JP3237767B2 - Stabilized vitamin B2 composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a vitamin B _2, which is stabilized by adding an antioxidant activity substances contained in green leaf component of green plants.

[0002]

2. Description of the Related Art Vitamin B ₂ is a bioactive substance that plays an important role in the oxidation-reduction mechanism in living cells, and is widely used in foods, pharmaceuticals, quasi-drugs and the like.

[0003] However, vitamin B ₂ is a substance which is slightly unstable against oxidation, and its stabilization is desired. Thus, the present inventors have tried to stabilize vitamin B ₂ by blending tocopherols, BHA, BHT, vitamin C, caffeic acid, etc., which are conventionally known as antioxidants, with vitamin B _2. The results were not satisfactory.

On the other hand, the present inventors have found that a wheat plant has been found to contain a number of physiologically active components such as antitumor, antihyperlipidemic, hypoglycemic and antiviral activities. About the green leaf components of green plants, including
As a result of examining the components from the standpoint of antioxidant properties, it was found that the green leaf component contained a component having a strong antioxidant activity equivalent to or higher than that of α-tocopherol, and proposed the same to the source. (Japanese Patent Application 3-59374)
issue).

Further, the present inventors have conducted various studies on the application of the antioxidant active substance derived from the green leaf component of the green plant. As a result, the antioxidant active substance is safe, and the stability of vitamin B ₂ is stable. The present inventors have found that the present invention is also extremely effective, and have completed the present invention.

[0006]

DISCLOSURE OF THE INVENTION Thus, according to the present invention, vitamin B ₂ compositions stabilized, characterized in that it contains antioxidant active substance from green leaf component of green plants is provided.

Hereinafter, the antioxidant active substance derived from the green leaf component of the green plant used for stabilizing vitamin B ₂ in the present invention will be described in more detail. In addition, the water content% of the water-containing alcohol in this specification is based on v / v%.

As a green plant to be used as a raw material, a wheat plant is preferred, but in addition, clover, alfalfa, kale, spinach, lettuce, parsley, celery, cabbage, Chinese cabbage, mizuna, green pepper, carrot green leaf, radish Pastures, vegetables, and wild grass plants such as green leaves, sasa, and ashitaba; freshwater or marine green algae such as spirulina, chlorella, wakame, and blue seaweed can also be used.

Barley is the most suitable wheat plant for use in the present invention.
Barley, oats, oats, corn, millet, Italian diegrass and the like can also be used.

[0010] In the present invention, fresh stems and / or leaves of these green plants, particularly young plants harvested before the maturity stage among the wheat plants (these are collectively referred to as "green leaves" in the present specification). ) Are particularly suitable.

The green leaves of a green plant, for example, a wheat plant,
The juice is squeezed by a mechanical crushing means such as a mixer, a juicer, etc., and, if necessary, the squeezed liquid (hereinafter referred to as "green juice") is removed by removing coarse solids by means such as sieving and filtration. Prepare.

The green juice prepared in this manner or the green juice can be further dried by a suitable drying means such as freeze-drying or spray-drying to obtain green juice.

The green juice or green juice powder obtained as described above is subjected to an extraction treatment as described below to obtain an antioxidant active substance which can be used for stabilizing vitamin B _{2 in} the present invention. it can.

First, the green juice or green juice powder obtained as described above is extracted with a sufficient amount of water or n-hexane.
This extraction treatment can be usually performed at room temperature, and may be repeated twice or more depending on the case, thereby separating and recovering a water-soluble component or a component substantially insoluble in n-hexane. At this stage, the recovered extract can be dried and solidified in the same manner as described above.

The water-soluble component or n-hexane-insoluble component thus obtained is then subjected to a water-containing ethanol having a water content of 0 to 80%, preferably 10 to 70%, more preferably 15 to 50%, for example, a water-containing ethanol having a water content of 20%. An extraction treatment is performed with ethanol, and components soluble in the aqueous ethanol are separated and collected.

