JP3851625B2 - Whitening agent containing phenolic compound rutinose glycoside as an active ingredient - Google Patents

Description

The present invention relates to a whitening agent containing a rutinose glycoside of a phenol compound as an active ingredient .

  The glycosides of phenolic compounds are widely present in plants even in small amounts, and have low physiological toxicity despite their excellent physiological activity. Therefore, development of food materials, food additives, pharmaceuticals, cosmetics, etc. Is attracting attention as a target.

  For example, arbutin is a type of conventionally known phenol compound glycoside. Its chemical name is hydroquinone β-D-glucopyranoside, which is a glucose glycoside having hydroquinone as an aglycone. Arbutin is widely used as a cosmetic because it has whitening effects, melanin production inhibition and active oxygen inhibition. In addition, glycosides containing catechol, resorcinol and the like as aglycones are also known to be effective in preventing and treating skin pigmentation.

  When the glycosides of phenolic compounds having excellent pharmacological effects as described above are examined in more detail, the aglycones of each glycoside, that is, hydroquinone, catechol, and resorcinol itself are excellent for each glycoside. Have pharmacological effects. However, the effects of these aglycones themselves are small compared to the respective glycosides. Therefore, it is not only forced to be used at a high concentration, but also has side effects such as vitiligo on the skin when used over a long period of time.

  From the above facts, it is expected that a phenol compound having a useful pharmacological effect generally improves its pharmacological effect and reduces side effects by making it a glycoside. Furthermore, it is known that glycosides are generally more water soluble than the aglycone itself.

  On the other hand, for example, ellagic acid, which is a dimer of gallic acid, has excellent pharmacological effects such as an antioxidant action and a melanin inhibitory action, but is easily oxidized and unstable, and is soluble in water. Because of its extremely low nature, there is a problem that it is inconvenient to handle. Therefore, with respect to such a phenol compound, it is expected that the problem of side effects and solubility can be improved by making this a glycoside without impairing its physiological activity.

  However, because there is no efficient and simple method for converting phenolic compounds to glycosides, certain phenolic compounds are not fully utilized despite having useful activity. This is the actual situation.

  Moreover, the sugar part in the conventional phenol glycoside is limited to several kinds, such as glucose and ribose, and the combination of an aglycone and sugar was comparatively limited. Therefore, even if the same aglycone is used, if a different sugar can be bound, it is expected that a useful novel phenol glycoside can be obtained.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel whitening agent .

The present invention is a whitening agent containing a rutinose glycoside of a phenol compound as an active ingredient .

The rutinose glycoside of a phenolic compound contained in the whitening agent of the present invention as an active ingredient is obtained by reacting a rutin-degrading enzyme on rutin in the presence of the phenolic compound, thereby causing a sugar moiety contained in rutin, that is, rutinose. It can be produced by transferring to the phenol compound, and this transfer reaction is represented by the following formula.

  The phenol compound in the present invention is not particularly limited as long as it acts as a receptor for rutinose in the above enzyme reaction. However, preferred phenolic compounds include hydroquinone, pyrogallol, veratryl alcohol, gallic acid, catechol, ellagic acid, benzyl alcohol and the like.

  Rutin used in the present invention is a glycoside in which rutinose (a disaccharide composed of glucose and rhamnose) is O-glycosidically bonded to quercetin, which is an aglycon, as shown in the above formula. This rutin is a kind of flavonol glycoside widely distributed in natural plants such as Enju (legaceae), buckwheat (Tapaceae), and Henruda (Rutaceae), and is useful for preventing arteriosclerosis and hypertension. Known as active substance.

  The rutin-degrading enzyme in the present invention may be any as long as it can decompose rutin to cut out rutinose and transfer it to the oxygen atom of the phenol compound. Examples thereof include enzymes derived from plants such as Enju, oats, and Henruda, or enzymes derived from microorganisms such as Penicillium and Aspergillus bacteria. Among these enzymes, particularly preferred is a rutin-degrading enzyme contained in the seeds of dartane buckwheat. This is because this rutin-degrading enzyme has high activity, and tartane buckwheat is still widely used for food and is highly reliable in terms of safety. In addition, a rutin degrading enzyme obtained from Aspergillus niger is preferable in that it has glycosylation ability for a wide range of phenolic compounds.

  In the present invention, the rutin-degrading enzyme is preferably a purified product, but depending on the strength and content of its activity, it is not necessarily a purified product. That is, plant or microorganism extracts containing these enzymes may be used as they are, or in some cases, plant bodies or microorganisms may be used as they are.

