JP2016222581A - Precursor of vitexin 2" sulfate or isovitexin 2" sulfate, melanogenesis inhibitor and food and drink comprising the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a melanogenesis inhibitor comprising tea leaves-derived skin-whitening components with a high level of safety as an active ingredient, and food and drink comprising the skin-whitening components in large quantities.SOLUTION: A melanogenesis inhibitor comprises vitexin 2" sulfate represented by formula (8) as an active ingredient, the vitexin 2" sulfate having a compound X represented by formula (1) and the like as a precursor.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a highly safe tea leaf-derived melanin production inhibitor and food and drink containing the same.

  In pigmentation such as spots and freckles, melanocytes activated by UV irradiation (= pigment cells existing in the basal layer of the epidermis) produce excessive melanin, and the melanin accumulates in adjacent keratinocytes. It occurs in. As a method for preventing and improving pigmentation, oral ingestion of a component that can suppress melanocyte activation and suppress melanin production (ie, whitening component) and / or application of a skin external preparation containing the component is common. It is. However, some of the compounds (especially chemically synthesized products) used as the whitening component are worried about safety, so many consumers demand a whitening component derived from a highly safe plant.

  As such a plant, tea is a very promising plant. Tea leaves have been used for drinking since ancient times, and safety is widely recognized along with various effects. Then, chafuroside A (chafuroside A, formula (1)) and chafuroside B (chafuroside B, formula (2)), which are flavone C glycosides (compounds in which anomeric carbon of sugar and flavone are covalently bonded) contained in tea leaves, Has been reported to suppress the activation of melanocytes by ultraviolet irradiation (Patent Documents 1 and 2). Chafloside A and B are structural isomers but are not tautomers, and it has been clarified that chafloside B is more effective than chafloside A (Patent Document 2).

  Thus, Chafloside A and B (especially Chafloside B) are plant-derived and highly safe whitening components, but have a problem that the content in tea leaves is very low. In particular, the content of chafloside B is very low (usually about 1/3 that of chafloside A). The most abundant oolong tea leaves are 2-3 μg / 1 g tea leaves, and the green tea leaves are about 2-30 ng / 1 g tea leaves. Therefore, since the isolation of chafuroside A and B from tea leaves is low in yield and cost and poor in practicality, chafroside A is obtained from the related flavone C glycosides isovitexin and vitexin by Mitsunobu reaction. And a method for synthesizing B has been proposed (Patent Document 3). However, since this method requires an azo reagent with a high risk of explosion, it is not suitable for industrial mass production.

  In response to this problem, the present inventor paid attention to the synthesis route of chafuroside A and B, and as a precursor of chafuroside A, a sulfated form of isovitexin (isovitexin 2 ”-O-sulfate (sulfated isovitexin represented by Formula 3) was used. 2 ")) was identified as a precursor of chafuroside B, and a sulfated form of vitexin (vitexin 2" -O-sulfate (sulfated vitexin 2 ") represented by Formula 4) was identified (see Patent Document 4). And (1) the heat treatment conditions for increasing the content of sulfated vitexin 2 ″ in tea leaves and (2) the heat treatment conditions for promoting the conversion of sulfated vitexin 2 ″ to chafloside B are found to be different, It is shown that dry tea leaves containing a high concentration of chafuroside B can be produced by performing the heat treatment (2) after the heat treatment (1) on the tea leaves (Patent Document 5).

  However, the method of Patent Document 5 is a method of promoting the reaction in which chafuroside B is produced from sulfated vitexin. Therefore, tea leaves containing more “precursor of sulfated vitexin 2” are expected to significantly increase the content of sulfated vitexin 2 ”(or its downstream product, chafuroside B) by the method of Patent Document 5. It will be. However, research on the upstream of sulfated vitexin 2 ″ has not progressed, and the precursor was completely unknown.

JP 2012-219076 A WO2012 / 141255 JP 2005-289888 A WO2010 / 0776879 Japanese Patent No. 5229839

Journal of Agricultural and Food Chemistry, Vol. 57, 6779-6786 (2009)

  This invention is made | formed in view of the said situation, and it aims at provision of the food / beverage products which contain many whitening ingredients and the melanin production inhibitor which uses the whitening ingredient derived from tea leaves highly safe as an active ingredient.