The extraction treatment with aqueous ethanol is carried out by using a green juice prepared as described above, or a water-soluble component of green leaves from which water-insoluble components have been completely removed, or a suitable drying means such as freeze-drying or spray-drying. And drying can be carried out directly on the powder obtained.

The aqueous ethanol-soluble component thus recovered can be blended with vitamin B ₂ or a material containing vitamin B ₂ as it is or after concentration or evaporation of the solvent.

Further, according to the present invention, if necessary, the above-mentioned aqueous ethanol-soluble component can be added to a suitable adsorbent such as St.
yrene-DVB resin adsorbent (e.g., Rohm & Haas, Amberlite ^R adsorbent XAD-2)
Etc. and a water content of 0 to 80%, preferably 20 to 7
By performing the elution treatment with 0%, more preferably 30 to 60% aqueous methanol, a component soluble in the aqueous methanol can be recovered. As a result, a fraction having more excellent antioxidant activity can be obtained.

The aqueous methanol-soluble component thus recovered can be blended with vitamin B ₂ or a material containing vitamin B ₂ as it is or after concentration or removal of the solvent.

Furthermore, the water-soluble methanol-soluble component thus recovered from barley is purified by recrystallization using, for example, water-containing methanol having a water content of 30 to 70%, preferably 40 to 60%. The body of the antioxidant active substance can be obtained as slightly yellow crystals. The body of the antioxidant active substance thus isolated is NM
R, the result of analysis such as mass spectrometry, the following formula

[0021]

Embedded image 2'-O-glucosyl-isovitexin having the structure represented by the following formula (see Reference Example 1 described later). The antioxidant active substance thus isolated can be blended with vitamin B ₂ or a material containing vitamin B ₂ .

The antioxidant active substance having the above structural formula or a structure similar thereto is contained in the green leaves of green plants such as wheat plants, which is the active ingredient of the antioxidant active substance in the present invention. It is presumed that they do.

When the antioxidant active substance derived from the green leaf component of the green plant described above is used for stabilizing vitamin B ₂ ,
The blending amount is not strictly limited, and can be changed according to the use of vitamin B _{2 and} the like. Generally, the amount of the above-mentioned 2′-O-glucosyl-isovitexin is 0.01 per mole of vitamin B _2. ~ 5 mol, preferably 0.05
Suitably, it is in the range of ~ 2 mol.

The thus stabilized vitamin B ₂ composition can be advantageously used in foods, medicines, quasi-drugs and the like.

Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples.

[0026]

Reference Example 1 (Fractionation and preparation method of active ingredient) 500 ml of n-hexane was added to 20 g of lyophilized powder of barley green juice before maturation, and the mixture was stirred well at room temperature for about 5 minutes. Centrifugation (80
(00 rpm, 10 min), and 500 ml of n-hexane was added to the separated insoluble component, and the same operation was repeated to obtain an n-hexane insoluble component.

To this insoluble component, 500 ml of ethyl alcohol having a water content of 20 v / v% is added, and the mixture is thoroughly stirred at room temperature for about 5 minutes, and the insoluble component is filtered off. The filtered insoluble matter is again treated similarly with 500 ml of ethyl alcohol having a water content of 20 v / v%. The obtained filtrates are combined, and the solvent is distilled off under reduced pressure. This resulted in 13.0 ethyl alcohol extract.
g was obtained.

After the ethyl alcohol-soluble matter was adsorbed on an Amberlite XAD-2 column, it was successively separated with distilled water, water-containing methanol having a water content of 80, 60, 40, 20, and 0 v / v%, and acetone, respectively. After dissolution, an eluate was obtained.

The solvent was distilled off from each eluate by distillation under reduced pressure. As a result, 4.77 g of a water extract, 20%, 180 mg of a methanol extract, 131 mg of a 40% methanol extract, 131 mg of a 6%
199 mg of 0% methanol extract, 32 mg of 80% methanol extract, 165 mg of 100% methanol extract,
0.87 mg of an acetone extract was obtained (where the% of methanol is the methanol concentration v / v% in the aqueous methanol).

