JP2008179598A - Melamine production inhibitor containing extract from thymelaea plant as active ingredient, and skin-lightening medicine composition, cosmetic composition, and food containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new melamine production inhibitor having a high melamine production-inhibiting activity. <P>SOLUTION: This melamine production inhibitor is characterized by containing an extract from a Thymelaea plant as an active ingredient. The melamine production inhibitor can exhibit high safety and excellent skin functionality, and can further exhibit a decarcinogenesis action for melanoma cells. The medicine composition and the cosmetic composition each comprises the melamine production inhibitor and pharmacologically acceptable additives, and a food comprises the melamine production inhibitor and food raw materials. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a melanin production inhibitor. More specifically, the present invention relates to a melanin production inhibitor extracted from a plant belonging to the genus Thymellaa, and a whitening pharmaceutical composition, a cosmetic composition, and a food containing the same.

  Skin pigmentation such as sunburn, spots, and buckwheat occurs when cellular melanocytes present in epidermal cells proliferate and pigment melanin produced in the proliferated melanocytes diffuses to adjacent cells. Various drugs are known that suppress melanin production and exert a whitening effect by suppressing the expression of the enzyme tyrosinase, which plays a central role in the production of melanin in melanocytes, or by inhibiting the activity of the enzyme tyrosinase. Yes. Representative examples of such drugs include kojic acid and arbutin.

  In addition, melanin is produced by enzymatic or non-enzymatic oxidation from dopa and dopaquinone produced by the action of the enzyme tyrosinase. Various drugs are also known that inhibit the process and thereby suppress whitening. ing. Representative examples of such drugs include ascorbic acid and hydroquinone.

  Of these, kojic acid and its derivatives have been used as whitening cosmetic compositions and skin external preparations as melanin production inhibitors (see Patent Documents 1-3). However, in recent years, the possibility that kojic acid has carcinogenicity and genotoxicity has been pointed out, and a safer melanin production inhibitor is demanded instead.

Furthermore, in the whitening cosmetic composition, an attempt has been made to find a plant extract having an inhibitory action on melanin production, reflecting the demand for low skin irritation and natural orientation (patents). Reference 4-6).
Japanese Examined Patent Publication No. 56-18869 Japanese Patent Publication No. 60-9722 Japanese Patent Publication No. 60-10005 Japanese Examined Patent Publication No. 61-10442 Japanese Patent Publication No. 5-27605 Japanese Patent Publication No. 5-87482

  In view of the present situation as described above, the main object of the present invention is to provide a novel melanin production inhibitor excellent in safety and skin functionality and having high melanin production inhibitory activity.

  In order to solve the above-mentioned problems, the present inventors have searched for various natural products, found for the first time that a Thymelea genus plant extract has a melanin production inhibitory effect, and have completed the present invention.

  Therefore, the present invention provides a melanin production inhibitor containing a Thymelea plant extract as an active ingredient. This extract contains one or more selected from Genkwadaphnin, Gnidicin, Gnitilatidin.

  Among the plants belonging to the genus Thymelaea, particularly an extract from Thymelaea hirsuta has a remarkable melanin production inhibitory activity.

  In addition, these melanin production inhibitors also exert a decancerating action on melanoma cells.

  A melanin production inhibitor containing a Thymelia plant extract as an active ingredient can be mixed with a pharmacologically acceptable additive to obtain a whitening pharmaceutical composition and a whitening cosmetic composition. It can also be mixed with food ingredients to make a whitening food.

  These pharmaceutical compositions, cosmetic compositions and foods have the following advantages.

  Since the melanin production inhibitor according to the present invention is a plant-derived component, the pharmaceutical composition is highly likely to be continuously applied over a long period of time, and is highly likely to have few side effects. Further, for example, it is considered that an excellent whitening effect can be obtained by continuously applying the melanin production inhibitor according to the present invention as a cosmetic, health supplement or the like for a long period of time.