  As a result of intensive studies by the inventor in order to solve the above problems, it has been found that not only chafuroside B but also its precursor sulfated vitexin 2 ″ can function as a melanin production inhibitor. From the hot water extract of tea leaves, a novel compound (= Compound X) was successfully identified as a precursor capable of producing the sulfated vitexin 2 ″, and the present invention was completed.

That is, according to the present invention, a compound X represented by any one of the following chemical formulas (5) to (11) is provided as a precursor that produces sulfated vitexin 2 ″. Are all tautomers of the formula (5), that is, the compound represented by the formula (6) and the compound represented by the formula (7) are enolated between two benzene rings. The compound represented by the formula (5) has a β-diketone structure, the compound represented by the formula (6) and the compound represented by the formula (7) and the formula (5). A compound having a keto-enol tautomer relationship, a compound represented by Formula (8), Formula (9), or Formula (10) and Formula (11). The compound is represented by the compound represented by the formula (5), the compound represented by the formula (6), or the formula (7). A compound produced by a cycloaddition reaction in a compound, and since a ring-closing (cyclization) reaction and a ring-opening reaction occur reversibly, the compound represented by formula (5), the compound represented by formula (6) And a compound compatible with the compound represented by formula (7).

The present invention provides a melanin production inhibitor containing sulfated vitexin 2 ″ as an active ingredient.
Furthermore, food / beverage products for ingesting 1 mg / kg body weight or more per day of the melanin production inhibitor in terms of sulfated vitexin 2 ″ amount are provided.

  According to the present invention, a plant-derived highly safe melanin production inhibitor containing sulfated vitexin 2 ″ as an active ingredient and compound X which is a precursor of the sulfated vitexin 2 ″ are provided. And by using the tea leaf which contains many compounds X, the melanin production inhibitor and food-drinks containing the said sulfated vitexin 2 '' can be easily manufactured now.

This is a result of measuring the number of activated melanocytes (= DOPA-positive melanocytes) by irradiating a mouse orally administered with sulfated isovitexin 2 ″ or sulfated vitexin 2 ″. FIG. 2 is a diagram showing a mechanism for producing sulfated isovitexin 2 ″ and sulfated vitexin 2 ″ from compound X. FIG. 3 is a diagram showing a mechanism in which seven tautomers of Compound X are generated with each other. It is a figure showing the structure of compound X (7 types of tautomers) suggested from the NMR analysis result. 2 is a diagram showing the structures of two isomers obtained as a methylated product of Compound X. FIG. 1 is a diagram showing a reaction mechanism in which a methylated product is generated from compound X.

  Hereinafter, preferred embodiments of the present invention will be described.

[Precursor of sulfated vitexin 2 "]
The precursor of sulfated vitexin 2 ″ found in the present invention is a novel compound X represented by any one of the following chemical formulas (5) to (11).

This compound X is efficiently converted to sulfated vitexin 2 ″ and / or sulfated isovitexin 2 ″ by appropriate heat treatment (FIG. 2). That is, Compound X is a common precursor of sulfated vitexin 2 ″ and sulfated isovitexin 2 ″ present in tea leaves, which was first discovered by the present inventors.
The present inventor has already reported that the content of sulfated vitexin 2 ″ can be remarkably increased by maintaining the dry tea leaves in the range of 120 to 170 ° C. for 1 minute 30 seconds to 4 minutes 30 seconds. Therefore, it is considered that a tea leaf containing a large amount of sulfated vitexin 2 ″ can be obtained by using this method for a tea leaf containing a large amount of Compound X.
In addition, as described in Patent Document 5, as tea leaves that can be suitably used in that case, green tea that is non-fermented tea and tea leaves for roasted tea (for example, Karabe ni tea, Yabukita tea, etc.), Examples include tea leaves for oolong tea that are semi-fermented teas (for example, junran incense, martial narcissus, daffodil, narcissus, etc.), tea leaves for tea that are fermented teas (for example, kimmon), and the like.