  Taking rutin-degrading enzyme contained in the seeds of tartane buckwheat as an example, a buffer is added to the seeds of tartnut buckwheat or the buckwheat flour prepared by grinding the seeds to obtain an extract, which is further purified. It is preferable to use the purified enzyme obtained in this way. However, since the enzyme contained in tartane buckwheat has high activity, it functions well even if the extract is used as it is. Furthermore, the seeds of tartane buckwheat contain a very large amount of rutin compared to the seeds of normal buckwheat. That is, it is about 14 mg% in the normal seed of buckwheat noodles, whereas it is about 1300 mg% in the seed of buckwheat noodles (mg% represents the amount of rutin contained in 100 g of buckwheat seeds). Therefore, even if the seed seeds of tartane buckwheat or its flour are used as they are, the intended transfer reaction of rutinose can be sufficiently performed.

  Further, when using a rutin-degrading enzyme derived from a microorganism, the enzyme may be extracted from the microorganism itself. However, when the microorganism secretes the enzyme outside the cells, the culture solution in which the microorganism is cultured can be used as a crude enzyme solution as it is. Of course, it is also possible to further extract and purify this culture solution and use it as a purified enzyme.

  Extraction of rutin-degrading enzyme from plants and microorganisms can be usually performed by a method used for enzyme extraction. For example, the extraction may be performed by stirring in an acetate buffer.

  According to the method of the present invention, a phenol compound can be converted into its rutinose glycoside in a single step reaction. Therefore, the purified rutin-degrading enzyme can be reacted by adding it directly to rutin. However, if the bioreactor is constructed by fixing it to an appropriate carrier, it is possible to continuously produce the desired glycoside. , Efficiency can be improved dramatically.

  The method for producing a rutinose glycoside according to the present invention can be carried out under any conditions as long as the rutin degrading enzyme is not inactivated. However, it is preferably performed at a pH of 3 to 9 and a temperature of 80 ° C. or less, and most preferably at a pH of 5 and a temperature of 40 ° C.

<Example 1>
(1) Preparation of crude enzyme solution After adding 1.5 liters of 20 mM acetate buffer (pH 5) to 50 g of tartane buckwheat flour, the mixture was stirred and extracted for 1 hour. By filtering with 1 filter paper, 1.41 liters of crude enzyme solution was obtained.

(2) Production of Glycoside 4 g of rutin, 400 mg of hydroquinone, and 20 ml of the crude enzyme solution obtained above were mixed and reacted by stirring at 40 ° C. for 24 hours. Next, 20 ml of water was added and centrifuged at 10,000 rpm and filtered. Subsequently, the filtrate was applied to an activated carbon column and then washed with 10% ethanol to remove rutinose. Furthermore, after eluting with 100% ethanol and distilling off under reduced pressure, ethyl acetate was added and washed to remove hydroquinone, and then subjected to HPLC under the following conditions to obtain 24.7 mg of a purified product.

Column; Asahipak NH2 P-50 4.6 × 250 mm
(Showa Denko Co., Ltd.)
Detector: RI detector, UV detector
Eluent; acetonitrile: water = 75: 25
Flow rate: 1.0 ml / min
The results of HPLC analysis of the purified product obtained above are shown in FIG. 1 and FIG. FIG. 1 shows the result of the UV detector, and FIG. 2 shows the result of the RI detector.

  Further, the UV absorption spectrum of the purified product obtained above is shown in FIG. 3, and the UV absorption spectrum of hydroquinone is shown in FIG.

Furthermore, when the purified product obtained above was subjected to ¹³ C-NMR analysis, the results shown in FIG. 5 were obtained.

From these results, it was found that the product obtained by the above reaction was a hydroquinone rutinose glycoside represented by the following formula (hydroquinone rutinoside: hereinafter referred to as HQR).

<Example 2>
Enju was used instead of tartane soba, and everything else was performed in the same manner as in the example. As a result, 6.5 mg of purified HQR was obtained.

<Example 3: Acid decomposition of HQR>
1 mg of the purified product (HQR) obtained in Examples 1 and 2 was dissolved in 1.2 ml of 2N hydrochloric acid solution, heated to 100 ° C. with a block heater, and subjected to a decomposition reaction for 2 hours. Then, it distilled off under reduced pressure, adding ethanol. The obtained decomposition product was subjected to the same HPLC analysis as that performed in Example 1. The results are shown in FIG. 6 (UV detector) and FIG. 7 (RI detector).

  This result also supports that the purified product obtained in Examples 1 and 2 is HQR.

<Example 4>
In order to examine the tyrosinase inhibitory activity of the purified HQR obtained above, the following test solution was prepared.

