JPH0565480A - Antioxidizing active substance - Google Patents

Abstract

PURPOSE:To obtain an antioxidizing active substance, composed of a specific ingredient available from a green plant, having an antioxidizing activity equal to or higher than that of alpha-tocopherol and useful as an antioxidant in the field of food and medicines. CONSTITUTION:An antioxidizing active substance composed of an ingredient which is a leaf ingredient of a green plant, insoluble in n-hexane and soluble in hydrous ethanol with 0-80% water content, e.g. new 2'-O-glycosyl-isovitexin expressed by the formula obtained from barley, Leaves and/or stems of the young barley before the maturation period are preferred as the green plant. The above-mentioned antioxidizing active substance is obtained by squeezing the leaves of the green plant with a mixer, etc., subsequently drying the obtained squeezed juice according to a freeze-drying method, etc., then subjecting the resultant green juice powder to extracting treatment with water or n-hexane, subsequently recovering a water-soluble ingredient or an ingredient insoluble in n-hexane and extracting the recovered ingredient with hydrous ethanol.

Description

Detailed Description of the Invention

The present invention relates to an antioxidant component having a strong antioxidant activity, which is derived from green leaves of barley plants, especially young green leaves of barley.

As antioxidants derived from natural sources or chemically synthesized and used mainly in the fields of foods, pharmaceuticals, etc., for example, natural antioxidants such as α-tocopherol and ascorbic acid, and butylhydroxyaniril. (B
HA), dibutylhydroxytoluene (BHT) and other phenolic synthetic antioxidants are known.

On the other hand, the inventors of the present invention have revealed that the barley plant contains components having many physiological actions such as antitumor action, antihyperlipidemic action, blood glucose lowering action and antiviral action. Focusing on the green leaves of the plant, the components were examined from the standpoint of antioxidant properties.

As a result, it has now been found that the green leaf component of a barley plant contains a component having a strong antioxidant activity equal to or higher than that of α-tocopherol, and the present invention was completed. It was

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

According to another aspect of the present invention,
A green leaf component of a barley plant, which is substantially insoluble in n-hexane and soluble in water-containing ethanol having a water content of 0 to 80%, and further soluble in water-containing methanol having a water content of 0 to 80%. There is provided an antioxidant active substance comprising components.

The antioxidant active substance of the present invention will be described in more detail below. The water content% of hydrous alcohol in the present specification is based on v / v%.

As a green plant which is a raw material, wheat plants are preferable, but in addition to them, clover, alfalfa, kale, spinach, lettuce, parsley, celery, cabbage, Chinese cabbage, water leaf, bell pepper, carrot green leaf. Further, grasses, vegetables, and wild grass plants such as radish green leaves, sasa, and ashitaba; and freshwater or seawater green algae such as spirulina, chlorella, seaweed, and green seaweed can also be used.

Barley is most suitable as the barley plant preferably used in the present invention.
Barley, oats, pigeons, corn, millet, Italian diegrass and the like can also be used.

In the present invention, fresh stem and / or leaf parts of these green plants, especially young plants among wheat plants that have been harvested before maturity (these are collectively referred to as "green leaves" in the present specification). ) Is particularly suitable.

First of all, the green leaves of green plants such as barley plants are
Juice is squeezed by mechanical crushing means such as a mixer, juicer, etc., and if necessary, coarse solids are removed by means such as sieving and filtration to obtain squeezed juice (hereinafter referred to as "green juice"). Prepare.

Then, the green juice is obtained as it is, or the green juice powder obtained by drying it 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 process can usually be performed at room temperature, and may be repeated twice or more times depending on the case, whereby the water-soluble component or n-
A component which is substantially insoluble in hexane is separated and collected. The recovered extract components can be dried and solidified at this stage in the same manner as described above.

The water-soluble component or the n-hexane-insoluble component thus obtained is then mixed with water having a water content of 0 to 80%, preferably 10 to 70%, more preferably 15 to 50%, for example, a water content of 20%. Extraction treatment is carried out with ethanol to separate and collect the water-soluble ethanol-soluble component.

This extraction treatment with water-containing ethanol is carried out by a suitable drying means such as freeze-drying or spray-drying the green juice prepared as described above or the water-soluble components of green leaves from which water-insoluble components have been completely removed. It can also be carried out directly on the powder obtained by drying at.

The water-containing ethanol-soluble component thus recovered can be used as the antioxidant substance of the present invention as it is or by concentrating or distilling off the solvent.