[Melanin production inhibitor]
The melanin production inhibitor according to the present invention contains sulfated vitexin 2 ″ represented by the following chemical formula (4) as an active ingredient.

As the melanin production inhibitor according to the present invention, sulfated vitexin 2 ″ isolated from tea leaves according to a known method (for example, the method described in Non-Patent Document 1) can be used. As shown in Fig. 5, a compound X isolated from tea leaves and converted to sulfated vitexin 2 "by heat treatment may be used. The compound X may be ingested / administered in any of the seven types of tautomers described above and is effective for the living body.
The melanin production inhibitor according to the present invention is 1 mg / kg body weight or more per day, more preferably 5 mg / kg body weight or more, most preferably 10 to 30 mg / kg body weight, in terms of the content of the sulfated vitexin 2 ″. If the dose is less than 1 mg / kg body weight, a sufficient whitening effect may not be observed, and even if it is ingested in excess of 30 mg / kg body weight, the whitening effect commensurate with the increase in the intake is not so much. Because it is not expected.

[Food and drink containing melanin production inhibitor]
The melanin production inhibitor according to the present invention can be suitably blended in a beverage or food that is expected to have a whitening effect. In addition to sulfated vitexin 2 ″, which is an active ingredient, these foods and drinks can optionally contain additives as necessary. As additives, excipients, flavoring agents, coloring agents, Examples include preservatives, thickeners, stabilizers, gelling agents, antioxidants, and functional materials.

  Examples of the functional materials include various vitamins, pantothenic acid, folic acid, biotin, zinc, calcium, magnesium, amino acids, oligosaccharides, propolis, royal jelly, EPA, DHA, coenzyme Q10, chondroitin, lactic acid bacteria, lactoferrin, isoflavone, prune, Examples include chitin, chitosan, and glucosamine.

  The excipient is not particularly limited as long as it is usually used when a desired dosage form is used. For example, powders such as fine particle silicon dioxide, sucrose fatty acid ester, crystalline cellulose / sodium carboxymethylcellulose, Calcium hydrogen phosphate, wheat starch, rice starch, corn starch, potato starch, dextrin, cyclodextrin and other starches, crystalline celluloses, lactose, glucose, sugar, reduced maltose, starch syrup, fructooligosaccharide, emulsified oligosaccharide Sugar alcohols such as sorbitol, erythritol, xylitol, lactitol, and mannitol.

Examples of the flavoring agent include bontang extract, lychee extract, apple juice, orange juice, yuzu extract, peach flavor, ume flavor, acesulfame K, erythritol, oligosaccharide, mannose, xylitol, isomerization, which are sweeteners. Examples include sugars, tea components such as green tea, oolong tea, banaba tea, tochu tea, iron kannon tea, pearl barley tea, amacha tea, macomo tea, kelp tea, and yogurt flavor.
The colorant, preservative, thickener, stabilizer, gelling agent, and antioxidant can be appropriately selected from known materials used for food and drink.

As the form of the melanin production inhibitor according to the present invention, any form such as ampoules, capsules, pills, tablets, powders, granules, solids, liquids, gels or bubbles, creams and the like can be selected.
Examples include beauty / health drinks, foods, pharmaceuticals, confectionery, Japanese confectionery, general confectionery such as gum, candy, caramel, etc., general soft drinks such as fruit juice, processed fishery products such as kamaboko and chikuwa, sausages, Livestock products such as ham, raw noodles, boiled noodles, buckwheat noodles, sauces, soy sauce, sauce, sugar, honey, powdered candy, condiments such as syrups, spices such as curry powder, mustard powder, pepper powder, jam, Examples include dairy products such as marmalade, chocolate spread, cheese, butter and yogurt. Preferably, from the viewpoint of good ingestion efficiency, orally administered beauty / health drinks or beauty / health foods and the like can be mentioned.
Moreover, these can be manufactured by a conventionally well-known method.

