JP2010285364A - Singlet oxygen scavenger - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a singlet oxygen scavenger containing a carotenoid as an active ingredient and having a more improved effect thereof. <P>SOLUTION: The singlet oxygen scavenger includes (A) crocetin and (B) xanthophyll as active ingredients in a molar ratio (A)/(B) of (A) and (B) of 0.4-20. The singlet oxygen scavenger has excellent singlet oxygen-scavenging ability and is used for preventing various disorders or diseases associated with singlet oxygen. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a singlet oxygen scavenger.

In recent years, attention has been focused on singlet oxygen ( ¹ O ₂ ), which is a kind of active oxygen, involved in aging and various diseases. For example, singlet oxygen is particularly likely to be generated at sites that are directly exposed to sunlight, such as the eyes and skin, so singlet oxygen generated at such sites may cause cataracts, age-related macular degeneration, skin aging, etc. It is thought to cause. Therefore, several methods for eliminating singlet oxygen have been proposed for the purpose of preventing such diseases and disorders.

  For example, a singlet oxygen scavenger comprising coenzyme Q10 (see Patent Document 1), a singlet oxygen scavenger containing an organic solvent extract of a roasted plant fiber material hot water extract (see Patent Document 2), A singlet oxygen production inhibitor (see Patent Document 3) comprising D-cystenolic acid as an active ingredient has been proposed.

  On the other hand, it is known that crocetin, which is a kind of carotenoid and contained in gardenia fruit or saffron pistil, exhibits an effect as a singlet oxygen scavenger (see Patent Document 4). However, such a singlet oxygen scavenger is not always satisfactory in its effect.

JP 2006-213660 A JP 2005-325034 A JP 2001-288078 A JP 05-320036 A

  An object of the present invention is to provide a singlet oxygen scavenger containing a carotenoid as an active ingredient, the effect of which is further improved.

As a result of intensive studies to solve the above problems, the present inventors have found that remarkable singlet oxygen scavenging ability is exhibited when crocetin, which is a kind of carotenoid, and lutein are used in combination. Completed the invention.
That is, the present invention
[1]. (A) A singlet oxygen scavenger containing crocetin represented by the following formula (1) and (B) xanthophyll as active ingredients, wherein the molar ratio (A) / (A) and (B) Singlet oxygen scavenger, wherein B) is 0.4-20,

［２］．前記キサントフィルが、ルテイン、ゼアキサンチン、アスタキサンチン、β-クリプトキサンチン、フコキサンチン、フコキサンチノール、カプサンチン、カプソルビン、ビキシン及びノルビキシンの群から選ばれる一種又は二種以上である［１］に記載の一重項酸素消去剤、
を提供するものである。

[2]. The singlet according to [1], wherein the xanthophyll is one or more selected from the group consisting of lutein, zeaxanthin, astaxanthin, β-cryptoxanthin, fucoxanthin, fucoxanthinol, capsanthin, capsorubin, bixin and norbixin. Oxygen scavenger,
Is to provide.

The singlet oxygen scavenger of the present invention has an excellent singlet oxygen scavenging ability.
The singlet oxygen scavenger of the present invention can be used for prevention of various disorders or diseases in which singlet oxygen is involved.

  Crocetin used as the component (A) in the present invention is a compound (molecular weight 328.40) represented by the following formula (2).

  This crocetin is usually obtained by hydrolyzing crocin (a digentiobiose ester of crocetin), which is a carotenoid yellow pigment. Crocin is contained in the fruits of Rubiaceae gardenia (Gardenia augusta MERILIL var. Grandiflora HORT., Gardenia jasminoides ELLIS), dried saffron stigmas, and the like. Gardenia fruit is preferably used as an industrial raw material for obtaining crocin.

  There is no restriction on the method of extracting crocin from the gardenia fruit, for example, a dried gardenia fruit is pulverized and extracted using water, alcohol (for example, methanol, ethanol, etc.) or a mixture thereof. The method is used. For example, when the water / alcohol mixed solution (1: 1) is used, the extraction condition is preferably room temperature (about 0 to 30 ° C.) to 50 ° C. for about 1 to 18 hours, and about 30 to 40 ° C. for about 2 to 4 hours. Is more preferable. In order to further increase the extraction rate of crocin from the pulverized dried fruit, the extraction operation is usually repeated a plurality of times. The extract containing crocin is concentrated by a method known per se, and is usually stored refrigerated as a concentrated solution.

