JP7069270B2 - AGE-derived melanin production method and composition - Google Patents

Description

The present invention relates to an AGE-derived melanin production inhibitor and an AGE-derived melanin production method.

The reaction in which proteins and lipids and sugars bind non-enzymatically is called saccharification, and the reaction produces advanced glycation end products (AGE). Specifically, Nε-carboxymethyl lysine (CML), Nε-carboxyethyl lysine (CEL), pentosidine (pentsidine), pyralin, crosulin, GA-pyridine, Nω-carboxymethyl arginine (CMA), floylfuranylimidazole ( There are many compounds such as 2- (2-furoyl) -4 (5)-(2-furanyl) -1H-imidazole) and glucospan. Since it has become clear that AGE is involved in various chronic diseases, reports on its anti-glycation effect have been increasing in recent years.

Furthermore, since the accumulation of AGE can be an exacerbating factor of aging such as dullness of the skin and decrease of skin elasticity, it is attracting attention from the viewpoint of beauty. It is well known that UV rays generally exacerbate skin aging, and there are reports that collagen saccharification is involved in wrinkle formation. On the other hand, the direct relationship between saccharification and AGE for age spots has not been clarified.

Stain is an α-melanocyte stimulating hormone (α-MSH) or endothelin that promotes melanocyte production from keratinocytes present in the epidermis when stress stimuli such as ultraviolet rays and inflammation are applied to the skin. -1, ET-1), various cytokines such as stem cell factor (SCF) are secreted, and when melanocytes receive these active factors, they are activated by intracellular signal transduction and melanin is synthesized. It is known to be caused by. Melanin is converted from L-tyrosine in melanocytes to dopaquinone by being catalyzed by a tyrosinase enzyme, and is further biosynthesized through oxidation and polymerization processes.

As a mechanism of action of whitening so far, those that inhibit tyrosinase enzyme activity are generally used, evaluation of tyrosinase enzyme activity in vitro, and activity of tyrosinase in melanoma cells stimulated by melanin stimulating agents such as α-MSH. It has been listed as a candidate for whitening agent by evaluating the evaluation, gene expression, protein amount, and melanin amount actually released from cells. For example, there have been reports on the suppression of melanin production in Morinda citrifolia (also known as noni), and although no whitening effect was observed in the fruit and leaf extracts of Morinda citrifolia, the whitening effect of seed extract of Morinda citrifolia was observed. Has been found to have a tyrosinase inhibitory effect and an antioxidant effect (DPPH radical capture effect) in an in vitro test (Non-Patent Document 1). Furthermore, it has been reported that it also has an inhibitory effect on melanin production using α-MSH-stimulated mouse B16 melanoma cells and an intracellular tyrosinase inhibitory effect (Non-Patent Document 2).
However, the mechanism of action of whitening has not been sufficiently elucidated, and new findings are desired.

Masuda M., et al .; Inhibitory effect of constituents of Morinda citrifolia seed on elastase and tyrosinase. J. Nat. Med., 63, 267-273 (2009) Masuda M., et al .; Inhibitory effect of Morinda citrifolia extract and its constituents on melanogenesis in murine B16 melanoma cells. Biol. Pharm. Bull., 35, 78-83 (2012)

The present inventor aims to provide new insights into the mechanism of action of whitening, and based on the hypothesis that AGE and age spots may have different mutual effects from the known mechanism, B16F10 cells (B16). Using murine melanoma cells), various AGEs were added instead of melanin promoters, and the effect on melanin production was investigated.

experimental method
1. 1. Creation of AGE-Collagen
In a 1.5 ml tube, 200 mM phosphate buffer (PB; pH 7.4), 250 μl and 2 M glucose (Glu) or fructose (Fru) sugar solution and 3 mg / ml collagen (collagen type I, bovine skin, pepsin-solubilized) ) Was added in an amount of 200 μl each, and the total volume was adjusted to 1 ml with purified water. As a control, one without collagen and one without sugar were prepared. The reaction solutions were incubated at 60 degrees for 0, 1, 10, 24, 72, 168, 240 hours (h) at different times. In the cell test, each reaction solution was diluted 20-fold with DMEM before use.

2. Melanin production test
The effect of AGE on melanin production was investigated using B16F10 cells.
900 μl of cells adjusted to 2 × 104 cells / well with DMEM without phenol red was seeded in 24-well plates, and after 24 hours, AGE-ized collagen or α-MSH (final concentration;) as a positive control was inoculated. 0.1 μM) was added in 100 μl each. After another 72 hours, transfer the supernatant of the cell culture solution to a 96-well plate in 100 μl increments, wash the cell pellet with phosphate buffer (PBS, pH 7.4), add 1 N NaOH (150 μl), and add 1 N NaOH (150 μl) for 1 hour at 60 ° C. Incubated and dissolved. The protein content of the lysate was measured using a DC protein assay reagent (Bio-Rad Laboratories) using BSA (bovine serum albmin) as a standard product. In addition, 100 μl each was transferred to a 96-well plate, the absorbance of each of the supernatant (release) and the cell pellet (intracellular) was measured at 405 nm, and the amount of melanin produced was calculated using the calibration curve of synthetic melanin.
AGE collagen was prepared by changing the reaction time (0, 1, 10, 24, 72, 168, 240 hours), and 20-fold diluted with DMEM (final concentration; 200-fold diluted) was used in the experiment. The results are shown in Table 1 below.

