JP2551607B2 - Melanin production inhibitor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a melanin production inhibitory substance present in the placenta of a cow during pregnancy.

[Conventional technology]

It is known that the placenta extract is used as an external preparation for skin application and a whitening cosmetic composition for suppressing the production of melanin pigment in the skin and depigmenting the pigment because there is a substance having an inhibitory effect on melanin production in the placenta. (Japanese Patent Publication No. 48-30370). Further, a method for extracting a substance that suppresses melanin production from the placenta is disclosed in Japanese Examined Patent Publication (Kokoku) No. 48-30370 and Japanese Unexamined Patent Publication (Kokai) No. 58-177918.

Furthermore, JP-A-60-255730 discloses a substance having an action of suppressing melanin production in human placenta.

[Problems to be solved by the invention]

The present invention is a substance contained in the placenta of humans and animals and having a melanin production inhibitory action.Exploring a substance having a melanin production inhibitory action different from a known substance, isolating the substance, and clearly confirming its properties By doing so, it is intended to obtain a more useful substance.

[Means for solving problems]

The present inventors, for the melanin production inhibitor contained in the placenta of various mammals including humans, the substance was isolated from the placenta extract, purified, and after repeated studies on its mechanism of action, bovine It was found that the substance contained in the placenta during pregnancy is unique in that the mechanism of melanin production suppressing action is different from the substance contained in the term placenta of known humans.

Therefore, this substance was isolated and purified, and the substance was confirmed, thus completing the present invention.

The present invention provides total nitrogen 105 mg / min obtained from the pregnant placenta of cattle.
100 ml, infrared absorption spectrum (as shown in Fig. 1),
It is a melanin production inhibiting substance having a characteristic of an ultraviolet absorption spectrum (as shown in FIG. 2).

The substance of the present invention is obtained by cutting the placenta during pregnancy of a cow into small pieces and removing blood, and mechanically destroying this with a mixer or the like.
Extract with water at about 40-45 ° C for 3-5 hours, and then centrifuge.
The crude bovine placenta extract obtained was adjusted to pH 4-5 and adjusted to 50-6.
It can be obtained by heating at 0 ° C. for about 10 minutes, filtering the filter paper to remove the formed precipitate, collecting the filtrate, and adjusting the pH to 6.0 to 7.5.

 Next, production examples of the substance of the present invention will be shown.

Production example The placenta of a cow during pregnancy is washed with water, shredded into 1 cm square pieces, and squeezed in water to remove blood, and drained thoroughly. This placenta (40.00 Kg) is further finely chopped, and purified water (80.0 L) and methyl paraoxybenzoate (240 g) are added thereto, and the mixture is treated with a mixer to give a gruel. Extraction is performed while gently stirring this solution at 40 to 45 ° C. for 3 hours. Centrifuge this extract (8000r.pm)
The supernatant is taken, the pH is adjusted to 4.5, the mixture is stirred at 50 ° C. for 10 minutes, and the protein having an isoelectric point in this pH region is precipitated, and this is filtered with a filter paper. The filtrate was adjusted to pH 7.0 with a 10% aqueous sodium hydroxide solution and further filtered to obtain 80 kg of the substance of the present invention.

Next, the test and the result of the melanin production inhibitory action and fibroblast proliferation action of the substance of the present invention are shown.

1. Melanin production inhibitory activity test The substance of the present invention is an eagle containing 10% fetal bovine serum.
l16) was dissolved in MEM medium and B16 mouse melanoma cells (hereinafter referred to as B16 cells) were cultured in this medium. After 5 days of culture, B16 cells were almost completely whitened. This whitened B16
Light-microscopic observation of cells showed a decrease in dopa-positive cells and premelanosome-positive cells, and electron-microscopic observation of melanosome (melasome).
Nosome) was markedly reduced, and many premelanosomes with morphological changes were observed.

The activity of tyrosinase isozyme of B16 cells cultured in the medium to which the substance of the present invention was added gradually decreased with the culture period, and the activity of electrophoretic tyrosinase isozyme of each fraction was T ₁ , T ₂ , T ₃
Both saw a general decline.

