JP2817809B2 - Antioxidant active substance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to an antioxidant component having strong antioxidant activity derived from green leaves of wheat plants, particularly barley young green leaves.

[0002] Conventionally, antioxidants derived from natural sources or chemically synthesized and mainly used in the fields of foods, pharmaceuticals and the like include, for example, natural antioxidants such as α-tocopherol and ascorbic acid, and butylhydroxyanilyl. (B
HA) and phenolic synthetic antioxidants such as dibutylhydroxytoluene (BHT) are known.

On the other hand, the present inventors have found that a wheat plant has been found to contain many physiologically active components such as antitumor, antihyperlipidemic, hypoglycemic and antiviral activities. Focusing on the green leaves, we examined its components from the standpoint of antioxidant properties.

As a result, it has now been found that the green leaf component of a wheat plant contains a component having a strong antioxidant activity equal to or higher than that of α-tocopherol, thereby completing the present invention. Was.

[0005] Thus, according to one aspect of the present invention,
An antioxidant active substance comprising a green leaf component of a wheat plant, which is substantially insoluble in n-hexane and soluble in water-containing ethanol having a water content of 0 to 80%.

[0006] According to another aspect of the present invention,
A green leaf component of a wheat plant that is substantially insoluble in n-hexane and soluble in water-containing ethanol having a water content of 0 to 80%, and is further soluble in water-containing methanol having a water content of 0 to 80%. An antioxidant active comprising the components is provided.

Hereinafter, the antioxidant active substance of 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 barley plant is preferable, but in addition to the above, clover, alfalfa, kale, spinach, lettuce, parsley, celery, cabbage, Chinese cabbage, water leaf, bell pepper, carrot green leaf Grasses, vegetables, wild grass plants such as radish green leaves, sasa, and ashitaba; and 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.

Next, the green juice obtained by drying the green juice as it is or by a suitable drying means such as freeze-drying or spray-drying is extracted with a sufficient amount of water or n-hexane. This extraction can be usually performed at room temperature, and may be repeated twice or more depending on the case, whereby the water-soluble component or n-
The component substantially insoluble in hexane is separated and recovered. 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 used as the antioxidant active substance of the present invention as it is or by concentrating or distilling off 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 Sty.
rene-DVB resin adsorbent (e.g., Rohm & Haas, Amberlite ^R adsorbent XAD-2) treated and moisture content 0-80% the like, preferably 20 to 70%
More preferably, by performing the elution treatment with 30 to 60% aqueous methanol, components soluble in the aqueous methanol can be recovered. As a result, a fraction having more excellent antioxidant activity can be obtained.

Furthermore, the water-soluble methanol-soluble component thus recovered from barley has, for example, a water content of 3%.
By recrystallization and purification using 0 to 70%, preferably 40 to 60% aqueous methanol, the main body of the antioxidant active substance can be obtained as slightly yellow crystals. The body of the antioxidant active substance isolated in this way is NMR,
As a result of analysis such as mass spectrometry, the following formula

[0018]

Embedded image 2'-O-glycosyl-isovitexin having the structure represented by the following formula (see Example 1 described later).

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

The antioxidant active substance of the present invention has a potent antioxidant activity equal to or higher than that of α-tocopherol, as is apparent from the examples described later.
It is useful as an antioxidant in the field of pharmaceuticals and the like.

For example, the antioxidant active substance of the present invention is obtained by removing various metal elements usually contained in green leaves as a raw material, substances that promote denaturation of foods, and the like, so that foods, pharmaceuticals and the like which require antioxidant properties are removed. And the like, can be advantageously blended with various inorganic or organic (composition) compositions in such fields. For example, the antioxidant active substance of the present invention, if necessary, after inclusion with cyclodextrin, crown ether or the like, sugars such as fructose, glucose, dextrin, starch; amino acids; citric acid, malic acid, tartaric acid, Organic acids such as succinic acid; various vitamins; coloring agents, fragrances, various thickeners and the like can be mixed. In particular, since the antioxidant active substance of the present invention has substantially no adverse effect on the water solubility and transparency of the compounded composition, the aqueous composition can be sterilized by filtration.

The antioxidant active substance of the present invention is mixed or sprayed with raw materials such as medicines such as talc, zinc white, sodium carbonate, sodium bicarbonate, titanium dioxide, kaolin and calcium phosphate, paints, cosmetics and foaming agents. Drying,
By blending as a powder by vacuum drying or the like, it is possible to produce a new industrial product, and further, there is an advantage that the quality of the product is not changed. Moreover, the water-soluble and alcohol-soluble antioxidant active substance is
Helps stabilize inorganic and organic compositions and enables the production of very good new products, such as antioxidants for polymer production; emulsion paints; cosmetics; paper products; food products; pharmaceuticals; Is what you do.

