JP3445110B2 - Melanin production inhibitor - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a melanin production inhibitor. Furthermore, the present invention relates to a fragrance composition containing the melanin production inhibitor and having a melanin production inhibitory effect, a skin external preparation, a bath preparation and a food blackening inhibitor.

[0002]

2. Description of the Related Art Pigments, freckles and pigmentation on the skin after sunburn are less likely to occur, increase or disappear with age, which is a problem for middle-aged and older people.
Although the mechanism of occurrence of such acquired pigment (melanin) deposition has not yet been elucidated, melanin is caused by hormonal abnormalities, external stimuli such as ultraviolet rays and oxygen from sunlight, and chemical substances. Pigments are formed and are believed to be abnormally deposited in the skin. The development of drugs that prevent the formation and deposition of melanin is strongly desired, and many drugs have been developed so far. Among these drugs are ascorbic acid, hydroquinone, kojic acid,
There are placenta extract, arbutin, and the like. Further, as a melanin production suppressing component of the plant extract component, a Lamiaceae plant (JP-A-7-187988), an extract of Cous Igaya, etc. (JP-A-7-82133), a saponin of a specific plant (special Kaihei 8-133955), an extract of Kyonin and Karin (JP-A-8-119843), a seed of cumin (JP-A-8-1119848) and the like have been reported.

[0003]

However, these conventional suppressions of melanin production are often insufficient in terms of stability, side effects and effects, and a new melanin production inhibitor has been desired. An object of the present invention is to solve such conventional problems and to develop a naturally occurring product having an excellent melanin production suppressing action.

[0004]

Means for Solving the Problems As a result of repeated studies to solve the above problems, the inventors of the present invention obtained an extract obtained from a specific plant using a specific solvent by a silica gel chromatography method. It was found that the residue of the treated fraction obtained after the solvent was distilled off strongly suppressed the melanin production of B-16 melanoma cells, and further studies were conducted to finally complete the present invention. That is, the present invention relates to a cypress (Cham).
aecyparis obtusa E. ) (Japanese cypress), Jasmine (Jasminum officin)
aleL. ) (Oleaceae), Penile royal or Mentha pulegium L.
(Lamiaceae), Green mint (Mentha spica)
taL. ) (Lamiaceae), Tagetes or Mexican marigolds (Tagetes glandullife)
ra S. ) (Asteraceae), tansy or tansy (Tanacetum vulgare L.) (Asteraceae), Artemisia absint
Hium L. ) (Asteraceae), Cistus or Labdanum (Cistus ladaniferus L., C.
istuscreticus L. ) (Henniaceae), Brunesia or Yusouboku (Bulnesia)
sarmienti L. ) (Heteringidae), Lavender (Lavandula officinalis)
C. ) (Lamiaceae), Smell (Thuja occi)
dentalis L. ) (Japanese cypress), tobacco (Ni
cotiana tabacum L. ) (Solanaceae) from one or more plants selected from the group consisting of water, lower alcohols, polyol-based organic solvents, petroleum ethers and hydrocarbons, and an extract with one or more solvents. A melanin production inhibitor containing, as an active ingredient, a fraction obtained by treating the extract by a chromatography method and then distilling off the solvent of the fraction.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The plant used to obtain the melanin production inhibitor of the present invention is the above-mentioned specific plant, and these are used alone or in combination of two or more. The site to be used is not particularly limited,
Use flowers, leaves, branches, stems, bark, etc. In addition, they may be used immediately after collection or after being dried. Extraction from the plant is carried out using one or more solvents selected from water, lower alcohol and polyol organic solvents, petroleum ethers and hydrocarbons. As the lower alcohol, an alcohol having 1 to 4 carbon atoms is used, and methanol and ethanol are particularly preferable. Further, specific examples of the polyol organic solvent include ethylene glycol, propylene glycol and the like. As the petroleum ether, those publicly known before this application can be used, but commercially available ones can also be used. Examples of the hydrocarbon solvent include aliphatic hydrocarbons, alicyclic hydrocarbons, and aromatic hydrocarbons that are liquid at room temperature, but particularly aliphatic hydrocarbons and aromatic hydrocarbons that are liquid at room temperature, especially n- Hydrocarbons such as hexane (hereinafter referred to as hexane) and toluene are preferable.