Further, according to the present invention, if desired, the hydrous ethanol-soluble component may be combined with a suitable adsorbent such as Sty.
treated with a rene-DVB resin adsorbent (for example, Amberlite ^R adsorbent XAD-2 manufactured by Rohm and Haas Co.) and the like, and has a water content of 0 to 80%, preferably 20 to 70%,
More preferably, the component soluble in the water-containing methanol can be recovered by performing the elution treatment with 30-60% water-containing methanol. As a result, it is possible to obtain a fraction further excellent in antioxidant activity.

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

[0018]

[Chemical 2] It was identified to be 2'-O-glycosyl-isovitexin having the structure shown in (see Example 1 below).

An antioxidant active substance having the above structural formula or a structure similar thereto is contained in the green leaves of green plants such as barley plants, and it contains the active ingredient of the antioxidant active substance of the present invention. It is presumed to have been done.

The antioxidant substance of the present invention has a strong antioxidant activity equal to or higher than that of α-tocopherol as will be apparent from the examples described below, and, for example, food,
It is useful as an antioxidant in the field of pharmaceuticals and the like.

[0021] For example, the antioxidant active substance of the present invention is free from various metal elements normally contained in the green leaf as a raw material, substances that promote the denaturation of foods, etc., and foods and pharmaceuticals that are required to have antioxidant properties. It can be advantageously mixed with various inorganic or organic (composition) materials in the fields such as. For example, the antioxidant active substance of the present invention, if necessary, after inclusion with cyclodextrin, crown ether, etc., sugars such as fructose, glucose, dextrin, starch; amino acids; citric acid, malic acid, tartaric acid, It can be mixed with organic acids such as succinic acid; various vitamins; coloring agents, flavors, various thickening agents, and the like. In particular, since the antioxidant active substance of the present invention does not substantially affect 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 talc, zinc white, sodium carbonate, sodium bicarbonate, titanium dioxide, kaolin, calcium phosphate and the like, medicines, paints, cosmetics, foaming agents and the like. Dry,
By blending as a powder by vacuum drying or the like, there is an advantage that a new industrial product can be manufactured and the quality of the product is not changed. Moreover, antioxidant substances that are water-soluble and have alcohol solubility are
It also helps to stabilize inorganic and organic compositions and enables the production of excellent new products such as antioxidants for polymer production; emulsion coatings; cosmetics; paper products; foods; pharmaceuticals; medical materials, etc. To do.

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

[0024]

Example 1 (Fractionation of Active Ingredients and Preparation Method) 20 g of freeze-dried powder of green juice of barley before maturity was added to 500 g of n-hexane.
After adding 5 ml and stirring well at room temperature for about 5 minutes, insoluble components are separated by centrifugation (8000 rpm, 10 min), and the separated insoluble components are further added with 500 ml of n-hexane.
Was added 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% was added and well stirred at room temperature for about 5 minutes, and then the insoluble component was filtered off. The filtered insoluble matter is treated again with 500 ml of ethyl alcohol having a water content of 20 v / v% in the same manner, and the obtained filtrates are combined and the solvent is distilled off under reduced pressure. This resulted in ethyl alcohol extract 13.
0 g was obtained.

The ethyl alcohol-soluble component was adsorbed on an Amberlite XAD-2 column and then sequentially separated with distilled water, water-containing methanol having a water content of 80, 60, 40, 20 and 0 v / v%, and acetone. It was made to melt | dissolve and the eluate was obtained.

The solvent of each eluate was distilled off under reduced pressure, and as a result, 4.77 g of water extract, 180 mg of 20% methanol extract, 131 mg of 40% methanol extract, 60
% Methanol extract 199 mg, 80% methanol extract 32 mg, 100% methanol extract 165 mg, acetone extract 0.87 mg were obtained (where% of methanol is the methanol concentration v / v% in hydrous methanol). ).

The 60% methanol extract obtained as described above was recrystallized with 60% methanol to give 1
80 mg of pale yellow crystals were obtained. The structure of this crystal was determined by mass spectrometry and NMR.

Mass spectrometry was conducted by FAB-MS: VG ZAB-
2F, (Xenon Gun) (Jon Tech) type mass spectrometer was used, and the results shown in FIG. 1 were obtained. From this mass spectrum, a peak of [M + H ⁺ ] is observed at m / z = 595,
The molecular weight of this substance was determined to be 594, and when considered together with the result of elemental analysis, the molecular formula of this 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 showed the absorptions of flavonoid glucosides as shown in FIGS. 2 and 3, respectively.

Infrared absorption spectrum of JASCO FT
The result measured by applying the KBr method with / IR-7000S is shown in FIG. The presence of OH groups is shown at 3422 cm ^-1 .

