, Zukon Epi Ti. # 711-708

Claims (6)

[Claims] 1. A grape seed extract having tyrosinase inhibitory activity, extracted using water and / or an organic solvent. 2. The extraction comprises ethanol as an organic solvent,
2. The grape seed according to claim 1, wherein at least one of methanol and acetone is used, and the grape seed powder is obtained from a grape seed powder under a hydrothermal condition or a heating condition in an organic solvent in a concentration range of 60 to 90%. Extract. 3. A cosmetic composition comprising the grape seed extract according to claim 1 or 2 as an active ingredient, which has a melanin production-inhibiting, skin aging-preventing, skin-protecting and whitening function. 4. A pharmaceutical composition for a melanogenesis inhibitor, comprising the grape seed extract according to claim 1 or 2 as an active ingredient. 5. The pharmaceutical composition according to claim 4, for use in the treatment of hyperpigmentation, including spots, freckles, spots or hyperpigmentation during pregnancy. 6. A food additive which suppresses browning of foods, including fruits, vegetables and fresh products, comprising a grape seed according to claim 1 or 2 as an extract. [0001]