Tylosinase is one of the important enzymes involved in melanin production, and this enzyme exists as three types of isozymes in vivo, namely T ₁ type, T ₂ type, and T ₃ type. This tylosinase is first produced in vivo by a T ₂ -type isozyme, which is sequentially modified and converted into T ₁ -type and T ₃ -type. Granules (premelanosomes) that ultimately produce melanin
The isozymes present in T are type T ₃ .

Thus, the characteristic of the substance of the present invention in suppressing melanin production is that tylosinase isozymes T ₁ , T ₂ , T ₃
The activity is generally decreasing with time.

FIG. 1 shows an infrared absorption spectrum of the melanin production inhibitor of the present invention, and FIG. 2 shows an ultraviolet absorption spectrum of the melanin production inhibitor of the present invention.

On the other hand, the known melanin production inhibitor present in human term placenta was tested for whitening of B16 cells in the same manner as the above-mentioned test, and the activity of tylosinase of B16 cells cultured in a medium containing the same substance was Early in the culture period, first T ₂
Type isozyme activity is markedly decreased, and then the T ₁ type and T ₃ type isozyme activities are decreased with an increase in the culture period.

 The results of this experiment are shown in FIGS. 3 and 4.

FIG. 3 is a drawing showing the tylosinase isozyme activity of bovine term placenta of the present invention, and FIG. 4 is a drawing showing the tylosinase isozyme activity of human term placenta extract (control).

From the above findings, it is clear that the substance of the present invention and the known substance in the human full-term placenta have different melanin production suppressing properties.

2. Fibroblast Proliferation Action Test A lyophilized product of 14 ml of the substance of the present invention was dissolved in 14 ml of Eagle MEM containing 3% calf serum to prepare a medium to which the substance of the present invention was added. This sample was used as one unit.

Put 1/4, 1/8, 1/16 unit of the above medium into a plastic dish separately, and inoculate 0.2 x 10 ⁵ cultured fibroblasts (mouse-derived Balb / 3TI2-3) into each medium, 3
The cells were cultured at 7 ° C in a 5% CO ₂ gas phase for 5 days.

After 5 days of culturing, cells were detached using trypsin solution and PB
After diluting with S, the cell number was measured.

As a control, the same test was performed on a medium to which the substance of the present invention was not added.

The number of fibroblasts in the added concentration of the substance of the present invention is shown in the table below.

Then, as a comparative example, the fibroblast proliferation effect of the extract of human term placenta was tested.

As a sample, human term placenta extract was prepared in the same manner as the bovine placenta extract.

The human term placenta extracts obtained were examined for fibroblast proliferation activity in the same manner as described above.

 The results are shown in Table 2.

As is clear from the results in Table 2, the substance of the present invention has a higher fibroblast-proliferating effect than that of human full-term placenta extract.

As is apparent from the above test results, the substance of the present invention exhibits a remarkable fibroblast proliferation effect at low concentrations.

〔The invention's effect〕

The substance of the present invention is different from the known melanin production inhibitor contained in human term placenta in the mechanism of melanin production inhibition, and tylosinase which is an enzyme essential for melanin production in vivo.
It is an extremely useful melanin production inhibitor that markedly reduces the activity of isozymes T ₁ , T ₂ , and T ₃ types in general. Further, it is a substance having a remarkable proliferative effect on fibroblasts used as an index for activating cells of human skin. The substance of the present invention having the above-mentioned unique action is a substance which is extremely useful as an active ingredient of an external preparation containing cosmetics. At this time, further effects can be expected when used in combination with known melanin production inhibiting substances such as kojic acid, vitamin C, vitamin E, hydroquinone and derivatives thereof, flavonoid compounds, and herbal medicines such as Reishi, licorice, cinnamon, and toki. .

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of the melanin production inhibiting substance of the present invention. FIG. 2 shows an ultraviolet absorption spectrum of the melanin production inhibiting substance of the present invention. FIG. 3 is a drawing showing the tyrosinase isozyme activity of the melanin production-inhibiting substance of the present invention. FIG. 4 is a drawing showing the tylosinase isoenzyme activity of human term placenta extract (control).

Claims (1)

(57) [Claims] 1. Total nitrogen 105m obtained from the placenta of a cow during pregnancy.
A melanin production inhibitor having properties of g / 100 ml, infrared absorption spectrum (as shown in FIG. 1) and ultraviolet absorption spectrum (as shown in FIG. 2).