The extraction operation is not particularly limited, and it varies depending on the plant and the solvent used, but it is usually carried out by immersing the plant in the solvent at a temperature of room temperature to 80 ° C. or gently stirring for extraction. Further, an extract can be efficiently obtained by using a device such as a Soxhlet extractor well known before the present application. The time required for extraction is usually 3
It is about 0 minutes to 12 hours. The two-stage extraction method known from before the present application may be adopted. The extract of the present invention, in addition to the extract obtained by the above method, an extract obtained by subjecting the extract to any treatment, for example, a concentrate obtained by further removing the solvent from the extract, a so-called extract or Those obtained by further removing a specific compound from the extract are also included. The extract of the present invention also includes those obtained by crushing leaves, branches or trunks of the above plants and then steam distilling. Further, a commercially available product obtained by using the above method for the above plant may be used.

The extract is then processed by chromatographic methods to obtain fractions. This means that the extract or extract-containing solvent is poured into a chromatography column that has been prepared and adjusted in advance (hereinafter sometimes referred to as development), and then the eluate composed of the solvent is poured into the column to temporarily This means that the retained solution is poured together with the solvent (hereinafter sometimes referred to as elution), and the outflowing solvent is divided into several by known means (the divided one is referred to as a fraction). Then, it means a series of operations in the usual way. The extract-containing solvent is usually 0.1 to 30 parts by volume, preferably 0.1 to 30 parts by volume, based on 1 part by weight of the extract.
It is prepared by making it 5 to 20 parts by volume. In the above chromatography method, silica gel chromatography is usually used. The solvent used is particularly preferably hexane, ethyl acetate or a mixed solvent thereof, and the amount ratio of hexane and ethyl acetate is not particularly limited. The elution temperature is usually room temperature, but may be low temperature. More specifically, 10 times the amount of silica gel (Merck, Silica gel 60, 70-2) of the extract or the solvent containing the extract to be developed.
(30 mesh) is slurried with hexane and poured into a clean column to prepare a column. The extract was dissolved in an appropriate amount of hexane and then poured into a column, then 5
0 ml hexane, 50 ml 5% ethyl acetate-hexane, 50 ml 10% ethyl acetate-hexane, 50 ml
20% ethyl acetate-hexane and 50 ml of ethyl acetate are sequentially added. More simply, the extract may be developed with hexane and then eluted with hexane first and then with ethyl acetate. Next, the solvent flowing out by the above method is fractionated by a known means to obtain a fraction. After combining each fraction or a plurality of fractions, the solvent is distilled off under reduced pressure to obtain a residue. The residue thus obtained has a melanin production inhibitory action, and particularly, the residue derived from the fraction from the ethyl acetate or the ethyl acetate-hexane mixed solvent has an excellent melanin production inhibitory action.

The melanin production inhibitory action test was conducted on B16 melanoma (B16 Mera) which is a melanoma cell derived from a mouse.
Noma 4A5). That is, first, B16 melanoma, which is a test cell, is cultured for 3 days in a medium that does not contain the above-mentioned residue (hereinafter sometimes referred to as a sample). The medium was discarded on the 3rd day, and the medium was replaced with a medium containing the samples having the concentrations shown in Tables 3, 4, and 5, and the cells were cultured for 3 days to determine the melanin production inhibitory effect. It was