Next, the present invention will be described more specifically with reference to examples.

[0024]

Example 1 (Active ingredient fractionation and preparation method) n-hexane 500 g was added to 20 g of lyophilized powder of barley green juice before maturation.
After stirring well for about 5 minutes at room temperature, the insoluble component was separated by centrifugation (8000 rpm, 10 min), and the separated insoluble component was added to 500 ml of n-hexane.
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 stirred well at room temperature for about 5 minutes, and then 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%, and the obtained filtrates are combined and the solvent is distilled off under reduced pressure. This gives the ethyl alcohol extract 13.
0 g was obtained.

After the ethyl alcohol-soluble matter was adsorbed on an Amberlite XAD-2 column, it was sequentially 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, 180 mg of a 20% methanol extract, 131 mg of a 40% methanol extract, and 60 mg of a 60% methanol extract were obtained.
% Methanol extract (199 mg), 80% methanol extract (32 mg), 100% methanol extract (165 mg) and acetone extract (0.87 mg) (where methanol is the methanol concentration v / v% in 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.

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

When the ultraviolet absorption spectrum of this substance was measured in H ₂ O and methanol, it was as shown in FIG. 2 and FIG. 3, respectively, indicating 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 .

This 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 substance was analyzed by using a GE OMEGA 300 type nuclear magnetic resonance spectrum absorption measuring apparatus using 25 mg of the purified antioxidant active substance to obtain tetramethylsilane [TMS, (CH
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 actives gives signals for 27 carbon atoms in MeOH-d _4, of isovitexin ¹³ C-
NMR standard values [Ramarathnam, N., Osawa, T., Namiki,
M. and Kawakishi, S .: J. Agric. Food Chem., 37 ,
316-319 (1989)].

[0034]

Embedded image The present invention is named 2'-O-glucosyl-isovitexin from this structural formula.

[0035]

Example 2 (Fractionation of Active Ingredient and Preparation Method) In the same manner as in Example 1, 20 g of a freeze-dried powder of wheat juice before maturation was treated to obtain 118 mg of a 60% methanol extract. The extract was further recrystallized with 60% methanol to obtain 106 mg of white crystals. This substance was the same as that obtained in Example 1.

[0036]

Example 3 (Fractionation and preparation method of active ingredient) 20 g of lyophilized powder of comfrey green juice before maturation was treated in the same manner as in Example 1 to obtain 40 mg of a 60% methanol extract.
The extract was further recrystallized with 60% methanol to obtain 37 mg of white crystals. This substance was the same as that obtained in Example 1.

[0037]

Example 4 (Measurement of Lipid Peroxide by TBA Method) To 7.5 mg of linoleic acid, 0.22 mg of α-tocopherol or 0.22 mg of each extract obtained in Example 1 was added, and the mixture was added to Fenton TS ( FeCl ₂ , H ₂ O ₂ ) 200 μl
And incubated at 37 ° C. for 16 hours (total volume 5).
ml).

In 0.2 ml of this solution, 8% SDS ¹⁾ 0.2% aqueous solution was added.
2 ml, 1.5 ml acetate buffer (PH 3.5) and 0.67%
1.5 ml of an aqueous solution of TBA ²⁾ was added, and the mixture was placed in a boiling water bath (95-1).
(00 ° C.) for 1 hour.

After cooling, 5 ml of butyl alcohol was added, shaken vigorously, and centrifuged (2000 rpm, 10 m
In), the butyl alcohol layer was separated, and the absorbance at 535 nm of the butyl alcohol layer was measured. FIG. 6 shows the results.

Note 1) SDS = sodium dodecyl sulfate 2) TBA = thiobarbituric acid

[0041]

Example 5 (Lipid Oxidation Product, MD ³⁾ and 4NH
Gas Chromatographic Analysis of (4-Hydroxynonenal) Microsomes and 7.5 mg of arachidonic acid
0.22 mg of tocopherol or 0.22 mg of the antioxidant active substance obtained in Example 1 was added thereto, followed by addition of Tris-HCl buffer (0.05 M Trizma HCl, PH 7.4; 0.15 M).
Add 5 ml of KCl (0.2% SDS), gently shake to suspend, and further add Fenton TS (FeCl ₂ , H ₂ O ₂ ) 2
After adding 00 μl, the mixture was reacted at 37 ° C. for 16 hours. 4%
BHT ⁴⁾ After stopping the reaction by adding 50 μl, N-
Add methylhydrazine (40 μl) and add
-Methylhydroazine derivative was formed, 15 ml of saturated saline was added thereto, and the mixture was extracted with 5 ml of dichloromethane for 3 hours.