  The present invention provides a novel melanin production inhibitor having high melanin production inhibitory activity. Moreover, the melanin production inhibitor can exhibit high safety and excellent skin functionality.

  The Thymelaea genus plant is a plant that grows along the Mediterranean coast.

  Examples of plants belonging to the genus Thymelaea include Thymelaea antiatlantica, T. et al. hymelea aucheri, T. et al. broteriana, T .; calicina, T .; cilicica, T .; coridifolia, T .; dioica, T .; granathis, T .; gussonei, T .; There are about 30 types such as hirsuta.

  The inventor of the present invention has newly found that the extract from the above-mentioned Thymelaea genus plant has melanin production inhibitory activity, and in particular, the extract from Thymelaea hirsuta has particularly remarkable melanin production inhibitory activity. Thymelaea hirsuta lives in a semi-arid region of North Africa and is used as an anti-inflammatory drug in the region as a folklore drug. However, the melanin production inhibitory action and the decancerous action have not been examined so far for the extract from Thymelea hirsuta.

  Hereinafter, preferred embodiments for carrying out the present invention will be described. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

  The melanin production inhibitor according to the present invention can be obtained by extracting the whole plant of the genus Thymelea using various solvents. Here, the whole plant of the genus Thymellaa means that any part of leaves, flowers, stems and roots can be used. Moreover, these may be used in a raw state, or a dried product may be used. As an extraction solvent, an organic solvent such as methanol, ethanol, propanol, butanol, propylene glycol, 1,3-butylene glycol, glycerin, acetone, methyl ethyl ketone, ethyl acetate, ethers, chloroform, and dichloromethane, and water can be used. it can. Among these, in the present invention, methanol, ethanol, ethyl acetate or a combination of these solvents and water is preferable, and in view of safety in vivo that toxicity is low, ethanol or a mixed solvent of water and ethanol is used. Further preferred. As a suitable extraction method, the whole plant of the genus Thymelea can be obtained by immersing it in 70% ethanol for 1 week and then removing insolubles using a microfilter having a pore size of 0.45 μm.

  The melanin production inhibitor according to the present invention can be processed into an appropriate form such as a whitening pharmaceutical composition, a whitening cosmetic composition, or a whitening food.

  When processed into a pharmaceutical composition or a cosmetic composition, a melanin production inhibitor and a pharmacologically acceptable additive are mixed. Examples of additives include surfactants, excipients, colorants, preservatives, coating aids, and combinations thereof. These additives are not particularly limited as long as they are additives in normal pharmaceutical production, and more specific examples include lactose, dextrin, sucrose, mannitol, corn starch, sorbitol, crystalline cellulose, polyvinyl pyrrolidone, flavoring agent, Sweeteners and combinations thereof are mentioned. In addition, general cosmetic raw materials such as oils, alcohols, humectants, ultraviolet absorption, fragrances, antioxidants, pigments and the like can also be contained. Furthermore, it is good also as a mixture with another chemical | medical agent as needed.

  The pharmaceutical composition may be used for either oral or parenteral administration. The dosage form is not particularly limited, and it is produced in an appropriate dosage form according to the Japanese Pharmacopoeia. Specifically, when it is intended for oral administration, it is produced in a dosage form such as a capsule, a tablet, a powder or a sustained release agent. When intended for parenteral administration, it is produced in dosage forms such as injections, infusions, and external preparations. In addition, the cosmetic composition is a lotion, emulsion, gel, cream, ointment, and more specifically, a makeup whitening makeup such as a makeup base lotion, makeup base cream, liquid, cream or ointment type foundation. It can also be provided as a whitening body cosmetic such as an up cosmetic, a sunscreen lotion, a sunscreen cosmetic such as a sunscreen cream, a hand cream, a leg cream, or a body lotion.