Furthermore, the melanin production inhibitor of the present invention can also be blended in a composition such as a skin external preparation in anticipation of the effect of percutaneous absorption. Examples of the external preparation for skin include cosmetics, patches, pharmaceuticals and the like.
In addition to sulfated vitexin 2 ", which is an active ingredient, external preparations for skin include ingredients usually used for external preparations for skin such as cosmetics and pharmaceuticals, such as moisturizers, antioxidants, oily ingredients, UV absorbers, interfaces Activators, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, and the like can be appropriately blended as necessary.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples. In addition, sulfated vitexin 2 ″ and sulfated isovitexin 2 ″ used in the following examples were isolated from tea leaves according to a known method (Non-patent Document 1).

[Test Example 1: Examination of inhibitory effect on activation of melanocytes by ultraviolet rays]
As described above, chaflosides A and B are known to have an inhibitory effect on activation of melanocytes induced by ultraviolet rays (Patent Documents 1 and 2). Therefore, it was decided to examine whether or not these precursors, sulfated vitexin 2 ″ and sulfated isovitexin 2 ″, had the effect.

<Test method>
Mice were forcibly orally administered a medium containing sulfated vitexin 2 "or sulfated isovitexin 2" into the stomach for 9 days (once per 1 mg sulfated vitexin 2 "or sulfated isovitexin 2" / 5 ml vehicle / kg body weight). /Day). As a medium, a 0.5% (w / v) methylcellulose 400 solution was used. As a mouse, a 5-week-old DBA / 2 mouse (male, manufactured by Charles River Japan Co., Ltd.) was purchased and used for the experiment after acclimation breeding for 1 week.
After 9 days of administration, the mice were irradiated with 120 mJ / cm ² of medium wavelength ultraviolet light (UVB), and the skin was collected the next day (= 10th day) to perform DOPA reaction. The skin was observed with a microscope, and the number of DOPA positive melanocytes per unit area (= number of activated melanocytes) was measured. In the non-administration group (= positive control), only the vehicle was administered for 9 days.
In each test group, 8 mice were used, and the average value and standard deviation were calculated. Significance with the non-administration group was tested by Student-t test, and a significance level of less than 5% was regarded as statistical significance. The results are shown in Table 1 and FIG.

<Result>

As shown in Table 1 and FIG. 1, the number of DOPA-positive melanocytes after ultraviolet irradiation was significantly reduced in mice that were orally administered sulfated vitexin 2 ″ compared to mice that were not administered (76.7). %, P <0.05) On the other hand, in the mice orally administered with sulfated isovitexin 2 ″, the number of DOPA-positive melanocytes tended to decrease, but was not statistically significant (p ≧ 0.05).
Thus, it was shown that sulfated vitexin 2 ″ can effectively suppress the activation of melanocytes induced by ultraviolet rays, that is, it can function as a safe plant-derived melanin production inhibitor. It was also revealed that sulfated isovitexin 2 ″, which is a precursor of A, is inferior to sulfated vitexin 2 ″ as a melanin production inhibitor.

[Test Example 2: Isolation of Compound X from tea leaves]
Next, an attempt was made to isolate a compound capable of producing sulfated vitexin 2 ″, that is, a precursor of sulfated vitexin 2 ″ from tea leaves.

-Isolation of C-glycoside sulfide compound from oolong tea leaves (Milan incense tea leaves) Add 400% fine powder of tea leaves (variety: Miran incense) to a 50% methanol aqueous solution and stir with heating at 50 ° C for 15 minutes. Obtained an extract of honey orchid. The resulting extract was diluted with water to give a 5% aqueous methanol solution, and then subjected to aromatic Diaion (registered trademark) HP-20 column chromatography (1000 ml, 5% aqueous methanol solution) while gradually increasing the methanol content. Elute. Only the fraction containing a large amount of the target product obtained by this method was concentrated at 40 ° C. or lower. Subsequently, the obtained concentrate was again subjected to HP-20 column chromatography (300 ml, 5, 10 and 15 and a 20% aqueous methanol solution were used as a developing solvent). Furthermore, the fraction containing a large amount of the desired product was subjected to Sephadex LH-20 column chromatography (3 × 150 cm, developing solvent methanol). After the above operation was performed twice, high-performance liquid chromatography (HPLC) (Develosil C30-UG-5 (250 × 20 mm) or Cadenza CD 18 (250 × 10 mm) 16 was obtained from the high content fraction of the obtained target product. About 3 mg of the target compound X was obtained using% CH ₃ CN-20 mM CO ₂ NH ₄ (4:21)). The target compound was a compound that gave a chafuroside precursor (sulfated isovitexin 2 ″ and / or sulfated vitexin 2 ″ by dilute formic acid treatment.