  The hydrolysis of crocin may be performed according to a conventional method, and is usually performed using an acid, an alkali or a suitable hydrolase. Examples of the acid include hydrochloric acid, sulfuric acid, and phosphoric acid. Examples of the alkali include sodium hydroxide, potassium hydroxide, sodium carbonate and potassium carbonate. Examples of hydrolases include β-glucosidase.

  Industrially, crocin is usually hydrolyzed using alkali. For example, an excess amount of an aqueous sodium hydroxide solution is added to the concentrate containing crocin, and preferably at room temperature (about 0 to 30 ° C.) to 70 ° C. for about 1 to 24 hours, preferably about 40 to under stirring. React at 60 ° C. for about 3-5 hours.

  After completion of hydrolysis with an alkali, an appropriate amount of an aqueous solution of an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid such as citric acid is added to the reaction solution, and the liquidity is about pH 4.0 or less, preferably about pH 1. Crystals of crocetin are precipitated by adjusting to 0 to 3.0. Alternatively, the reaction solution may be added to an aqueous solution of an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid such as citric acid to precipitate crocetin crystals. Thereafter, the liquid mixture containing the crocetin crystals is subjected to solid-liquid separation to obtain a suspension or slurry containing the crocetin crystals.

  When crocin is hydrolyzed using an acid, crocetin crystals are usually precipitated simultaneously with the hydrolysis, so that the reaction solution is obtained as a suspension containing crocetin. After completion of the reaction, the resulting suspension is subjected to solid-liquid separation to obtain a suspension or slurry containing crocetin crystals.

  The suspension or slurry containing crocetin crystals obtained as described above is mixed with crocetin crystals together with acids, neutralized salts and impurities derived from the raw materials. A cleaning process is performed. The treatment may be performed by a known method such as washing the suspension or slurry with a sufficient amount of water, alcohol or a mixture thereof. The washing process is usually repeated several times until the desired purity is obtained. After the washing treatment, the suspension or slurry containing crocetin crystals is dried at a temperature not exceeding about 50 ° C. using, for example, a vacuum dryer to obtain purified crocetin. The purified crocetin is preferably sealed and stored in a container substituted with an inert gas such as nitrogen gas.

  The content of crocetin used in the present invention is calculated based on the following formula from the color value of the sample containing crocetin.

  In addition, the said color value is measured based on the following [color value measuring method].

[Color value measurement method]
1) A sample is accurately weighed so that the absorbance to be measured is in the range of 0.3 to 0.7, and dissolved in dimethyl sulfoxide (DMSO) to make exactly 100 ml.
2) Weigh exactly 5 ml, and add Kolthoff buffer solution (50 mM Na ₂ B ₄ O ₇ .10H ₂ O-50 mM Na ₂ CO ₃ , pH 10.0) to make 50 ml.
3) Weigh exactly 5 ml of the solution, and add Kolthoff buffer (pH 10.0) to make 50 ml.
4) Weigh exactly 5 ml, add Kolthoff buffer (pH 10.0) to make 50 ml, and use this solution as the test solution.
5) Using a Kolthoff buffer solution (pH 10.0) as a control, measure the absorbance A at the maximum absorption portion near 420 nm with a liquid layer length of 1 cm, and obtain the color value by the following formula.

  As the component (A) of the present invention, a pharmacologically acceptable salt of crocetin may be used. Examples of such salts include pharmaceutically acceptable salts of Group 1 elements such as sodium and potassium, salts of Group 2 elements such as magnesium and calcium, pyridine, dimethylamine, diethylamine and ethanolamine. Examples thereof include salts of organic amino compounds.

  As the xanthophyll used as the component (B) in the present invention, for example, one or two selected from the group of lutein, zeaxanthin, astaxanthin, β-cryptoxanthin, fucoxanthin, fucoxanthinol, capsanthin, capsorbine, bixin and norbixin The above is preferable, and lutein is particularly preferable.

  The xanthophyll may be chemically synthesized as long as it meets the object of the present invention, or may be derived from a natural product. The chemically synthesized xanthophyll is not particularly limited as long as it is synthesized by a method known per se. As a xanthophyll derived from a natural product, for example, lutein that uses flowers of Tagetes erecta WILLD as an industrial raw material can be mentioned.

  There are two types of xanthophyll, one in which a fatty acid is ester-bonded to a hydroxyl group (for example, lutein fatty acid ester) and the other in a free form in which no fatty acid is bound. Used. The fatty acid constituting the ester is, for example, palmitic acid, myristic acid, arachidonic acid, stearic acid, lauric acid, linolenic acid, oleic acid or linoleic acid.