  When processed into food, a melanin production inhibitor and an appropriate food material are mixed. This suitable food ingredient is generally one that can be used as a food material. Examples include flours such as rice, wheat, corn, potatoes, sweet potatoes, soybeans, kelp, wakame, tengusa; chickenpox; lactose; glucose; fructose; sucrose; mannitol; and combinations thereof. Furthermore, spices, colorants, sweeteners, edible oils, vitamins and the like may be added. These suitable food ingredients are used alone or in combination. Furthermore, you may add water as needed and process it into a desired shape. In this way, health functional foods (including specific health functional foods, nutritional functional foods, and beverages), so-called health foods (including beverages), and feed can be obtained. These foods can also be fed to animals as feed.

<Extraction of melanin production inhibitor from Thymelaea plant>
Inhibition of melanin production used in the following examples by immersing 100 g of dried Thymelaea hirsuta in 1000 ml of 70% ethanol for 1 week and then removing insolubles using a microfilter with a pore size of 0.45 μm An agent (hereinafter also referred to as “Thymelaea plant extract”) was obtained. This Thymelea genus plant extract was dried under reduced pressure at 50 ° C. with a rotary evaporator to obtain a slightly greenish light brown powder (hereinafter also referred to as Thymelea genus plant extract dried product). The yield was 0.8%.

<Examination of melanin synthesis inhibitory action using melanoma cells>
About the melanin production inhibitor prepared in Example 1, the melanin synthesis inhibition test using B16 cell strain was done. B16 cells are a cell line established from mouse melanoma (melanoma). Since it is a pigment cell that produces melanin pigment, it is widely used in medicinal effects tests on melanin synthesis.

A 10 cm petri dish for cell culture containing 15 ml of DMEM medium containing 10% fetal bovine serum was inoculated so that the cell density would be 1 × 10 ⁶ cells / dish, and cultured overnight at 37 ° C. under 5% CO _2. The melanin production inhibitor prepared in Example 1 was added to the petri dish so as to have a 1/1000 concentration (administration group), and further cultured under the same conditions for 3 days. Only the solvent in which the melanin production inhibitor was dissolved was added to the control group.

  After completion of the culture, the cells were washed twice with 10 ml of PBS (−) buffer, cells were collected by 0.25% trypsin treatment, and then the membrane was lysed with PBS (−) containing 0.1% Triton X100. FIG. 1 shows a solution obtained by extracting and purifying melanin with acetic acid and dissolving melanin in sodium hydroxide. Moreover, the result of having measured melanin content (microgram) with the existing method is shown in FIG.

  In FIG. 1 and FIG. 2, in the administration group indicated by symbol S, marked whitening of the cell suspension was observed as compared to the control group indicated by symbol C (see FIG. 1). In addition, the amount of melanin synthesis in the cells was 92.81 μg in the control group, and 51.89 μg in the administration group, a significant decrease was confirmed (see FIG. 2). This result has shown that the melanin production inhibitor which concerns on this invention has the whitening effect | action with respect to B16 cell, and suppresses the melanin pigment production in a pigment cell.

  Next, the phosphorylation state of ERK1 / 2 belonging to the MAP kinase cascade was examined in these cells. Conventionally, it is known that melanin synthesis is suppressed when ERK1 / 2 is phosphorylated by activating the MAP kinase cascade.

  Proteins are extracted from cells in the administration group and the control group by a conventional method, separated by SDS-PAGE, blotted on a membrane, and an antibody (SIGMA) that recognizes ERK1 / 2 and phosphorylation (p-ERK1 / 2). Western blotting was performed. The results are shown in FIG. Symbol S represents the administration group, and symbol C represents the control group.

  In the administration group, a marked increase in p-ERK1 / 2 was observed, confirming that melanin synthesis suppression accompanying activation of the MAP kinase cascade occurred.

  Next, in the administration group and the control group similarly treated with the melanin production inhibitor, the cell viability 48 hours after the treatment was measured by a flow cytometry method.