The melanin inhibitor of the present invention has many uses, and examples thereof include a fragrance preparation, a skin external preparation, a bath preparation, and a food blackening inhibitor. The blending amount thereof is 1 to 50% by weight, preferably 5 to 40% by weight for the prepared fragrance, 0.01 to 10% by weight, preferably 0.05 to 5% by weight for the skin external preparation, and 0.1 for the bath preparation. -10% by weight,
It is preferably 0.2 to 5% by weight, and 0.1 to 10% by weight, and preferably 0.2 to 5% by weight for the blackening inhibitor for foods. When it is mixed with, for example, a lotion, an emulsion or a cream, it is usually 0.05 to 10% by weight, preferably 0.0.
It is 5 to 2% by weight. How to mix melanin suppressor
Although blended as it is, a single or a mixed solution of a polyol or a lower alcohol, which is an organic solvent usually used for perfumes,
It is diluted with a mixed solution with other surfactants and blended,
It may be mixed with a commonly used flavoring material and blended as a blended flavoring composition.

[0010]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. The plant extracts used in this example are all obtained by using at least one of the various solvents specified by the present invention.

[0011]

[Example] 20 g of dried jasmine flowers was set in a Soxhlet extractor, and 100 ml of hexane was added to 70
Reflux extraction was performed at ℃ for 3 hours. The extract was concentrated to obtain 0.71 g of jasmine concrete. 9 on this concrete
After adding 7 g of 9% ethanol and heating and dissolving, the mixture was left at 5 ° C. overnight, the precipitated wax was removed by filtration, and ethanol was removed by concentration under reduced pressure to remove jasmine absolute 0.
5 g was obtained. The yield from dried flowers was 2.5%. This operation was repeated and 1 g of the obtained extract was dissolved in 10 ml of hexane and subjected to silica gel column chromatography. After developing this hexane solution on a silica gel column, 50 ml of hexane (called Y-HC fraction,
The same shall apply hereinafter) and 50 ml of ethyl acetate (Y-ET fraction) were successively eluted. Each effluent was separated and the solvent was distilled off under reduced pressure to obtain a residue. The recovery rate of the residue obtained from each fraction was as follows: Y-HC fraction 3.0%, Y-ET fraction 9
It was 5.1%.

[0012]

Example 2 100 g of lavender dried flowers were set in a steam distillation apparatus, and 2 l of water was added to perform steam distillation. The oil separated after the distillation was removed to obtain 2.64 g of lavender oil. The yield from dried flowers is 2.64%. 1 g of this oil was weighed and the same operation as in Example 1 was carried out to obtain a residue from each fraction. The recovery rate of each residue was Y-HC fraction 11.2%, Y-HC fraction.
The ET fraction was 85.8%.

[0013]

Example 3 The plant-derived extracts shown in Table 1 (including commercially available extracts) were treated in the same manner as in Example 1 to obtain a residue from each fraction. Table 1 shows the yield (%) of the residue from each fraction. Note that there is a loss during sample processing, and the total is 100.
It may not be%.

[Table 1]

[0014]

[Example] 1 g of commercially available cistus extract was added to 10 m of hexane.
It was dissolved in 1 and subjected to silica gel column chromatography. After developing this hexane solution on a silica gel column, 50 ml of each hexane (hereinafter referred to as HC fraction, the same applies), 5
% Ethyl acetate-hexane (5E fraction), 10% ethyl acetate-hexane (10E fraction), 20% ethyl acetate-hexane (20E fraction), ethyl acetate (E fraction). Each effluent was separated and the solvent was distilled off under reduced pressure to obtain a residue. The recovery rate of the residue obtained from each fraction was as follows: HC fraction 22.5%, 5E fraction 37.1%,
The 10E fraction was 13.4%, the 20E fraction was 5.9%, and the E fraction was 9.0%.

[0015]

Example 5 Using the plant-derived extracts (including commercially available ones) shown in Table 2 below, the same procedure as in Example 4 was carried out to obtain a residue from the fractions. Table 2 shows the yield (%) of the residue from each fraction. Note that there is a loss during sample processing, and the total may not reach 100%.