The dichloromethane layer was separated, a certain amount of an internal standard solution for gas chromatography (IS) was added, and the mixture was made exactly 10 ml with dichloromethane to obtain a sample for gas chromatography analysis, which was subjected to chromatography under the following conditions. Was.

Capillary column: DB-WAX 25 m × 0.25 mm Column temperature: 35 ° C. (hold 1.0 minutes) 分 190 ° C. (hold 20 minutes) Temperature rise 40 ° C./min Inlet temperature: 250 ° C. Detector temperature: 300 ° C. Detector: NPD (nitorogen-phosphorus detector) Carrier gas: helium The results are shown in FIGS. From these chromatographic charts, it can be seen that the antioxidant active substance of the present invention having a structure in which one glucose molecule is bound to isovitexin not only produces MA but also significantly suppresses the production of 4HN (4-hydroxynonenal). -It has been found to exhibit a stronger antioxidant effect than tocopherol.

Note) 3) MAD = malondialdehyde 4) BHT-butylhydroxytoluene

[0045]

Example 6 To 100 g of the green juice powder obtained in Example 1 was added 2.5 l of n-hexane, and the mixture was stirred at room temperature for about 5 minutes, and then the insoluble components were centrifuged (8000 rpm, 10 mi).
n. ) And n-hexane 2.
Five liters were added, and the same operation was repeated to obtain an n-hexane insoluble component.

To this insoluble component was added 2.5 liters of ethanol having a water content of 20 v / v%, and the extraction was repeated by the same operation. The ethanol was distilled off under reduced pressure from the soluble component in the resulting ethanol having a water content of 20 v / v%. Leave the water content 2
72 g of the fraction extracted with 0 v / v% ethanol
Obtained.

60 g of this fraction was applied to Amberlite XAD-2.
After being adsorbed on the column, distillation was carried out, and the
Eluted sequentially with 0, 20 and 0 v / v% methanol and acetone.

The eluate was distilled under reduced pressure to remove the solvent, and 27 g of a water extract, 1.1 g of a 20% methanol extract and 40 g of a 40% methanol extract were extracted.
680 mg of a 60% methanol extract, 1.5 g of a 60% methanol extract, 170 mg of an 80% methanol extract, and 5.3 mg of an acetone extract were obtained. Separately, 1.5 g of a 60% methanol extract prepared in the same manner was recrystallized to give 2 ′.
1.2 g of -O-glucosyl-isovitexin were obtained.

A model diuse containing the composition shown in Tables 1 and 2 containing β-carotene was prepared and prepared at pH 3 and p
60% methanol extract in H5 and 2'-O-
The antioxidant activity of glucosyl-isovitexin on β-carotene was measured using water and vitamin C as controls.

The determination of β-carotene was carried out in accordance with the Japanese Society of Pharmaceutical Sciences: Sanitary Test Methods and Comments, p. 347-349 (1990, Kanehara Publishing Co., Ltd.).

[0051]

[Table 1] Make up to 100 ml with H ₂ O. The pH was adjusted to 3.0.

* Carotene base: Sanei Chemical Co., Ltd.

[0053]

[Table 2] Make up to 100 ml with H ₂ O. The pH was adjusted to 5.05.

* Carotene base: manufactured by Sanei Chemical Co., Ltd. Tables 3 and 4 show the antioxidant activity of each fraction against β-carotene. The reaction temperature was 18 ° C.

[0055]

[Table 3]

[0056]

[Table 4]

[0057]

Example 7 After washing the green leaves of barley young leaves, 10 kg of green juice obtained by squeezing the green juice obtained by squeezing and drying it by a drying method such as spray drying or freeze drying is extracted twice with hexane 2001. The water-soluble component was spray-dried by repeatedly adding water 1001 to the hexane-insoluble portion to obtain 3.8 kg of a spray-dried product. Subsequently, 1001 of water-containing ethanol 1001 having a water content of 20% was added to the dried powder to obtain 2.7 kg of a water-soluble ethanol-soluble component having a water content of 20%, and ethanol was distilled off. Further, 70 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 2 kg of a water-soluble methanol-soluble component having a water content of 40%. This component is referred to as a substance A. A suspension was prepared by adding 400 g of talc to 100 g of the obtained substance A, and the intake temperature was 180 ° C. and the exhaust temperature was 12
Spray drying was performed at 0 ° C. to produce 470 g of a powder raw material.