  In the flow cytometry method, cells are stained with a fluorescent dye (PM2) that stains dead cells and a fluorescent dye (PM1) that stains nuclear DNA, and further, aggregated cells and apoptotic cells depend on the magnitude of forward scattered light. This is a method for distinguishing between necrotic cells and distinguishing live cells from dead cells. Therefore, it is possible to measure the cell viability by counting the number of cells excluding aggregated cells from those having low PM2 staining and strong PM1 staining.

  The results are shown in FIG. Symbol S represents the administration group, and symbol C represents the control group.

  The cell survival rate in the administration group was 94.6%, which was equivalent to the cell survival rate of 98.3% in the control group. This indicates that the melanin production inhibitor according to the present invention has no cytotoxicity.

<Examination of decancerous action using melanoma cells>
Next, in the administration group and the control group similarly treated with the melanin production inhibitor, the cell morphology was observed for 48 hours to 72 hours after the treatment.

  The results are shown in FIG. Symbol S represents the administration group, and symbol C represents the control group.

  The control group of B16 cells has a fibroblast-like cone shape, and there are scattered cells having abundant melanin pigment granules. In contrast, in the administration group, the cells changed to an epithelial cell-like form, and no melanin pigment granules were observed in the cells. That is, it can be seen that the melanin production inhibitor according to the present invention induces transformation of B16 cells and exerts a decancerating action on melanoma cells.

  The above strongly suggests that the melanin production inhibitor according to the present invention may be applied as a therapeutic agent for melanoma (melanoma) due to the action of promoting decanceration of melanoma cells.

<Identification of active ingredient in plant extract of genus Thymelaea>
By the same method as in Example 1, a genus Thymelea plant extract was prepared. However, in this example, methanol was used for extraction. The extract was partitioned between ethyl acetate and water. Next, for each of the ethyl acetate soluble part and the water soluble part, a melanin synthesis inhibition test using the B16 cell line was performed in the same manner as in Example 2. As a result, the melanin synthesis inhibitory effect depending on the dose was observed in the ethyl acetate soluble part.

  Fractionate ethyl acetate soluble fraction using normal phase column chromatography (silica gel column: φ2.2 × 33cm, solvent: hexane / ethyl acetate 5: 1, 2: 1, chloroform / methanol 8: 1, 0: 100) And 17 fractions (Th-EtOAc-1 to 17) were obtained. Each fraction was subjected to a melanin synthesis inhibition test using the B16 cell line in the same manner as in Example 2. As a result, the strongest melanin synthesis inhibitory action was observed in the Th-EtOAc-11 fraction.

Fractionation of Th-EtOAc-11 fraction using C ₁₈ Sep-Pak cartridge (solvent: methanol / water 50:50, 100: 0, chloroform / methanol 1: 6, 100: 0, water) (Th-EtOAc-11-1 to 6) were obtained. Each fraction was subjected to a melanin synthesis inhibition test using the B16 cell line by the same method as in Example 2. As a result, the strongest melanin synthesis inhibitory action was observed in the Th-EtOAc-11-3 fraction.

Using a _C18 reverse phase column (Intersil ODS3: φ1.0 × 25 cm, solvent: acetonitrile / water 65:35, flow rate: 2.0 ml / mis), the Th-EtOAc-11-3 fraction was further subjected to high performance liquid chromatography ( Purification by HPLC gave three compounds (Th-EtOAc-11-3-6, Th-EtOAc-11-3-8, Th-EtOAc-11-3-14).

¹ H NMR spectrum and EIMS spectrum were measured for three compounds of Th-EtOAc-11-3-6, Th-EtOAc-11-3-8, and Th-EtOAc-11-3-14. For the measurement, Brucker AVANCE 500 spectrometer and MS-6890 were used, respectively.