[Test Example 3: Structure determination of compound X]
Since it was a novel compound, its structure was determined by the following method.
Estimated structure 1) From the high resolution mass spectrum, the molecular formula of Compound X was determined as C ₂₁ H ₂₂ O ₁₄ S.
_{Negative mode for C 21 H 21 O} 14 S -
Actual value 529.0673 Theoretical value 529.0657
2) Compound X was dissolved in MeOH at room temperature or acid-treated with formic acid-MeOH (1: 1000) at the same temperature. As a result, almost equimolar sulfated isovitexin 2 ″ and sulfated vitexin 2 "Generated.
From 1) and 2), Compound X was considered to be a compound represented by the following formula (5).

That is, in Compound X, a cyclization reaction occurs at a rate similar to that of the two routes (A and B) as shown in FIG. 2, followed by a dehydration reaction to produce sulfated isovitexin 2 ″ and sulfated vitexin 2 ″. It was inferred that

Therefore, the following examination was added to this estimation.
Compound X was subjected to ¹ H-NMR, ¹³ C-NMR, DEPT, HH-COSY, HMBC and NEOSY measurements. The analysis results of the data are shown in Tables 2 and 3. The assignment of each signal in the table is based on the numbering shown in FIG.

Comparison of this result with chafuroside precursors, sulfated isovitexin 2 "and sulfated vitexin 2" and hydroxynaringin derivatives (non-patent literature H, Wakimoto T, Kitao Y, Tanaka S, Miyase T, Nukaya H., J. Agric. Food Chem., 57, 6779-86, 2009, and Kawakami et al., J. Nat. Med (2009) 63, 46- 51), the sugar part of this compound is glucose, the hydroxyl group at the 2-position is sulfated, and the anomeric carbon at the 1-position is β-glycoside-bonded to the carbon atom of the benzene ring of the aglycon part. I can see. And the aglycone part is not cyclized as shown in FIG. 3 under the conditions of the NMR measurement temperature (25 ° C.) and the solvent (d ₆ -DMSO solution). It is presumed to exist in the structure represented by the chemical formula 2-3 in which the 2-position is enolated and the chemical formula 2-4 in which the 4-position is enolized without being cyclized. B and Chemical Formulas 2-2A and B (each A and B are isomers having different OH groups at the 2-position), Chemical Formula 2-3) and Chemical Formula 2-4 (each having different enolization specifications) ) Is considered to occur at high speed.

  However, as shown in Tables 2 and 3, from this result, the structure of the thick line portion of the compound represented by Compound X was confirmed (FIG. 4). Moreover, complete assignment of all signals to each isomer was not possible. For these reasons, this measurement was carried out with the highest level of equipment, but under the measurement conditions, the compound undergoes fast tautomerization in solution, so broadening occurs in many signals. For this reason, it is presumed that the correlation observation in the signal that is easily affected by this becomes extremely difficult.

Therefore, diazomethane and CH ₂ N ₂ were allowed to act on this compound to induce methylation, and the structure was determined by tautomerization.
The isolated compound (about 1 mg) is allowed to react with an excess amount of diazomethane (CH ₂ N ₂ ) at room temperature in a mixture of methanol and ether (condition: excess CH ₂ N ₂ is allowed to act overnight at room temperature), and then slightly. Excess N (CH ₃ ) ₃ was allowed to act overnight at room temperature. One of the obtained methylation products was subjected to high performance liquid chromatography (HPLC) (Develosil C30-UG-5 (250 × 20 mm), Cadenza CD C18 (250 × 10 mm), MeOH-10 mHCO ₂ NH ₄ mixed solvent) ) To obtain about 0.5 mg.

