JP5286531B2 - Extraction method of tyrosinase inhibitory activator from tishima zasa - Google Patents

Description

  The present invention relates to a method for extracting a tyrosinase-inhibiting activator extracted from an apple secondary residue or Tichamasa and the tyrosinase-inhibiting activator.

  In recent years, among the actions required for cosmetics, there is a growing demand for a whitening action that prevents pigmentation, spots, freckles, and the like that occur on the skin as melanin settles on the skin tissue.

  In this regard, tyrosinase is known as one of the enzymes that change tyrosine, which is a kind of amino acid, into melanin, which is a generation factor of skin pigmentation, spots, buckwheat, and the like.

  Examples of tyrosinase inhibitory active substances that are used in cosmetics to suppress the activity of tyrosinase and thereby have a skin whitening effect are disclosed in, for example, JP-A-8-231343 (Patent Document 2) and JP-A-9-315928. As shown in (Patent Document 3), it is widely known.

  However, when a tyrosinase inhibitory active substance is produced from a synthetic substance, there are not a few people who cause an allergic reaction due to this, and therefore a whitening agent is sought from a highly safe natural material-derived raw material that avoids allergies. The fact is that the voice is expanding rapidly.

  In response to the demands of this field, for example, as shown in JP-A-2000-319192 (Patent Document 4) and JP-A-2001-335498 (Patent Document 5), a tyrosinase-inhibiting active substance is produced from a raw material derived from a natural product. What to do has been proposed. The former uses a rhizome part of birch, especially a maitake rhizome part, and the latter uses a white birch extract extracted from birch bark.

  Such rhizome rhizomes are low in yield and expensive, and the birch bark has a specific route of acquisition (see JP 2001-335498 A [0008]), and there is a difficulty in lack of easy availability of raw materials. is there. For this reason, both of them have a difficulty in lacking a stable supply of raw materials for tyrosinase-inhibiting active substances.

  As one that overcomes these conditions, the one disclosed in JP-A-6-305978 (Patent Document 1) has been proposed. It is a tyrosinase inhibitor containing guava or Erica extract as an active ingredient.

In addition to the above, Japanese Patent Publication No. 2006-199618 (Patent Document 6) containing a shochu extract residue or a shochu extract as an active ingredient is manufactured from a natural product-derived raw material. Japanese Unexamined Patent Application Publication No. 2006-199678 (Patent Document 7) containing as an active ingredient, Japanese Unexamined Patent Application Publication No. 2006-232232 (Patent Document 8) containing a horseradish extract, an extract of an active ingredient from vegetable Hoso Babadan (Nigana) or Hoso Baba Dan JP-A-2007-70228 (Patent Document 9) containing baked chestnut skin extract, JP-A-2007-126406 (Patent Document 10) containing an extract of calcined chestnut skin, Amaria spp. Japanese Unexamined Patent Application Publication No. 2007-204369 (Patent Document 11), Japanese Unexamined Patent Application Publication No. 2007-2 using a material such as rose hip as a raw material. 1987 is (Patent Document 12).
JP-A-6-305978 JP-A-8-231343 JP 9-315928 A JP 2000-319192 A JP 2001-335498 A JP 2006-199618 JP 2006-199678 A JP 2006-232806 A JP 2007-070228 A JP 2007-126406 A JP 2007-204369 A JP 2007-261987 A

  The present invention relates to an apple secondary residue (apple juice) which is a natural product material that can be supplied in large quantities at low cost, in addition to guava and Erica used as raw materials in JP-A-6-305978. And a tyrosinase-inhibiting activator and a method for extracting the tyrosinase-inhibiting activator using apples or tishima zasa as a raw material, which can be extracted from squeezed squeezed or tishimazasa

  This invention is made | formed in view of the above viewpoints, Comprising: A tyrosinase inhibitor activator is obtained from the extract extracted by giving an apple secondary residue and Tichishimasa high pressure.

  The raw material used in the present invention is native to Yamano in Japan, and its growth and regeneration speed is fast and it is easy to secure it as a resource because it does not require cultivation effort. In addition, apples, regardless of their type, have been processed in large quantities as industrial waste until now. Since it is a residue that has no way to be used outside of processing as industrial waste, it is easy to secure its amount, while its use also leads to reduction of industrial waste, and also From the aspect of pollution measures such as effective use and odor prevention, it is extremely meaningful as pollution prevention technology.