^{In the 1} H NMR spectrum, 5.12 ppm and 4.92 ppm oxymethine signals and 5.02 ppm methylene signals were observed. Detailed measurement data for the three compounds are as follows.
(1) Th-EtOAc-11-3-6
EIMS m / z 602 (M +), ¹ H NMR (CDCl ₃ , 500MHz): δ7.60-7.75 (m, aromatic-H), 7.50 (s, 1H), 5.28 (s, 1H), 5.05 (s, 2H), 4.95 (d, J = 2.5 Hz, 1H), 4.25 (s, 1H), 3.90 (m, 1H), 3.85 (d, J = 5.4 Hz, 2H), 3.63 (br s, 1H), 2.63 (q, J = 7.3 Hz, 1H), 1.88 (s, 3H), 1.75 (d, J = 2.6 Hz, 3H), 1 and 0.88 (m, 3H).
(2) Th-EtOAc-11-3-8
EIMS m / z 628 (M +), ¹ HNMR (CDCl ₃ , 500MHz): δ7.60-7.75 (m, aromatic-H), 7.50 (s, 1-H), 6.37 (d, J = 16.0 Hz, 1H ), 5.18 (s, 1H), 5.05 (s, 2H), 4.95 (d, J = 2.5 Hz, 1H), 4.25 (s, 1H), 3.90 (m, 1H), 3.85 (d, J = 5.4 Hz , 2H), 3.63 (br s, 1H), 2.52 (q, J = 7.3 Hz, 1H), 1.88 (s, 3H), 1.75 (d, J = 2.6 Hz, 3H), and 0.88 (m, 3H) .
(3) Th-EtOAc-11-3-14
ESIMS m / z 671 (M + Na ⁺ ); ¹ H NMR (CDCl ₃ , 500MHz, TMS): δ7.90-7.40 (5H, m, aromatic-H), 7.50 (1H, s,), 6.76-5.53 (4H, m, olefinic-H), 5.21 (1H, s), 5.01 (2H, s), 4.90 (1H, d, J = 2.50), 4.16 (1H, s), 3.90 (1H, s), 3.85 (2H, br s), 3.63 (1H, s), 3.60 (1H, d, J = 2.46), 2.55 (1H, q, J = 7.34 Hz), 1.88 (3H, s), 1.75 (3H, br s ), 1.36 (3H, d, J = 7.26Hz), 1.33 (3H, m), 0.88 (3H, m) ppm.

  From the above measurement data, Th-EtOAc-11-3-6 was identified as Genkwadaphnin (see Kasai R, et al. Genkwadaphnin, a potent antileukemic diterpene from Daphne genkwa. Phytochemistry 1981: 20: 2592-2593.). Also, Th-EtOAc-11-3-8 is Gnidicin (see Brooks G, et al. Daphnanediterpenes of T. hirsuta. Phytochemistry 1990: 29: 2235-2237.), Th-EtOAc-11-3-14 is Gnidilatidin (see Borris RP, et al. Studies of the Thymelaeaceae II. Antineoplastic principles of Gnidiakraussiana. Journal of Natural Products. 1984: 47: 270-278). Genkwadaphnin, Gnidicin, and Gnidilatidin are compounds having a daphnane skeleton. Each structural formula is shown in FIGS.

<Study of melanin synthesis inhibitory action using melanoma cells 2>
Genkwadaphnin, Gnidicin, and Gnidilatidin were tested for inhibition of melanin synthesis using the B16 cell line.

By inoculating the B16 cell line in a petri dish by the above-mentioned method, culturing it overnight at 37 ° C. under 5% CO ₂ , Genkwadaphnin, Gnidicin and Gnidilatidin purified in Example 4 were 0.1 μM, 0.3 μM and 1 μM, respectively. It added to the petri dish by the density | concentration (administration group), and also culture | cultivated for 2 days. Only ethanol was added to the control group.

  FIG. 7 shows the results of measuring the melanin content and the cell viability after completion of the culture by the same method as in Example 2. 7A to 7C show the results of Genkwadaphnin, Gnidicin, and Gnidilatidin, respectively. The melanin content and cell viability were shown as 100 for the control group.