Table 1 Effect of AGE-ized collagen on melanin production in B16F10 cells

As shown in Table 1, AGE did not affect cell proliferation (WST method), and melanin production was observed depending on the length of incubation time. In addition, it can be understood that glycated collagen (particularly saccharified by monosaccharides) has a melanin-producing effect. Among them, more melanin is produced in AGE-modified collagen by glucose than in AGE-modified collagen by fructose. Was admitted.

3. 3. Tyrosinase activation test (effect of AGE-formed collagen on tyrosinase activity)
900 μl of cells adjusted to 2 × 104 cells / well with DMEM was seeded in a 24-well plate, and 100 μl of AGE-ized collagen or α-MSH (final concentration; 0.1 μM) was added as a positive control 24 hours later. After an additional 72 hours, the supernatant was removed, the cell pellet was washed with cold PBS (pH 7.4), 200 μl of PB (pH 6.8) containing 0.1% triton was added, thawed at -80 ° C for 30 minutes, frozen and then thawed. It was transferred to a tube and centrifuged at 10000 rpm for 10 minutes. Determine the amount of each protein using the DC protein Assay, adjust to 50 μg / 100 μl, transfer to a 96-well plate, add the same amount of 0.1% L-DOPA solution, incubate at 37 ° C for 30 minutes, and incubate at 475 nm. The absorbance was measured. The results are shown in Table 2 below.

Table 2 Effect of AGE-formed collagen on tyrosinase activity

In conclusion, no activity was observed in any advanced glycated collagen. In addition, no cytotoxicity due to AGE was observed.
From the above results, new findings were obtained that AGE-ized collagen directly produces melanin without activating tyrosinase.
That is, α-MSH promotes oxidation by activating tyrosinase, whereas AGE does not activate tyrosinase, so it is considered that melanin production is promoted by promoting oxidation by itself. Therefore, it means that the suppression of melanin production is insufficient only by suppressing the activity of tyrosinase.

4. Effects of CML, CEL, pentosidine, methylglyoxal (MGO), glyoxal (GO) and 3-DG (3-deoxyglucosone) on melanin production As mentioned above, melanin production was observed in advanced glycation end collagen. We proceeded to investigate the effects of AGE (CML. CEL and pentosidine) and their intermediates (MGO, GO, 3-DG) on melanin production using B16F10 cells. The results are shown in Table 3 below.

As shown in Table 3, melanin-producing effects were observed on the intermediates MGO, GO and 3DG without affecting cell proliferation. However, Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine (CEL), and pentosidine, which are AGE compounds alone, did not have a sufficient melanin-producing effect.

5. Since melanin-producing action was observed in the measurement intermediates of fluorescent AGE, 3DG, MGO, and GO in AGE-formed collagen, the reaction time (0, 1, 10, 24, 72, 168, 240 hours) was changed. The fluorescent AGE of the prepared AGE-modified collagen and 3DG, MGO, and GO were measured. In all the indicators, glucose-induced AGE-collagen showed a lower value than fructose-induced AGE-collagen, and it was found that it took a long time to produce an intermediate or AGE, and the amount produced was small (Table 4 below). ..

Table 4 Measurement of fluorescent AGE, 3DG, MGO, GO in AGE-ized collagen

From this result, although it is possible that the intermediate contributes to the melanin-producing action, the effect cannot be correlated with the concentration contained in AGE-ized collagen, so it is produced by the collagen glycation process. It is possible that the interaction of many intermediates and AGEs enhances the melanin-producing effect. Furthermore, it is considered that the intermediate enhances the action on AGE-ized collagen. In any case, it is considered that the production of melanin is promoted by the promotion of oxidation by the advanced glycation end collagen itself.

As described above, AGE-ized collagen (particularly glycation by glucose or fructose), which is glycated collagen, promotes melanin production by a mechanism different from the mechanism of melanin production by α-MSH stimulation and various cytokines derived from ultraviolet rays. It was shown to let. In the present application, the melanin production by this mechanism is referred to as the AGE-derived melanin production mechanism. In addition, the applicant defines AGE-like pigmented spots and the like due to melanin produced by this mechanism.