Test solution Control solution 0.05% L-tyrosine solution 1.0 ml 1.0 ml
HQR solution (5 mM) 1.0 ml
50 mM acetate buffer (pH 6.8) 0.9 ml 0.9 ml
Tyrosinase solution (500 mU / ml) 0.1 ml 0.1 ml
As tyrosinase, mushroom-derived tyrosinase (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

  The above test solution and control solution were incubated at 25 ° C., and the absorbance at 470 nm was measured every 30 minutes. The result is shown in FIG.

  From the results of FIG. 8, it was found that HQR inhibits tyrosinase activity and suppresses tyrosine degradation.

<Example 5>
Aspergillus niger received from IF0 was cultured at 30 ° C. for 8 days in a liquid medium containing 2% rutin, and the culture solution was recovered.

  This culture solution was directly used as a rutin-degrading enzyme, and a reaction between rutin and hydroquinone was carried out in the same manner as in Example 1. That is, rutin, hydroquinone and the above culture filtrate were mixed and reacted at 30 ° C. for 2 days. Unreacted rutin, produced quercetin and the like were removed as precipitates by centrifugation and suction filtration. The supernatant at that time was collected, ethyl acetate was added thereto, and unreacted hydroquinone was removed as an upper layer. On the other hand, the lower layer was subjected to activated carbon chromatography, and the fraction eluted with 30% 1-propanol was collected.

The ¹³ C-NMR spectrum of the product thus obtained was the same as that shown in FIG. From this NMR data, it was found that the product was HQR as in Example 1. From the ¹ H-NMR spectrum (data not shown), the anomeric structure was found to be β.

  Further, pyrogallol, veratryl alcohol, and gallic acid were used in place of hydroquinone, and the same reaction and purification as described above were performed. As a result, the production of rutinose glycosides was similarly observed for these phenol compounds other than hydroquinone.

<Example 6: Antioxidant ability of HQR>
In order to investigate the antioxidant property of hydroquinone rutinoside (HQR) obtained in the above-mentioned Examples, the reducibility was measured by high performance liquid chromatography (HPLC) / electrochemical detector (ECD). The electrochemical detector applies a constant potential between electrodes and measures a change in current caused by a sample passing therethrough. If the potential applied between the electrodes is a positive potential, it can be said that the substance detected under this condition has a reducing power, that is, an antioxidant ability.

5 mM HQR was subjected to high performance liquid chromatography (column: YMC-Pack NH ₂ 250 × 4.6 mm ID manufactured by YMC, eluent: 40 mM ammonium acetate, 17.5 mM acetic acid / 80% methanol, flow rate: 1 ml / Min) was injected at 20 μl, and the eluted sample was detected by an electrochemical detector (amperometric electrochemical detector manufactured by Shiseido Co., Ltd., applied voltage +800 mV).

  For comparison, the same concentration solution of arbutin and kojic acid, which are said to have antioxidant activity, was measured by the same method.

  The current change value (calculated from the peak height) of each substance measured by this method was as follows.

Sample Current change value
Example 5 mM HQR 1040 nA
Comparative Example 1 5 mM Arbutin 1150 nA
Comparative Example 2 5 mM Kojic acid 300 nA

  From the above results, it was found that HQR is approximately the same as arbutin and has a reducing power 3 to 4 times stronger than kojic acid, indicating that HQR has antioxidant ability.

[The invention's effect]
As described above in detail, according to the present invention, the rutinose glycoside can be produced from a phenol compound simply and efficiently. Moreover, the rutinose glycoside of the phenol compound obtained by the present invention is a novel compound because the sugar moiety is a disaccharide rutinose. In addition, many glycosides obtained by the method of the present invention have useful pharmacological actions such as tyrosinase inhibitory activity, whitening effect, melanin production inhibition, and active oxygen inhibition, and have few side effects and poor solubility. Therefore, the present invention greatly contributes to society as providing such useful compounds.

It is a figure which shows the result of the HPLC analysis (UV detector) of the refinement | purification thing (HQR) obtained in Example 1. FIG. It is a figure which shows the result of the HPLC analysis (RI detector) of the purified product (HQR) obtained in Example 1. It is a figure which shows the UV absorption spectrum of the refinement | purification thing (HQR) obtained in Example 1. FIG. It is a figure which shows UV absorption spectrum of hydroquinone. 1 is a diagram showing a ¹³ CNMR spectrum of a purified product (HQR) obtained in Example 1. FIG. It is a figure which shows the result of the HPLC analysis (UV detector) of the decomposition product of HQR obtained in Example 3. It is a figure which shows the result of the HPLC analysis (RI detector) of the decomposition product of HQR obtained in Example 3. It is a figure which shows the tyrosinase inhibitory activity of HQR.

Claims (1)

  A whitening agent containing a hydroquinone rutinose glycoside as an active ingredient.

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