  In all of Genkwadaphnin, Gnidicin, and Gnidilatidin, a marked decrease in the amount of melanin synthesis in the cells was confirmed at each administration concentration. In Genkwadaphnin and Gnidicin, melanin synthesis was suppressed to around 60% of the control group, and in Gnidilatidin to around 40%. In addition, no decrease in cell viability was observed in any of Genkwadaphnin, Gnidicin, and Gnidilatidin. These results indicate that Genkwadaphnin, Gnidicin, and Gnidilatidin have a whitening effect on B16 cells, suppress melanin pigment production in pigment cells, and have no cytotoxicity.

  From the above experiments, it was proved that the melanin production inhibitor according to the present invention has a remarkable melanin synthesis inhibitory activity and exhibits an excellent whitening effect. Moreover, since cytotoxicity was not recognized, it was considered to be a highly safe melanin production inhibitor.

  Next, the formulation example of the pharmaceutical composition containing the melanin production inhibitor of this invention is demonstrated in detail. According to the composition shown in Table 1, an ointment (formulation example) was prepared as an example of a pharmaceutical composition containing a Thymelia plant extract.

  Next, formulation examples of cosmetics containing the melanin production inhibitor of the present invention will be described in detail. According to the composition shown in Table 2, a lotion (formulation example) containing a Thymelaea plant extract was prepared.

Moreover, the emulsion (formulation example) containing a Thymelea genus plant extract was prepared according to the composition shown in Table 3.

(Note 1) “Lipidure-PMB” manufactured by NOF Corporation

  Next, the formulation example of the food containing the melanin production inhibitor of this invention is demonstrated in detail. According to the formulation shown in Table 4, tune gum (formulation example) containing a dried product of the genus Thymelea was prepared.

Moreover, according to the compounding composition shown to Table 5 and 6, the cookie and soft drink (prescription example) containing a Thymelea genus plant extract dried material were adjusted, respectively.

  Melanin production inhibitor comprising the extract of the genus Thymelia according to the present invention as an active ingredient is a whitening pharmaceutical composition, cosmetic composition, and food for preventing skin pigmentation such as sunburn, spots and buckwheat For example, it can be suitably used.

It is a figure which shows the whitening of the cell suspension of B16 cell which treated the melanin production inhibitor which concerns on this invention. It is a figure which shows the reduction | decrease in the amount of melanin synthesis | combination of the B16 cell which treated the melanin production inhibitor which concerns on this invention. It is a figure which shows activation of the MAP kinase cascade in B16 cell which treated the melanin production inhibitor which concerns on this invention. It is a figure which shows the cell survival rate of the B16 cell which treated the melanin production inhibitor which concerns on this invention. It is a figure which shows the decancerating effect | action of the melanin production inhibitor which concerns on this invention. It is a figure which shows the structural formula of Genkwadaphnin (A), Gnidicin (B), and Gnidilatidin (C). It is a figure which shows the reduction | decrease of the melanin synthesis amount of B16 cell which treated Genkwadaphnin (A), Gnidicin (B), and Gnidilatidin (C), and a cell survival rate.

Claims (7)

  A melanin production inhibitor comprising, as an active ingredient, an extract from a genus Thymelea.   The melanin production inhibitor according to claim 1, wherein the extract contains one or more selected from Genkwadaphnin, Gnidicin, and Gnitilidin.   The melanin production inhibitor according to claim 1 or 2, wherein the plant belonging to the genus Thymelea is Thymelea hirsuta.   The melanin production inhibitor according to any one of claims 1 to 3, which has a decancerating action on melanoma cells.   A whitening pharmaceutical composition comprising the melanin production inhibitor according to any one of claims 1 to 4 and a pharmacologically acceptable additive.   A whitening cosmetic composition comprising the melanin production inhibitor according to any one of claims 1 to 4 and a pharmacologically acceptable additive.   The food for whitening containing the melanin production inhibitor as described in any one of Claim 1 to 4, and a food raw material.