Therefore, the present invention comprises an AGE-derived melanin-producing composition composed of AGE-modified collagen in which collagen is saccharified. It also comprises an AGE-derived melanin-producing agent containing the composition. Further, the composition comprises methylglyoxal, glyoxal, or 3-deoxyglucosone. It also comprises an AGE-derived melanin production method comprising administering AGE-ized collagen to a subject. It is preferable that the saccharification is due to monosaccharides. Monosaccharides include at least glucose, galactose, mannose and fructose. Glucose and fructose are suitable as monosaccharides for saccharification, but glucose is particularly preferable. Further, as collagen, type I collagen is preferable among type I-IV. Type I collagen is fibrous collagen and is formed by aggregating two α1 chains (type I) and one α2 chain (type I). Since the composition and method are useful for acquiring melanin produced by a new mechanism, they are also useful for the development of new materials.

Furthermore, based on the above findings, in order to provide a new whitening material, we investigated the effect of suppressing melanin production derived from AGE on the fruits, leaves and seeds of the iridoid-containing plant Morinda citrifolia, and the Sanshuyu fruit and olive leaves. bottom.

experimental method
1. 1. Preparation of experimental materials and subjects Morinda citrifolia fruits and leaves collected in French Polynesia were used. The original plant was identified by Tropical Resources Inc. (UT, USA). The fruits were divided into pulp and seeds, the pulp was freeze-dried, and the leaves and seeds were hot-air dried. A 50% ethanol reflux extract (MCF (flesh), MCL (leaf), MCS (seed) extract) of flesh, leaves and seeds of Morinda citrifolia was prepared. In addition, Cornus officinalis fruits from China were extracted with aqueous ethanol (COF extract), and Olea europaea leaves from Morocco were extracted with ethanol from France (OEL extract).

2. 2. Creation of advanced glycated collagen
To a 1.5 ml tube, add 200 μl each of 200 mM PB (pH 7.4), 250 μl and 2 M glucose solutions and 3 mg / ml collagen (collagen type I, bovine skin, pepsin-solubilized), and add 1 ml of purified water to the total volume. Adjusted to be. The reaction solutions were incubated at 60 degrees for 24 hours. In the cell test, each reaction solution was diluted 5-fold with DMEM before use.

2. Melanin production test The melanin production test was performed using B16F10 cells.
800 μl of 2 × 104 cells / well cells adjusted to 2 × 104 cells / well with DMEM without phenol red was seeded on a 24-well plate, and after 24 hours, sample (100 μl) and AGE collagen (100 μl) were seeded. Was added. The sample was dissolved in DMSO and diluted with DMEM to the specified concentration (DMSO final concentration: 0.1%). 0.1% (v / v) DMSO / DMEM was used for the Control (Cont) and vehicle control (V-cont) groups. Advanced glycation end collagen was added to the V-cont group and the sample group. 72 hours after the addition of the sample, 100 μl of the supernatant of the cell culture medium was transferred (released) to a 96-well plate, the cell pellet was washed with PBS, 1 N NaOH (150 μl) was added, and the cells were incubated at 60 ° C. for 1 hour to dissolve them. rice field. After that, transfer 100 μl of the lysate to a 96-well plate (intracellular), measure the absorbance of each of the supernatant (release) and cell pellet (intracellular) at 405 nm, and measure the amount of melanin produced using the calibration curve of synthetic melanin. Calculated. The results are shown in Table 5.

Table 5 Melanin production test

As a result, the extracellular release and intracellular melanin amount increased by AGE were suppressed in all the samples, and among them, the seed extract of Morinda citrifolia showed a very strong melanin production inhibitory effect. Therefore, it was shown that the seed extract of Morinda citrifolia is very useful as a material having an AGE-derived melanin production inhibitory effect.

Therefore, the present invention comprises an AGE-derived melanin production-suppressing composition containing an extract of Morinda citrifolia seeds. The extract (extract) can be either liquid or powdery (individual) and can be contained in the composition in various known embodiments. Further, it comprises an AGE-derived melanin production inhibitor containing the composition. The means for administering the inhibitor may include not only oral but also parenteral means such as injection. Further, it comprises a food for suppressing melanin production derived from AGE containing the composition. Foods broadly include known forms known as health foods, including various oral forms such as food forms including beverages and supplement forms. Further, it comprises a method for suppressing AGE-derived melanin production, which comprises administering the composition to a subject. Further, it is preferable that the extraction solvent contains an organic solvent such as ethanol or methanol, and a mixed solvent of water and an organic solvent can be adopted. As the composition, various known embodiments such as liquid, powder, and tablet can be adopted.

Claims (4)

A composition for melanin production that does not activate tyrosinase and comprises type I collagen AGE-modified with glucose or fructose. The composition for producing melanin according to claim 1, which comprises at least one of methylglyoxal, glyoxal, and 3-deoxyglucosone. A melanin-producing agent containing the composition according to claim 1 or 2. A method for producing melanin that does not activate tyrosinase , which comprises adding type I collagen AGE-modified with glucose or fructose to B16 cells .