[0058]

Example 8 A solution prepared by adding 400 g of dextrin to 100 g of the substance A obtained in Example 7 was spray-dried at an intake air temperature of 190 ° C. and an exhaust air temperature of 120 ° C.
30 g of powder raw material was obtained.

[0059]

Embodiment 9 Lindex-P (Sanraku Corporation) 100
g to 300 ml of water and mixed to form a slurry,
The methanol fractionation in Example 6 was accurately performed stepwise, a substance which was a water-containing methanol fraction having a water content of 40% was separated, and the substance was obtained by recrystallization from water-containing tamenol having a water content of 40%. After adding 40 g of 2'-O-glucosyl-isovitexin and stirring at room temperature for 90 minutes, the mixture was spray-dried at a concentration of 30% at an intake air temperature of 170 ° C. and an exhaust air temperature of 110 ° C., and 127 g of a cyclodextrin clathrate powder was used. Body material was manufactured.

[0060]

Example 10 100 g of the substance A obtained in Example 7 was mixed with 200 g of kaolin to prepare a 30% suspension.
Spray drying was performed at an intake temperature of 170 ° C. and an exhaust temperature of 110 ° C. to produce 270 g of a powder raw material.

[0061]

Example 11 After desalting 100 ml of a 4% solution of sodium silicate, the pH was adjusted to 9 with 1% potassium hydroxide.
5 ml was heated at 95 ° C. for 15 minutes. Then, 10 g of 2'-O-glucosyl-isovitexin obtained in Example 8
Was added to the remaining sodium silicate solution (85 ml), and the mixture was successively added and concentrated at 90 ° C. for 8 hours to produce spherical silica containing an antioxidant active substance.

[Brief description of the drawings]

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

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

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

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

FIG. 5 is a view showing the result of ¹³ C-NMR measurement of 2′-O-glycosyl-isovitexin.

FIG. 6 is a view showing the result of ¹³ C-NMR measurement of 2′-O-glycosyl-isovitexin.

FIG. 7 is a view showing the result of ¹³ C-NMR measurement of 2′-O-glycosyl-isovitexin.

FIG. 8 is a graph showing the measurement results (measurement result of absorbance at 535 nm) of each extract obtained in Example 1 and α-tocopherol in lipid peroxide by TBA method.

FIG. 9 shows a gas chromatographic chart of the lipid peroxidation product of arachidonic acid, MA and 4HN using Fuenton's reagent.

FIG. 10 shows a gas chromatographic chart of the lipid peroxidation product of arachidonic acid, MA and 4HN using Fuenton's reagent.

FIG. 11 shows a gas chromatographic chart of the lipid peroxidation product of arachidonic acid, MA and 4HN using Fuenton's reagent.

──────────────────────────────────────────────────の Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C09K 15/08 C09K 15/34 A23L 3/3436 C07H 17/04 CA (STN) REGISTRY (STN)

Claims (7)

(57) [Claims] 1. A fraction soluble in water or insoluble in n-hexane and in water-containing ethanol having a water content of 80% or less of green leaves of wheat in the early maturation, comprising 2'-O-glucosyl-isovitexin. An antioxidant active fraction of barley green leaves, comprising: 2. The antioxidant-active fraction according to claim 1, wherein the barley is barley. 3. A water-containing ethanol having a water content of 15 to 50%.
The antioxidant-active fraction according to claim 1 or 2, wherein 4. The antioxidant-active fraction according to claim 1, which is soluble in aqueous methanol having a water content of 80% or less. 5. A water-containing methanol having a water content of 20 to 70%.
The antioxidant active fraction according to claim 4, which is: 6. A dried powder of green juice obtained by squeezing green leaves of wheat at an early stage of maturation is subjected to extraction treatment with water or n-hexane, and a water-soluble or n-hexane-insoluble fraction obtained. Is further extracted with water-containing ethanol having a water content of 80% or less, and a fraction soluble in the water-containing ethanol is recovered, whereby the antioxidant activity of green leaves of barley containing 2'-O-glucosyl-isovitexin is characterized in that Method for producing active fraction. 7. The method according to claim 6, wherein the fraction soluble in aqueous ethanol is further extracted with aqueous methanol having a water content of 80% or less.