When free form lutein (molecular formula C ₄₀ H ₅₆ O ₂ , molecular weight 568.87) is used as the component (B) of the present invention, its content is determined by carrying out the following method on a sample containing free form lutein. Can be measured.

[Method for measuring free lutein content]
About 0.1 g of a sample is accurately weighed and dissolved in ethanol to make 100 ml. This is diluted with ethanol so that the absorbance is 0.3 to 0.7, the absorbance at the maximum absorption around 445 nm of this diluted solution is measured with a spectrophotometer, and the content is calculated by the following formula.

  In the above formula, “2550” is the extinction coefficient of free lutein at 445 nm.

  When using ester lutein (lutein fatty acid ester) as the component (B) of the present invention, the content of lutein can be measured by carrying out the following method on a sample containing the ester lutein. According to the following method, since the ester lutein is hydrolyzed to a free lutein by saponification, the content of the free lutein can be measured.

[Method for measuring free lutein content by saponification]
About 0.1 g of a sample is accurately weighed and placed in a 100 mL volumetric flask, and 30 mL of n-hexane / acetone / ethanol / toluene mixture (10: 7: 6: 7) is added and mixed. 2 mL of 40% potassium hydroxide-methanol solution is added to this and mixed, and then the measuring flask is placed in a 70-degree water bath and heated for 20 minutes. After heating, remove the volumetric flask from the water bath, cool to room temperature, and leave it in the dark for 1 hour. Next, 30 mL of n-hexane is added and mixed, and a 10% sodium sulfate solution is further added to make a total volume of 100 mL. After stirring the obtained liquid mixture, it is left still in a dark place for 1 hour, and the contents of the volumetric flask are separated into an upper layer (50 mL) and a lower layer (50 mL). Dilute the upper layer with ethanol so that the absorbance is 0.3 to 0.7, measure the absorbance at the maximum absorption near 437 to 445 nm of this diluted solution with a spectrophotometer, and calculate the content by the following formula To do.

  In the above formula, “2550” is an extinction coefficient of free lutein.

  In the singlet oxygen scavenger of the present invention, the above components (A) and (B) are used as they are, or pharmaceutical additives, food additives, food materials and the like are appropriately blended, and according to a conventional method, for example, a liquid (for example, a drink) ), Powders, granules, tablets, microcapsules, soft capsules, hard capsules, oil / fat compositions, O / W emulsions, W / O emulsions or solubilized liquids.

  Examples of pharmaceutical additives, food additives, and food materials used in the preparation of the above preparations include excipients (lactose, dextrin, corn starch, crystalline cellulose, etc.), lubricants (magnesium stearate, sucrose fatty acid ester, glycerin). Fatty acid esters, etc.), disintegrating agents (carboxymethylcellulose calcium, anhydrous calcium hydrogen phosphate, calcium carbonate, etc.), binders (starch glue solution, hydroxypropylcellulose solution, gum arabic solution, etc.), solubilizers (gum arabic, polysorbate 80) Etc.), sweeteners (sugar, fructose glucose liquid sugar, honey, aspartame, etc.), coloring agents (β-carotene, edible tar dye, riboflavin, etc.), preservatives (sorbic acid, methyl parahydroxybenzoate, sodium sulfite, etc.), Thickener (alginic acid Thorium, sodium carboxymethylcellulose, sodium polyacrylate, etc.), antioxidants (BHT, BHA, ascorbic acid, tocopherol, etc.), flavors (mint, strawberry flavor, etc.), acidulants (citric acid, lactic acid, DL-malic acid, etc.) ), Seasoning (DL-alanine, sodium 5'-inosinate, sodium L-glutamate, etc.), emulsifier (glycerin fatty acid ester, sucrose fatty acid ester, etc.), pH adjuster (citric acid, trisodium citrate, etc.), Vitamins, minerals, amino acids and the like can be mentioned.

  In 100% by mass of the preparation, the total content of the component (A) and the component (B) is usually about 0.0001 to 50% by mass, preferably about 0.001 to 20% by mass, more preferably about 0. 0.01 to 10% by mass.

  Furthermore, the singlet oxygen scavenger of the present invention can take the form of food and drink. Examples of the food and drink include soft drinks, drops, candy, chewing gum, chocolate, gummy, yogurt, ice cream, pudding, jelly confectionery, and cookies.

  When the singlet oxygen scavenger of the present invention is orally ingested, the daily dose of the above component (A) for adults is in the range of about 0.1 to 500 mg, and the above component (B) for adults per day. The dose is in the range of about 1-100 mg. However, the actual dose should be determined in consideration of the purpose and the situation of the intaker (gender, age, health status, etc.).