  In addition, Ticimasasa and secondary apple residue are natural materials that are not synthetic products, and in addition to them, as is clear from the above points, they can be supplied in large quantities, so conventional tyrosinase inhibitor activators Concerns about synthetic products and concerns about the supply amount will be resolved.

Example 1
1. Tyrosinase inhibitory activity test using apple extract and Tichum extract 1 Sample 1) was ground leaves, branches,稈部high pressure squeezing apple juice apples secondary material (residue is discharged upon manufacturing regular apple juice) 20 kg and Sasa about 1mm~50mm, 220kg / cm ² of about 10 kg of long fibers after being subjected to the high pressure squeezing were mixed and subjected to a high pressure squeezing of about 220 kg / cm ^{2 at} room temperature. By doing so, the juice from which the secondary apple raw material has been squeezed is not spilled, but is retained with surface tension between the fibers of the long fibers of the long fibers mixed by high-pressure pressing. 10 L of the obtained fruit juice was concentrated to Brix 40.0 ± 1.0 under reduced pressure of about 76 mmHg at 50 ° C. to 60 ° C., preferably 55 ° C. to obtain 2 L of concentrated fruit juice. This concentrated fruit juice was designated as AH-3 and used as a sample.

(Example 2)
2) subjected to high pressure squeezing of about 220 kg / cm ² without heating pressate ground to non heated to about the length 1mm~50mm by all grinder leaves, branches,稈部150kg of Sasa native to Aomori Prefecture Sasa A green pressed solution 35L was obtained. The obtained compressed solution is heated at 90 ° C. to 100 ° C., preferably 95 ° C. for 2 to 4 minutes, preferably 3 minutes to remove the solidified part, and the heated liquid is 60 ° C. to 80 ° C., preferably 70 ° C. The solution was concentrated under reduced pressure of about 76 mmHg until Brix was about 40. The obtained distillate 25L was K-2 (colorless and transparent, liquid), the concentrated liquid was allowed to stand for about 24 hours and separated into two layers, and the upper layer 1.5L was obtained as K-3 (black brown, liquid), The lower layer 150 g was K-4 (brown, mud).

  In addition, 10 kg of dried Chishimazasa residue after pressing is added with 130 L of water and extracted with hot water at 170 ° C. to 180 ° C., preferably 175 ° C., 20 minutes to 40 minutes, preferably 30 minutes. Liquid 110L was obtained. This extract was distilled under reduced pressure at 60 ° C to 80 ° C, preferably 70 ° C and about 76 mmHg until Brix was about 40. 85 L of the resulting distillate was added to NK-2 (colorless and transparent, liquid), and the concentrated solution was The liquid 5L from which the precipitate was separated after standing for about 24 hours was designated as NK-3 (black brown, liquid).

  Five types of fractions from K-2 to K-4 and NK-2 and NK-3 were used as samples.

3) Preparation of each solution Tyrosinase (Tyrosinase Mushroom, SIGMA Co. Ltd.) was dissolved in 0.1M phosphate buffer (pH keeping constant) at pH 6.80 to obtain 93 μg / ml enzyme solution. The substrate is L-DOPA (3,4-Dihydroxy
-L-phenylalanine, SIGMA Co. Ltd.) was used to dissolve in 0.1 M phosphate buffer to give a 2 mM substrate solution. As inhibitors, AH-3, K-3, K-4 and NK-3 were diluted with 0.1 M phosphate buffer, and K-2 and NK-2 were used as they were. Arbutin (SIGMA Co. Ltd.) was used as a positive control.

2. Method ¹⁾
Make two rows of 96-well microplates (plastic containers that can process and measure multiple samples at once) with 2 mM substrate solution and 50 μl inhibitor, and preincubate the plates at 37 ° C for 5 minutes (pre-incubation) )did. After pre-incubation, 50 μl of 93 μg / ml enzyme solution was added to one row and 50 μl of 0.1 M phosphate buffer was added to the other row. After incubating (incubating) the plate at 37 ° C. for 15 minutes, the absorbance at a wavelength of 450 nm was measured using a plate reader (spectrophotometer for microplate measurement), and the average of one row was taken as the measured value.