The 60% methanol extract obtained as described above is further recrystallized using 60% methanol,
180 mg of slightly yellow crystals were obtained. The structure of this crystal was determined by mass spectrometry and NMR.

Mass spectrometry was performed by FAB-MS: VG ZAB-
2F, using a (Xenon Gun) (Jon Tech) type mass spectrometer, and the results shown in FIG. 1 were obtained. From this mass spectrum, [M + H] at m / z = 595
⁺ ] Was observed, the molecular weight was determined to be 594, and when considered in conjunction with the results of elemental analysis, the molecular formula of the substance was determined to be C ₂₇ H ₃₀ O ₁₅ .

When the ultraviolet absorption spectrum of the essential substance was measured in H ₂ O and methanol, the results were as shown in FIGS. 2 and 3, respectively, and showed the absorption of flavonoid glucoside.

The infrared absorption spectrum was measured by JASCO FT
FIG. 4 shows the results obtained by applying the KBr method according to / IR-7000S. The presence of an OH group is shown at 3422 cm ^-1 .

The essential substance liberated one molecule of glucose when hydrolyzed with hydrochloric acid-methanol in a conventional manner to produce isovitexin.

Further, the ¹³ C-NMR spectrum (500 MHz) of the essential substance was analyzed by using a GE OMEGA 300 type nuclear magnetic resonance spectrum absorption measuring apparatus with 25 mg of the purified antioxidant active substance to obtain tetramethylsilane [TMS, (C
H ₃ ) ₄ Si] as an internal standard, and the results shown in FIG. 5 were obtained. In FIG. 5, the chemical shift is indicated by δ. Purification Antioxidant active substance 2 in MeOH-d ₄
Gives a signal to 7 carbon atoms,
¹³ C-NMR standard value [Ramarathnam,
N., Osawa, T., Namiki, M .; and K
awasakishi, S .; : J. Agric. Fo
od Chem., 37 , 316-319 (198
9)] and the following structural formula was measured.

[0036]

Embedded image According to this structural formula, the substance is represented by 2′-O-glucosyl-
It is named Isovitexin.

REFERENCE EXAMPLE 2 (Fractionation of Active Ingredient and Preparation Method) 20 g of freeze-dried powder of wheat green juice before maturation was treated in the same manner as in Reference Example 1 to obtain 118 mg of a 60% methanol extract. Further extract 6
Recrystallization was repeated with 0% methanol to obtain 106 mg of white crystals. This substance had the same structure as that shown in Reference Example 1.

Reference Example 3 (Fractionation and Preparation of Active Ingredient) 20 g of freeze-dried powder of comfrey green juice before maturation was treated in the same manner as in Reference Example 1.
40 mg of a 0% methanol extract was obtained. The extract was further recrystallized with 60% methanol to give 37 mg.
Was obtained as white crystals. This substance had the same structure as that shown in Reference Example 1.

[0039] Example 1 Vitamin B ₂ and poured a solution 5ml to vial containing 8 mg / 100 ml, pH 5 of 0.5M acetate buffer 5m containing water content of 20% ethanol extract 2.5% to this
l, caffeic acid, vitamin C and vitamin E [E mix P20, Shinei Sangyo Co., Ltd.]
Irradiation was performed by standing still at 60 cm under a W daylight lamp, and the residual amount of vitamin B ₂ was measured over time by high performance liquid chromatography. Table 1 shows the results. In this experiment, the amount of the ethanol extract having a water content of 20% was added as 2'-O-glucosylisovitexin to 0.17 mM, and the other components were also adjusted to 0.17 mM.

[0040]

[Table 1] Vitamin B ₂ was remarkably stabilized by the 20% water content ethanol extract. The quantification of vitamin B ₂ is described in the Japanese Society of Pharmaceutical Sciences: Sanitary Test Methods and Comments, pp. 363-365 (19)
1990, Kanehara Publishing Co., Ltd.).

