JP5583422B2 - Crocetin composition - Google Patents

Description

  The present invention relates to a composition containing crocetin.

In recent years, attention has been focused on singlet oxygen ( ¹ O ₂ ), which is a kind of active oxygen, being involved in aging and various diseases and disorders. For example, singlet oxygen is likely to be generated in parts such as eyes and skin that are directly exposed to sunlight, and the singlet oxygen thus generated is considered to cause cataracts, age-related macular degeneration, skin aging, etc. Yes. Thus, for the purpose of preventing such diseases and the like, the use of coenzyme Q10, D-cystenoic acid, tocophenol and other substances having the ability to scavenge singlet oxygen has been studied.

  On the other hand, it is a kind of carotenoid, and crocetin obtained by hydrolyzing crocin extracted from gardenia fruit or saffron pistil is also known to exhibit singlet oxygen scavenging ability (see Patent Document 1). ). However, the conventional crocetin is not always satisfactory in terms of the singlet oxygen scavenging ability.

JP 05-320036 A

  An object of the present invention is to provide a composition of crocetin with further improved singlet oxygen scavenging ability.

As a result of intensive studies to solve the above problems, the present inventors have found that crocetin compositions prepared so that the proportion of the 13-ciscrocetin content falls within a certain range are prominent singlet oxygen. The erasing ability was found and the present invention was completed.
That is, the present invention
[1]. Following formula (1)

    13-ciscrocetin represented by the following formula (2)

  A composition containing transcrocetin represented by the formula: wherein the ratio of the content of 13-ciscrocetin is 3 to 10% in the total content of 13-ciscrocetin and transcrocetin; and

[2]. A singlet oxygen scavenger comprising the crocetin composition according to [1] as an active ingredient,
Is to provide.

The crocetin composition of the present invention has excellent singlet oxygen scavenging ability.
The crocetin composition of the present invention can be used as a singlet oxygen scavenger for the prevention of various disorders or diseases involving singlet oxygen.

  Crocetin in the present invention refers to 13-ciscrocetin (molecular weight 328.40) represented by the following formula (1), transcrocetin (molecular weight 328.40) represented by the following formula (2), and these Refers to all isomers.

  This crocetin is usually a carotenoid yellow pigment, crocin (a digentiobiose ester of crocetin), which is a carotenoid yellow pigment contained in the fruits of Rubiaceae (Gardenia augusta MERILIL var. Grandiflora HORT., Gardenia jasminoides ELLIS). Can be obtained. Moreover, crocetin is marketed and a commercial item can also be used.

  The crocetin composition of the present invention is a composition containing the 13-cis crocetin and trans crocetin described above, and the ratio of the content of 13-cis crocetin in the total content of 13-cis crocetin and trans crocetin (below) "13-ciscrocetin amount") is preferably about 3 to 10%, more preferably about 4 to 9.5%, and still more preferably about 5 to 9%.

  There is no particular limitation on the method for producing such a crocetin composition. For example, after washing crocetin produced by a conventional method with water, further washing with an aqueous ethanol solution to remove components soluble in the aqueous ethanol solution. After separating 13-ciscrocetin and transcrocetin from crocetin produced by a method for purification so that the amount of 13-ciscrocetin is contained in the range of about 3 to 10%, or crocetin produced by a conventional method, Examples thereof include a method of preparing a composition in which these are blended so that the amount of crocetin is included in the range of about 3 to 10%. Among these, in the latter method, since 13-ciscrocetin and transcrocetin are separately prepared from crocetin, for example, the following method can be used.

[Method for preparing 13-ciscrocetin]
Dimethylformamide is added to crocetin produced by a conventional method, and heated and dissolved at about 50 to 90 ° C. The obtained insoluble matter is filtered, and the filtrate is allowed to stand at about 1 to 30 ° C. for 3 to 9 days. Next, the mother liquor containing the produced transcrocetin crystals is filtered, and the step of concentrating the obtained filtrate and allowing to stand still is repeated 1 to 5 times (filtration, concentration, and standing step). In this step, concentration is preferably performed using a rotary evaporator at about 70 to 90 ° C. and about 0.5 to 3 kPa, and standing is preferably performed at about 9 to 11 ° C. for 3 to 9 days. . Next, the mother liquor containing transcrocetin crystals produced by the filtration / concentration / standing step is filtered, and the step of concentrating the obtained filtrate is repeated 1 to 5 times (filtration / concentration step). In this step, the concentration is preferably performed using a rotary evaporator under conditions of about 70 to 90 ° C. and about 0.5 to 3 kPa. In this step, methanol may be added to the concentrated solution obtained by concentration, and washing may be performed with an ultrasonic cleaner. Subsequently, the concentrate obtained by the filtration / concentration step is dissolved in methanol, and the resulting solution is supplied to a well-known preparative HPLC system to separate 13-ciscrocetin. Crocetin is prepared.