[Table 2]

[0016]

[Test Example 1] Visual determination test for inhibition of melanin production B16 Melanoma cells were placed in a cell culture bottle (manufactured by Iwaki Glass Co., Ltd.) having a bottom area of 25 cm ² at 8 × 10 ⁴ per bottle.
It is prepared by adding 8 ml of DMEM containing 10% FBS so as to be individual, and cultured at 37 ° C. under 5% CO _{2 for} 3 days. FBS is fetal bovine serum manufactured by GIBCO, and DMEM is Dulbecco's modified Eagle medium manufactured by Nissui Pharmaceutical. On the 3rd day, the old medium is discarded, and the medium is replaced with a new medium to which the concentration shown in Table 3 is added. After changing the medium, the cells are cultured under the same conditions for 3 days. After completion of the culture, the medium in the bottle was removed, washed once with 3 ml of PBS, 2 ml of 0.25% trypsin PBS solution was added, and the trypsin solution was removed after 20 to 30 seconds, and 37 ° C. 5%
CO left ₂ 5 minutes at below performs the separation of cells. In addition,
PBS means a physiological saline phosphate buffer solution, the composition of which is as follows. Centrifugal perform separation of cells repeatedly medium was added gently pipetting _{NaCl 0.8% KCl 0.02% Na 2} HPO 4 · 12H 2 O 0.29% KH 2 PO 4 0.02% cell detachment after 8ml Transfer to tube, 1
Centrifugation is performed at 000 rpm for 2 minutes, and the color tone and amount of the obtained cell pellet are visually determined.

The criteria for judgment are shown below. Judgment criteria Cell pellet color tone (melanin inhibition) -Equivalent color tone to control + Slightly lighter tone than control ++ Gray compared to control ++ Light gray to white cell pellet compared to control (Cell growth inhibition) -Equivalent to control Amount + Slightly less than control ++ Significantly less than control + Color of cell pellet is ++ or more, amount of cell pellet is +
In the following cases, it means that the safety is excellent and the melanin production inhibitory action is also excellent, and it is practical. Further, the cell pellet having a color tone of +++ is excellent in the melanin production suppressing action.

[Table 3]

[0018]

[Test Example 2] Melanin production suppression determination amount test Addition of sample under the same conditions as in Test Example 1 until visual determination,
Incubate. After visual inspection, remove the centrifugal supernatant and remove PBS4.
Add ml and gently pipette to disperse the cells.
100 μl thereof is taken and diluted with 9.9 ml of cellent (diluent for microcell counter), and the cell number is measured with a microcell counter (CC-130A manufactured by Toago Medical Electronics Co., Ltd.) to obtain the cell growth inhibition rate. The cell growth inhibition rate is calculated by the following formula. The remaining cell suspension was centrifuged again at 1000 rpm for 2 minutes to wash the cells, which was then washed with 1 ml of well-cooled 5% trichloroacetic acid, and the centrifugation was repeated 3 times, followed by ethanol / ethyl ether (3: 1 volume). 1% of the ratio), washed twice and centrifuged. Finally 1 ml of ethyl ether
Wash once with and dry the cells with warm air from a dryer.
After adding 1 ml of 2N-NaOH to the dried cells and lysing the cells (melanin in the cells) while heating to 70 ° C. and cooling, an ultraviolet-visible spectrophotometer (JASCO UVIDEC-4
Absorbance at 420 nm is measured using 30B). The measured value is converted from the calibration curve of synthetic melanin (Nacalai Tesque, Inc.) to calculate the amount of melanin per 106 cells to obtain the melanin suppression rate. When the melanin inhibition rate is 60% or more and the cell growth inhibition rate is 30% or less, it means that the safety is excellent and the melanin production inhibitory effect is also excellent, and it is practical.

[0019]

[Table 4]

[0016]

Example 6 Preparation of compounded fragrance containing melanin production inhibitor of the present invention 1) White rose-like compounded fragrance Table 5 shows components constituting the fragrance.