Example 2 In Example 1, the ethanol extract having a water content of 20% was replaced with 2 '.
Replaced by the amount of the -O- glucosyl isobutyl the Activity relaxin 0.17 mM, pH 3 to using 0.1M lactic acid buffer, to determine the effect on the stability of vitamin B ₂ other conditions in the same manner as in Example 1 . Table 2 shows the results.

[0042]

[Table 2] Vitamin B ₂ is stabilized by 2'-O- glucosyl isobutyl the Activity relaxin.

Example 3 After washing green leaves of young barley leaves, 3 kg of green juice powder obtained by pulverizing green juice obtained by squeezing by a drying method such as spray-drying or freeze-drying is twice with 60 liters of hexane. The extraction was repeated, and 30 liters of water was added to the hexane-insoluble portion to spray-dry the water-soluble component to obtain 1.4 kg of a spray-dried product.
Next, the dry powder was mixed with aqueous ethanol 3 having a water content of 20%.
By adding 0 liter, 900 g of a water-soluble ethanol-soluble component having a water content of 20% was obtained, and ethanol was distilled off. Further, 30 liters of water-containing methanol having a water content of 40% was added thereto to extract a water-soluble methanol-soluble component having a water content of 40%, and then methanol was distilled off to obtain 720 g of a water-soluble methanol-soluble component having a water content of 40%. This component is called substance A. Replacing the moisture content of 20% ethanol extract Tris buffer pH7 containing A material 2.5% in Example 1, to determine the effect on the stability of vitamin B ₂ other conditions in the same manner as in Example 1 . Table 3 shows the results.

[0044]

[Table 3] Vitamin B ₂ was significantly stabilized by substance A.

[Brief description of the drawings]

FIG. 1 shows a measurement chart of 2′-O-glucosyl-isovitexin obtained in Reference Example 1 by FAB-MS method.

FIG. 2 is an ultraviolet absorption spectrum of 2′-O-glucosyl-isovitexin in H ₂ O system.

FIG. 3 is an ultraviolet absorption spectrum of 2′-O-glucosyl-isovitexin in a MeOH system.

FIG. 4 is an infrared absorption spectrum of 2′-O-glucosyl-isovitexin.

FIG. 5 is a diagram showing the results of ¹³ C-NMR measurement of 2′-O-glucosyl-isovitexin.

Claims (9)

(57) [Claims] 1. The following formula Isovitexin vitamin B ₂ composition that has been stabilized, characterized in that contained as antioxidant active ingredient - in 2'-O- glucosyl shown. 2. The method according to claim 1, wherein n is extracted from a green leaf component of a green plant.
2. An extract fraction containing 2'-O-glucosyl-isovitexin as an antioxidant active ingredient, which is insoluble in hexane and soluble in aqueous ethanol having a water content of 80% or less. stabilized vitamin B ₂ composition. 3. An extraction comprising 2′-O-glucosyl-isovitexin, which is soluble in water and soluble in water-containing ethanol having a water content of 80% or less, extracted from green leaf components of green plants as an antioxidant active component. vitamin B ₂ stabilized as set forth in claim 1, characterized in that it contains a fraction
Composition. 4. A water-containing ethanol vitamin B ₂ composition stabilized according to claim 2 or 3 is ethanol moisture content 15% to 50%. 5. The green plant is a wheat plant.
Vitamin B ₂ compositions stabilized according to any one of. 6. n extracted from a green leaf component of a green plant
Contains 2'-O-glucosyl-isovitexin as an antioxidant active ingredient, which is insoluble in hexane and soluble in aqueous ethanol having a water content of 80% or less, and further soluble in water-containing methanol having a water content of 80% or less. vitamin B ₂ composition stabilized according to claim 1, characterized by containing extract fraction. 7. Vitamin B ₂ composition stabilized according to claim 6 aqueous ethanol is ethanol moisture content 15% to 50%. 8. The stabilized vitamin B ₂ composition according to claim 6, wherein the water-containing methanol is water-containing methanol having a water content of 10 to 70%. 9. The green plant is a wheat plant.
Vitamin B ₂ compositions stabilized according to any one of.