  Since the singlet oxygen scavenger of the present invention is excellent in singlet oxygen scavenging ability, it has various disorders or diseases involving singlet oxygen (for example, cataract, age-related macular degeneration, retinal vascular occlusion, etc.). Can be used for prevention.

  Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited thereto.

[Test example]
[Evaluation of singlet oxygen scavenging ability]

  The singlet oxygen scavenging ability when crocetin was used in combination with lutein and lutein fatty acid ester was evaluated by the ESR method.

(1) Preparation of sample solution (a) 3.3 mg of purified crocetin (crocetin content 99%) was dissolved in 10 mL of 100% DMSO to prepare a 1 mM crocetin solution.
(B) 5.7 mg of lutein (product name; lutein content 98%; manufactured by EXTRASYNTHESE) was dissolved in 10 mL of 100% DMSO to prepare a 1 mM lutein solution.
(C) The crocetin solution and the lutein solution were each mixed in the ratio shown in Table 1 to prepare sample solutions 1 to 8.

(2) Preparation of Evaluation Sample An evaluation sample was prepared by mixing the following 1) to 5). As a control, an evaluation specimen was prepared in the same manner using 50 μL of the crocetin solution instead of 50 μL of the sample solution of 4).
1) Phosphate buffer solution (pH 7.2) 700 μL
2) Rose Bengal (50 μM) 100 μL
3) 4-OH-TEMP 100 μL
4) Sample solution (any one of 1 to 8) 50 μL
5) Ultrapure water 50μL
Rose Bengal was used to generate singlet oxygen by light irradiation. 4-OH-TEMP (2,2,6,6-tetramethyl-4-hydroxyl-piperidine) was used as a supplement for singlet oxygen.

(3) Measurement by ESR apparatus The evaluation sample obtained in (2) was collected in a flat cell, and light irradiation (18000 lux, 5 minutes) was performed on the evaluation sample in the flat cell. Immediately after the light irradiation, this evaluation specimen was subjected to an ESR apparatus (model: JES-RE1X; manufactured by JEOL Ltd.), and the ESR signal intensity was measured under the following conditions.
[ESR measurement conditions]
Center Field: 335.9mT
Modulation Width: 79μT
Sweep Width: 5.0mT
Time constant: 0.03 sec
Sleep Time: 1.0min
Gain: 400

(4) Results The singlet oxygen scavenging rate (%) was determined based on the ESR signal intensity obtained by repeatedly performing the results (1) to (3) three times and the following formula, and the singlet oxygen scavenging rate (%) The mean value ± standard deviation of was calculated. The results are shown in Table 2.

In the above formula, S ₁ is the ESR signal intensity measured for the evaluation specimen, and S ₂ is the ESR signal intensity measured for the evaluation specimen prepared using 5% DMSO instead of the sample solution. is there.

  The results in Table 2 are statistically greater when (A) crocetin and (B) lutein are used in combination and the molar ratio (A) / (B) is 0.4 or more than when crocetin is used alone. It shows that significant singlet oxygen scavenging activity is obtained.

[Example]
Clobit P (product name; crocetin content 75%; manufactured by Riken Vitamin Co.) 50 g and lutein 20 (product name; lutein content 20%; manufactured by Kyowa Hakko Bio) MCT (product name; Actor M1; Riken Vitamin Co., Ltd.) In addition to 800 g, the mixture was dissolved by heating and stirring using a mixer (model Ultra Turrax T-25 Basic; manufactured by IKA Japan), and the resulting solution was vacuum degassed and cooled to room temperature. Next, according to a conventional method, this solution was wrapped with a film mainly composed of gelatin to produce a soft capsule-like singlet oxygen scavenger. This singlet oxygen scavenger contains about 7.5 mg (A) crocetin and 6 mg (B) lutein per capsule. The molar ratio (A) / (B) of the above (A) and (B) contained in this singlet oxygen scavenger is 2.2.

Claims (2)

(A) A singlet oxygen scavenger containing crocetin represented by the following formula (1) and (B) xanthophyll as active ingredients, wherein the molar ratio (A) / (A) and (B) A singlet oxygen scavenger, wherein B) is 0.4-20.

  The singlet according to claim 1, wherein the xanthophyll is one or more selected from the group consisting of lutein, zeaxanthin, astaxanthin, β-cryptoxanthin, fucoxanthin, fucoxanthinol, capsanthin, capsorubin, bixin and norbixin. Oxygen scavenger.