[Method for preparing transcrocetin]
Dimethylformamide is added to crocetin produced by a conventional method, and heated and dissolved at about 50 to 90 ° C. The obtained insoluble matter is filtered, and the filtrate is allowed to stand at about 1 to 30 ° C. for 3 to 9 days. Next, the mother liquor containing the transcrocetin crystals produced is filtered with a filter, the crystals remaining in the filter are washed with methanol, and further dried at about 30 to 80 ° C. to prepare transcrocetin.

The amount of 13-ciscrocetin in the crocetin composition of the present invention is determined from the peak area of transcrocetin and the peak area of 13-ciscrocetin in the HPLC chromatogram when the crocetin composition is analyzed by HPLC.
Here, the amount of 13-ciscrocetin for the crocetin composition of the present invention is measured by the following method.

[13-ciscrocetin content measurement method]
1) About 6 mg of a sample is accurately weighed and dissolved in 5 mL of dimethyl sulfoxide (DMSO), and methanol is added to make exactly 50 mL.
2) A solution obtained by filtering the solution through a 0.45 μm membrane filter is used as a test solution.
3) 10 μL of the test solution was injected into the HPLC, and the area of the peak around 7 minutes corresponding to transcrocetin and the peak around 13 minutes corresponding to 13-ciscrocetin was respectively measured by Empower Chromatography Manager. The 13-ciscrocetin amount (%) is calculated according to the formula.

  The HPLC analysis system and analysis conditions are shown below.

<HPLC analysis system>
2695 separation module (manufactured by Nippon Waters)
2996 Photodiode Array Detector (Nippon Waters)
Empower Chromatography Manager (Nippon Waters)
<HPLC analysis conditions>
Column: Wakosil 5C18 (inner diameter 4.6 mm, length 150 mm) (manufactured by Wako Pure Chemical Industries, Ltd.)
Mobile phase: 0.1% TFA aqueous solution-0.1% TFA methanol (25:75)
Flow rate: 1 mL / min
Detection: 430nm
Column temperature: 40 ° C

  The crocetin composition of the present invention is prepared by mixing the crocetin composition as it is or appropriately with a pharmaceutical additive, a food additive, a food material, etc., according to a conventional method, for example, a liquid (for example, a drink), a powder, a granule, It can be produced as a preparation in the form of a tablet, microcapsule, soft capsule, hard capsule, oil / fat composition, O / W emulsion, W / O emulsion or solubilized solution.

  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, hydroxypropyl cellulose 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 (Nalginate Sodium, sodium carboxymethylcellulose, sodium polyacrylate, etc.), antioxidants (BHT, BHA, ascorbic acid, tocopherols, etc.), flavors (mint, strawberry flavors, 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. These may be used alone or in combination of two or more. Although content of the crocetin composition in a formulation is not specifically limited, Usually, it is about 0.001-99 mass% with respect to a final formulation.

  Furthermore, the crocetin composition of this invention can take the form of food-drinks. Examples of the food and drink include soft drinks, drops, candy, chewing gum, chocolate, gummy, yogurt, ice cream, pudding, jelly confectionery, and cookies.

  Since the crocetin composition of the present invention is excellent in singlet oxygen scavenging ability, it can be used as a singlet oxygen scavenger containing the crocetin composition as an active ingredient. The intake of the singlet oxygen scavenger can be expected to prevent various disorders or diseases involving singlet oxygen (for example, cataract, age-related macular degeneration, retinal vascular occlusion, etc.).

  When the singlet oxygen scavenger of the present invention is taken orally, the daily dose for an adult is usually in the range of about 0.1 to 500 mg as the active ingredient crocetin composition. This amount is preferably taken in 1 to 3 divided doses. However, the actual dose should be determined in consideration of the purpose and the situation of the intaker (gender, age, health status, etc.).

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

[Production Example 1]
[Preparation of transcrocetin]
840 mL of dimethylformamide was added to 20 g of commercially available crocetin (trade name: clobit P; manufactured by Riken Vitamin Co., Ltd.), and heated and dissolved at 80 ° C. The obtained insoluble matter was filtered with a quantitative filter paper (No. 5C; manufactured by Advantech Toyo Co., Ltd.), and the filtrate was allowed to stand at 10 ° C. for 6 days.
The mother liquor containing the produced transcrocetin crystals was filtered with a glass filter No. 3, and the crystals remaining on the filter were washed with 1300 mL of methanol and further vacuum dried at 50 ° C. to obtain about 10.5 g of transcrocetin. It was. The amount of 13-ciscrocetin of this product was 0.2%.