3. Results A is the absorbance of those treated in phosphate buffer without tyrosinase inhibitory activity (the tyrosinase activity is not inhibited), and B is the absorbance of each inhibitor added to inhibit tyrosinase activity. The tyrosinase inhibition rate was calculated as inhibition rate (%) = {(A−B) / A} × 100. Table 1 and Table 2 summarize the concentration and inhibition rate of each inhibitor.

Table 1 Concentration and inhibition rate of each inhibitor

Table 2 Graph showing the relationship between inhibitor concentration and inhibition rate

4. 考察 Table 3 Approximate expression of inhibition rate of each inhibitor shown in Table 2

4. Discussion

[Explanation of Table 1]
Table 1 shows how much each inhibitor shown in each example inhibits the activity of tyrosinase (inhibition rate). “High inhibition rate” indicates “high whitening effect”, and “high inhibition rate even at low concentration” can be said to be “high whitening effect even with a small amount of use”. In this experiment, all inhibitors showed tyrosinase inhibitory activity. In particular, the effects of K-3 and K-4 were strong, with high inhibition rates of about 77% and 94%, respectively, at a concentration of about 66 mg / ml.

[Explanation of Table 2]
Table 2 is a graph of Table 1. As the slope of the straight line is steep (the straight line is vertical), the whitening effect is strong because the inhibition rate is high even at low concentrations. K-3 and K-4 are particularly strong, followed by NK-3 and AH-3. K-2 and NK-2 are somewhat weak, but show that they are effective.

  The approximate expression (y = ax + b) of the inhibition rate of each inhibitor in Table 3 is an equation of each linear function (straight line) in Table 2, and the case of several patterns of concentration per one inhibitor is measured. It is a mathematical formula derived from the measurement results of several points. And the value of the square of R of each inhibitor is a value that serves as an index indicating a correlation as to whether or not all measured data in one inhibitor is on the straight line of the above formula. If the R-square value is close to 1, each measurement data is arranged in a straight line. Conversely, if the R-square value is close to 0, each measurement data is scattered, and the correlation is small. Indicates. In the case of the present application, the closer the R-square value is to 1, the higher the correlation and the higher the reliability.

  As is clear from the above description, according to the present invention, the raw material is mainly a natural product material, and it is possible to provide a ticillonase activity inhibitor and a whitening agent excellent in safety. In addition, appropriate logging of firewood is expected to make a significant contribution to environmental protection because it helps to protect the nature of beech forests. Furthermore, it is expected to make a significant contribution to environmental protection from the use of apple juice residue that has been disposed of in large quantities as industrial waste. It contributes greatly to pollution control and effective use of nature.

Claims (2)

The leaf, branch, and buttocks of Chishimazasa are pulverized to a length of about 1 mm to 50 mm and compressed with high pressure without heating to obtain a green pressing solution. The resulting pressing solution is heated at 90 to 100 ° C. for 2 to 4 minutes. The solidified part is removed by heating to the extent that the liquid after heating is concentrated at a reduced pressure of 60 ° C. to 80 ° C. and about 76 mmHg until the Brix becomes 40. The remaining liquid is allowed to stand for about 24 hours and separated into two layers. A method for extracting a tyrosinase-inhibiting activator using Tichishimasa as a raw material, wherein a tyrosinase-inhibiting activator comprising a blackish brown liquid is obtained. The leaf, branch, and buttocks of Chishimazasa are pulverized to a length of about 1 mm to 50 mm and compressed with high pressure without heating to obtain a green pressing solution. The resulting pressing solution is heated at 90 to 100 ° C. for 2 to 4 minutes. The solidified part is removed by heating to the extent that the liquid after heating is concentrated under reduced pressure of 60 ° C. to 80 ° C. and about 76 mmHg until the Brix becomes 40. The remaining liquid is allowed to stand for about 24 hours and is separated into two layers. A method for extracting a tyrosinase-inhibiting activator using Tichishimaza as a raw material, comprising obtaining a tyrosinase-inhibiting activator comprising a portion of a brownish and muddy substance.