[Table 5]

2) Mugue-like compounding incense department Table 6 shows the components constituting the fragrance.

[Table 6]

With respect to this fragrance composition, the effective concentration of the melanin production inhibitory action was examined by the melanin production inhibitory quantitative method of Test Example 2. The results are shown in Table 7.

[Table 7]

[0019]

Example 7 Using the melanin production inhibitor of the present invention, a lotion, emulsion, cream, pack, bath salt and cream foundation were prepared according to the following formulations. 1) Lotion

[Table 8]

2) Emulsion

[Table 9]

3) Cream

[Table 10]

4) Pack

[Table 11]

5) Bath salt (granular type)

[Table 12]

6) Cream foundation

[Table 13]

[0025]

EFFECTS OF THE INVENTION According to the present invention, it was revealed that the chromatographically purified product of the extract from the specific solvent of the specific plant used as the raw material for the natural fragrance has excellent melanin production inhibitory activity. The melanin inhibitor composed of this purified product is not only excellent in the effect of improving or preventing pigmentation on spots, freckles and skin after sunburn, so-called whitening effect, but also excellent in stability and almost no side effects. Some are not. This melanin growth inhibitor can be added to basic cosmetics such as creams, lotions, milky lotions and packs, makeup cosmetics such as foundations, bath agents, external preparations for skin, and blackening inhibitors for foods.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI A61K 7/00 A61K 7/00 X 7/42 7/42 7/46 301 7/46 301 7/50 7/50 (72) Inventor Kazuhiko 1-4-11 Nishihachiman, Hiratsuka-shi, Kanagawa Takasago International Corporation (56) References JP-A-2-240009 (JP, A) JP-A-5-345719 (JP, A) ) JP-A-8-151319 (JP, A) JP-A-9-95441 (JP, A) JP-A-8-92057 (JP, A) JP-A-1-83013 (JP, A) JP-A-57- 83250 (JP, A) (58) Fields surveyed (Int.Cl. ⁷ , DB name) A61K ⁷ /00-7/50 A23L 1/272 A23L 3/3472

Claims (7)

(57) [Claims] 1. Jasmine (Jasminum officinale L.) (Oleaceae), Penny Royale or Mentha mentha (Menth)
a pulegium L.) (Lamiaceae), Mentha spic
ate L.) (Lamiaceae), Tagetes or Mexican marigold (Tagetes glandulifera S.) (Asteraceae), Tansy or tansy (Tanacetum vulgare L.) (Asteraceae), Artemisia absinthium L. (Asteraceae), Cistus Or Labdanum (Cistus ladaniferus
L., Cistus creticus L.) (Helianthaceae), Brunesia or Yuzoboku (Bulnesia sarmienti L.) (Lamiaceae) from one or more plants selected from water,
Fractions obtained by obtaining an extract with one or more solvents selected from lower alcohols, polyol-based organic solvents, petroleum ethers and hydrocarbon solvents, and treating the extract with a chromatography method A melanin production inhibitor containing, as an active ingredient, a solvent distilled off. 2. The melanin production inhibitor according to claim 1, wherein the extract is dissolved in n-hexane and then treated by a chromatography method. 3. When treated by a chromatographic method,
The melanin production inhibitor according to claim 1 or 2, wherein the eluate is n-hexane, a mixed solvent of n-hexane and ethyl acetate, or ethyl acetate. 4. A mixed perfume containing 1 to 50% by weight of the melanin production inhibitor according to claim 1, which is selected from claim 1, 2 or 3. 5. A skin external preparation containing 0.01 to 10% by weight of the melanin production inhibitor according to claim 1, which is selected from claim 1, 2 or 3. 6. A bath agent containing 0.1 to 10% by weight of the melanin production inhibitor according to claim 1, which is selected from claim 1, 2 or 3. 7. A black discoloration inhibitor for foods, which comprises 0.1 to 10% by weight of the melanin production inhibitor according to claim 1, which is selected from claim 1, 2 or 3.