[Production Example 2]
[Preparation of 13-ciscrocetin]
840 mL of dimethylformamide was added to 20 g of commercially available crocetin (trade name: clobit P; manufactured by Riken Vitamin Co., Ltd.), and heated and dissolved at 80 ° C. The obtained insoluble matter was filtered with a quantitative filter paper (No. 5C; manufactured by Advantech Toyo Co., Ltd.), and the filtrate was allowed to stand at 10 ° C. for 6 days. The resulting mother liquor containing transcrocetin crystals was filtered with a glass filter No. 3, and the filtrate was concentrated under the conditions of about 1 kPa and about 80 ° C. using a rotary evaporator until the weight of the obtained filtrate reached 150 g. The resulting concentrated solution was allowed to stand at 10 ° C. for 6 days. After standing, the mother liquor containing the transcrocetin crystals produced was filtered through a glass filter No. 3, and the obtained filtrate was used with a rotary evaporator at about 1 kPa and about 80 ° C. until the weight of the filtrate became 32 g. The filtrate was concentrated and the resulting concentrate was allowed to stand at 10 ° C. for 6 days. After standing, the mother liquor containing the produced 13-ciscrocetin crystals was filtered with a glass filter No. 3. The obtained filtrate was concentrated under the conditions of about 1 kPa and about 80 ° C. using a rotary evaporator until the weight of the filtrate became 5.3 g. 40 mL of methanol was added to the obtained concentrated liquid and suspended in an ultrasonic cleaner, followed by filtration with a glass filter No. 3, using a rotary evaporator until the weight of the obtained filtrate was 3.4 g. The solution was concentrated under the conditions of 1 kPa and about 80 ° C. 0.256 g of the resulting concentrate was dissolved in 12.8 mL of methanol, and a peak with a retention time of about 20 minutes was collected using the following preparative HPLC system and conditions. The separated liquid was concentrated using a rotary evaporator under the conditions of about 40 ° C. and about 2 kPa to obtain 20 mg of 13-ciscrocetin. The amount of 13-ciscrocetin of this product was 98.7%.

<Preparative HPLC system>
Pump: LC-6AD (manufactured by Shimadzu Corporation)
Detector: SPD-20A (manufactured by Shimadzu Corporation)
<Preparative HPLC conditions>
Column: μBONDASPHERE (inner diameter 19 mm, length 150 mm) (manufactured by Nippon Waters)
Column temperature: Room temperature Mobile phase: 0.05% TFA aqueous solution-TFA methanol (20:80)
Flow rate: 10 mL / min
Detection: 430nm

[Preparation of Crocetin Composition]
Transcrocetin obtained in Production Example 1 was dissolved in 100% DMSO (dimethyl sulfoxide) to prepare a 1 mM transcrocetin solution. Further, 13-ciscrocetin obtained in Production Example 2 was dissolved in 100% DMSO to prepare a 1 mM 13-ciscrocetin solution. Next, the trans crocetin solution and the 13-cis crocetin solution were mixed at the ratios shown in Table 1 to prepare 50.0 μL of crocetin compositions 1-7. Among these, crocetin compositions 4 to 6 are examples according to the present invention, and crocetin compositions 1 to 3 and 7 are comparative examples for them.

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

  The singlet oxygen scavenging ability was measured by the electron spin resonance (ESR) method shown below using the crocetin compositions 1 to 7 described above as sample solutions. For comparison, a known α-tocopherol (product name: (±) α-tocopherol; manufactured by Sigma) as a substance having a singlet oxygen scavenging ability was dissolved in 100% DMSO to obtain an α-tocopherol concentration of 1 mM. It measured similarly about the solution.

(1) Preparation of Evaluation Sample An evaluation sample was prepared by mixing the following 1) to 5).
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 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.

(2) Measurement by ESR apparatus The evaluation specimen obtained in (1) was collected in a flat cell, and light irradiation (18000 lux, 5 minutes) was performed on the evaluation specimen 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

(3) Results The singlet oxygen elimination rate (%) was calculated based on the ESR signal intensity obtained by carrying out (1) and (2) and the following equation. 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.

  From the results of Table 2, the crocetin compositions 4 to 6 of the present invention in which the amount of 13-ciscrocetin is included in the range of 3 to 10% are crocetin compositions 1 to 3 and 7 and α-tocopherol not included in this range. It is clear that it exhibits superior singlet oxygen scavenging ability compared to. This result also shows that the crocetin composition of the present invention can be used as an excellent singlet oxygen scavenger.

Claims (2)

Following formula (1)

13-ciscrocetin represented by the following formula (2)

A crocetin composition containing transcrocetin represented by the formula (1), wherein the ratio of the 13-ciscrocetin content is 3 to 9 % in the total content of 13-ciscrocetin and transcrocetin. A singlet oxygen scavenger that is taken orally as an ingredient.   The singlet oxygen scavenger according to claim 1, which is not in the form of a food or drink.