The molecular formula was determined to be C ₂₄ H ₂₈ O ₁₄ S from the high resolution mass spectrum of the obtained compound (the methylated product).
_{Negative mode for C 24 H 27 O} 14 S -
Actual value 571.1117 Theoretical value 571.1127

The ¹ H-NMR, ¹³ C-NMR, DEPT, HH-COSY, HMBC and NEOSY measurements were performed. The analysis results are shown in Table 4. The assignment of each signal in the table is based on the numbering shown in FIG.

From the analysis results of the above data, the methylated compound can be described as a compound having the structure shown in FIG. 5, Chemical Formula 3A, but Chemical Formula 3B cannot be denied. Therefore, as shown in FIG. 6, formic acid diluted with MeOH was allowed to act on this and cyclized, followed by dehydration to lead to a flavone body (Chemical Formula 4). Separately, two chafuroside precursors, sulfated isovitexin 2 "and sulfated vitexin 2", were allowed to react with an excess amount of diazomethane (CH ₂ N ₂ ) at room temperature in a mixture of methanol and ether (condition: excess amount). CH ₂ N ₂ was allowed to act overnight at room temperature), and then a slight excess of N (CH ₃ ) ₃ was allowed to act overnight at room temperature. In this manner, trimethylated compounds (Chemical 4A and Chemical Formula 4B) obtained by methylating the three phenolic hydroxyl groups of both chafuroside precursors were prepared. Subsequently, the structure of Chemical Formula 4 was determined to be a trimethylated form of sulfated isovitexin 2 ″ (Chemical Formula 4A in FIG. 5) by direct comparison between the trimethylated form of both chafuroside precursors and Chemical Formula 4 obtained.

From these results, Compound X could be determined as the compound represented on the left side of FIG. 2 (that is, the compound represented by the following chemical formula (5)).
In addition, it is thought that the compound X exists mainly as a cyclized C-glycoside compound represented on the right side of FIG. 3 in a highly polar solution. Therefore, each of the three phenolic hydroxyl groups of the cyclized compound was methylated with CH ₂ N ₂ to produce a trimethylated product. And it can be explained that one of them was isolated this time (Fig. 6).

In Chemical Formula 3, if there is a ketone group at the 4-position, chelate formation between two hydroxyl groups becomes possible (the following chemical formula (12), the structure on the left), which can greatly contribute to the stabilization of this structure.
On the other hand, in the structure on the right side of the chemical formula (12) having a ketone group at the 2-position, the 4-position is enolized, and a chelate is formed between the hydroxyl group formed at this time and the ketone group. Cannot exist on the same plane as the left pentasubstituted benzene ring. For this reason, it is placed at a position substantially perpendicular to this plane, and is more unstable than the case where the ketone group is at the 4-position. From this, it can be understood that the state of chemical 3 is mainly the state of chemical 3A (the above chemical formula (12), left structure).

  From the above results, it was strongly suggested that Compound X exists as a compound represented by any one of the following formulas (5) to (11).

[Composition example]
Although the example of a food / beverage product which mix | blended the melanin production inhibitor concerning this invention is given next, this invention is not limited by these.
Formulation Example 1 Soft capsule brown rice germ oil 710mg
Sulfated Vitexin 2 "100mg
Elastin 130mg
DNA 30mg
Folic acid 30mg

Formulation Example 2 Candy sugar 2300mg
Minamata 1486mg
Sulfated Vitexin 2 "38mg
Fragrance 38mg

Formulation Example 3 Sulfated Vitexin 2 ”High Content Tea Beverage Green Tea Extract (Supernatant obtained by extracting 20 g of dry tea leaves of green tea with 1 liter of hot water (80 ° C., 5 minutes) and then filtering) 1000 g
L-sodium ascorbate 200mg
Sulfated Vitexin 2 "100mg
Citric acid 10mg

Claims (3)

Compound X represented by the following chemical formula (1), and tautomers thereof represented by the following chemical formulas (2) to (7).
A melanin production inhibitor containing sulfated vitexin 2 ″ represented by the following chemical formula (8) as an active ingredient.
  A food or drink for ingesting the melanin production inhibitor according to claim 2 in an amount of 1 mg / kg body weight or more per day in terms of the amount of sulfated vitexin 2 ".