This substance liberated one molecule of glucose when hydrolyzed with hydrochloric acid-methanol according to a conventional method to produce isovitexin.

Further, the ¹³ C NMR spectrum (500 MHz) of this substance was analyzed by using GE OMEGA 300 type nuclear magnetic resonance absorption spectrometer using 25 mg of the purified antioxidative active substance, and tetramethylsilane [TMS, (CH
₃ ) ₄ Si] was used as an internal standard, and the results shown in FIG. 5 were obtained. In FIG. 5, chemical shifts are indicated by δ. The purified antioxidant active substance gives a signal for 27 carbon atoms in MeOH-d ₄ , and is ¹³ C-of isovitexin.
NMR standard values [Ramarathnam, N., Osawa, T., Namiki,
M. and Kawakishi, S .: J. Agric. Food Chem., 37 ,
316-319 (1989)], the following structural formula was estimated.

[0034]

[Chemical 3] According to this structural formula, the present invention names it 2'-O-glucosyl-isovitexin.

[0035]

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

[0036]

Example 3 Fractionation of Active Ingredient and Preparation Method 20 g of freeze-dried powder of comfrey green juice before maturity was treated in the same manner as in Example 1 to obtain 40 mg of a 60% methanol extract.
Further, the extract was repeatedly recrystallized with 60% methanol to obtain 37 mg of white crystals. This substance was the same substance as obtained in Example 1.

[0037]

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

To 0.2 ml of this solution, 0.2% of 8% SDS ¹⁾ solution was added.
2 ml, acetate buffer (PH3.5) 1.5 ml and 0.67%
TBA ²⁾ aqueous solution (1.5 ml ⁾ was added, and the mixture was put in a boiling water bath (95-1).
It heated at 00 degreeC) for 1 hour.

After cooling, 5 ml of butyl alcohol was added, and the mixture was shaken vigorously and then centrifuged (2000 rpm, 10 m
In), the butyl alcohol layer was collected and the absorbance of the butyl alcohol layer at 535 nm was measured. Results are shown in FIG.

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

[0041]

Example 5 (Lipid oxidizable product, MD ³⁾ and 4 NH
Gas Chromatographic Analysis of (4-Hydroxynonenal) Microsomes and arachidonic acid 7.5 mg α-
0.22 mg of tocopherol or 0.22 mg of the antioxidant active substance obtained in Example 1 was added, and to this was added Tris-hydrochloric acid buffer solution (0.05M Trizma HCl, PH7.4; 0.15M).
KCl; 0.2% SDS) (5 ml), gently shaken to suspend, and further Fenton's test solution (FeCl ₂ , H ₂ O ₂ ) 2
00 μl was added, and the mixture was reacted at 37 ° C. for 16 hours. 4%
After adding 50 μl of BHT ⁴⁾ to stop the reaction, N-
Methylhydrazine (40 μl) was added for 1 hour at room temperature, N
-Methylhydroazine derivative was produced, saturated saline (15 ml) was added thereto, and the mixture was extracted with dichloromethane (5 ml) for 3 hours.

The dichloromethane layer was separated, a predetermined amount of gas chromatography internal standard solution (IS) was added, and the mixture was further adjusted to exactly 10 ml with dichloromethane to prepare a sample for gas chromatography analysis, which was subjected to chromatography under the following conditions. It was

Capillary column: DB-WAX 25 m × 0.25 mm Column temperature: 35 ° C. (holding 1.0 min) -190 ° C. (holding 20 min) Temperature increase 40 ° C./min Injection port temperature: 250 ° C. Detector temperature: 300 [Deg.] C. Detector: NPD (nitorogen-phosphorus detector) Carrier-gas: Helium The results are shown in FIGS. From these chromatograph charts, the antioxidant active substance of the present invention having a structure in which one glucose molecule is bound to isovitexin significantly inhibits not only the production of MA but also the production of 4HN (4-hydroxynonenal). -It was revealed that it has 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, 2.5 liters of n-hexane was added, and the mixture was stirred at room temperature for about 5 minutes, and then the insoluble component was centrifuged (8000 rpm, 10 mi).
n. ) And n-hexane 2.
5 liters were added and the same operation was repeated to obtain an n-hexane insoluble component.

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

60 g of this fraction was added to Amberlite XAD-2
After being adsorbed on the column, it was distilled to obtain a water content of 80, 60, 4
Elution was performed sequentially with 0, 20 and 0 v / v% methanol and acetone.

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

Model diuses having the compositions shown in Tables 1 and 2 containing β-carotene were prepared to have pH of 3 and p.
60% methanol extract and 2'-O- in H5
The antioxidant activity of glucosyl-isovitexin against β-carotene was measured using water and vitamin C as controls.

The quantification of β-carotene was carried out in accordance with the Pharmaceutical Society of Japan: Hygiene Test Method / Comment, p347-349 (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 young barley leaves, the green juice obtained by squeezing the juice was powdered by a drying method such as spray drying or freeze drying, and 10 kg of green juice powder was extracted twice with hexane 2001. Repeatedly, 1001 of water was added to the hexane-insoluble portion and the water-soluble component was spray-dried to obtain 3.8 kg of a spray-dried product. Next, 1001 of hydrous ethanol having a water content of 20% was added to the dry powder to obtain 2.7 kg of a hydrous 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-containing methanol-soluble component having a water content of 40%, and then methanol was distilled off to obtain 2 kg of a water-containing methanol-soluble component having a water content of 40%. This component is referred to as substance A. A suspension was prepared by adding 400 g of talc to 100 g of the obtained substance A, and an intake temperature of 180 ° C. and an exhaust temperature of 12
Spray drying was performed at 0 ° C. to produce 470 g of a powder raw material.

[0058]

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

[0059]

[Embodiment 9] Lindex-P (Sanraku Co., Ltd.) 100
g to 300 ml of water and mixed to form a slurry,
The methanol fractionation in Example 6 was carried out accurately in a stepwise manner, a substance which was a water-containing methanol fraction having a water content of 40% was collected, and the substance was recrystallized from water-containing tamenol having a water content of 40% to obtain the substance. 40 g of 2'-O-glucosyl-isovitexin is added and stirred at room temperature for 90 minutes, then spray dried at a concentration of 30%, an intake temperature of 170 ° C. and an exhaust temperature of 110 ° C., and 127 g of a cyclodextrin inclusion compound powder. The 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 11 100 ml of a 4% solution of sodium silicate was desalted and then adjusted to pH 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 85 ml of sodium silicate solution, and the mixture was added successively and concentrated at 90 ° C. for 8 hours to produce spherical silica containing an antioxidant active substance.

[Brief description of drawings]

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

FIG. 2 is an ultraviolet absorption spectrum of 2′-O-glucosyl-isovitexin in the 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 ¹³ C-NMR measurement result of 2′-O-glucosyl-isovitexin.

FIG. 6 is a graph showing measurement results of lipid peroxides of each extract and α-tocopherol obtained in Example 1 by TBA method (measurement results of absorbance at 535 nm).

FIG. 7 shows a gas chromatographic chart of lipid peroxidation products of arachidonic acid, MA and 4HN using Fuenton's reagent.

FIG. 8 shows a gas chromatograph chart of lipid peroxidation products of arachidonic acid, MA and 4HN using Fuenton's reagent.

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

[Procedure amendment]

[Submission date] July 29, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief explanation of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 shows a chart of measurement 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 the 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 a ¹³ C-NMR measurement result of 2′-O-glycosyl-isovitexin.

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

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

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

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

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

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

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Correction target item name] All drawings

[Correction method] Change

[Correction content]

[Fig. 2]

[Figure 8]

[Figure 1]

[Figure 3]

[Figure 9]

[Figure 10]

FIG. 11

[Figure 4]

[Figure 5]

[Figure 6]

[Figure 7]

Claims (10)

[Claims] 1. An antioxidant active substance comprising a green leaf component of a green plant which is substantially insoluble in n-hexane and soluble in water-containing ethanol having a water content of 0 to 80%. 2. A water-soluble component of green leaf of a green plant, comprising:
An antioxidant active substance comprising a component soluble in water-containing ethanol having a water content of 0 to 80%. 3. The antioxidant active substance according to claim 1 or 2, wherein the water-containing ethanol is ethanol having a water content of 15 to 50%. 4. The antioxidant active substance according to claim 1, wherein the green plant is a wheat plant. 5. A green leaf component of a green plant, which is substantially insoluble in n-hexane and soluble in water-containing ethanol having a water content of 0 to 80%, and further has a water content of 0 to 80%. An antioxidant active substance consisting of a component soluble in water. 6. The antioxidant active substance according to claim 5, wherein the water-containing ethanol is ethanol having a water content of 15 to 50%. 7. The antioxidant active substance according to claim 5, wherein the water-containing methanol is a water-containing methanol having a water content of 10 to 70%. 8. The antioxidant active substance according to claim 5, wherein the green plant is a wheat plant. 9. The following formula: 2'-O-glycosyl-isovitexin represented by. 10. An antioxidant comprising 2'-O-glycosyl-isovitexin.