Antioxidation active substance and utilization thereof

An antioxidant substance which is a green leaf component in a green plant, comprising a component which is substantially insoluble in n-hexane but soluble in an aqueous ethanol solution having a water content of 0 to 80% by volume. The substance has an antioxidant activity as potent as or more potent than .alpha.-tocopherol, and is useful as an antioxidant for use in the field of foods, and medicines. Particularly, the antioxidant substance can be used for maintaining the freshness and quality of foods or storage thereof. The substance can be blended with cosmetics for skin and hair and are useful for the prevention of spots, freckles, chapping and sunburn.

The present invention relates to an antioxidant component having a potent 
antioxidant activity derived from green plants, preferably green leaves of 
cereal, especially young green leaves of barley and to utilization of the 
same as an additive to foods, cosmetics and the like. 
Hitherto, there have been known various antioxidants derived from natural 
sources or chemically synthesized and used in the field of foods 
medicines, cosmetics and the like which include natural antioxidants such 
as example, .alpha.-tocopherol, and ascorbic acid, and phenol type 
synthetic antioxidants such as butylhydroxyanisole (BHA), and 
dibutylhydroxytoluene (BHT). 
On the other hand, noticing that green leaves of green plants, particularly 
cereals, contain components having many physiological activities such as 
antitumor activity, antihyperlipemic activity, hypoglycemic activity, 
antiviral activity and the like, the present inventors have examined 
various components contained for their antioxidant activity. 
As a result, it has now been found that green leaf components in green 
leaves of green plants, for example, cereals such as barley and wheat, 
contain a component which has an antioxidant activity as potent as or more 
potent than .alpha.-tocopherol. The present invention has been achieved 
based on this discovery. 
According to one aspect of the present invention, there is provided an 
antioxidant substance derived from a green leaf component in a green 
plant, comprising a component which is substantially insoluble in n-hexane 
but soluble in an aqueous ethanol solution containing 0 to 80% by volume 
of water. 
According to another aspect of the present invention, there is provided an 
antioxidant substance derived from a green leaf component in a green 
plant, comprising a component which is substantially insoluble in n-hexane 
but soluble in an aqueous ethanol solution containing 0 to 80% by volume 
of water and in an aqueous methanol solution containing 0 to 80% by volume 
of water. 
Hereafter, the antioxidant substances of the present invention will be 
described in more detail. Herein, the water content % of aqueous alcohol 
solution is expressed in terms of v/v %. 
The green plants which can be used as raw materials may preferably be 
plants of Gramineae family, especially cereals such as barley and wheat. 
In addition, there can also be used meadow grasses such clovers, and 
alfalfa, vegetables such as kale, spinach, lettuce, parsley, cellery, 
cabbage, Chinese cabbage, mizuna (a kind of Japanese cabbage: Brassica 
rapa L. var. laciniifolia Kitam.), green pepper, green leaves of carrot, 
and green leaves of radish, non-cultivated vegetables which grow in fields 
or wastelands or mountains such as bamboo grass, and ashitaba (a kind of 
Japanese parsley: Angelica keiskei (Mig.) Koidz.); and further fresh-water 
or sea-water algae such as Spirulina, Chlorella, wakame (a Japanese sea 
weed: Undaria pinnatifida Suringar), and green laver (Enteromorpha). 
As the cereals which can be used favorably in the present invention, the 
most preferred one is barley. Besides, wheat, rye, oats, gromwell-reed, 
corn, millet, Italian dye-grass can also be used. 
In the present invention, fresh stems and/or leaves of young plants 
harvested before ripening of these green plants, especially cereals 
(herein these stems and/or leaves are called generally as "green leaves") 
are particularly suitable. 
Green leaves of green plants, for example, cereals, are first sucked by a 
mechanical crushing means such as a mixer, a juicer or the like and then 
crude solid contents are removed by sifting, filtration or the like, if 
desired, to prepare a sucked juice (hereinafter, referred to as "green 
juice"). 
Next, the green juice as is or green juice powder obtained by drying by a 
suitable drying means such as lyophilization, spray-drying or the like is 
extracted with a sufficient amount of water or n-hexane. This extraction 
treatment can be performed usually at room temperature and may be repeated 
twice or more, if desired, thereby separating and recovering a component 
which is soluble in water or substantially insoluble in n-hexane. The 
extract component recovered may be dried and solidified in this stage in 
the same manner as described above. 
The water-soluble component or n-hexane-insoluble component thus obtained 
is extracted with an aqueous ethanol solution having a water content of 0 
to 80%, preferably 10 to 70%, and more preferably 15 to 50%, for example, 
an aqueous ethanol solution having a water content of 20% to separate and 
recover a component soluble in that aqueous ethanol solution. 
The extraction with such aqueous ethanol solution may be performed directly 
on the green juice prepared as described above or water-soluble components 
of green leaves obtained by completely removing water-insoluble components 
from the green juice or powder prepared by drying such water-soluble 
components by a suitable drying means such as lyophilization or 
spray-drying. 
The aqueous ethanol-soluble component may be used as an antioxidant 
substance of the present invention as it is or after concentration or 
distilling off of the solvent. 
According to the present invention, the aforementioned aqueous 
ethanol-soluble component can be treated with a suitable adsorbent such as 
Styrene-DVB resin absorbent (for example, Amberlite Adsorbent XAD-2, 
registered trademark for a product by Rohm & Haas Co.) and eluted with an 
aqueous methanol solution having a water content of 0 to 80%, preferably 
20 to 70%, and more preferably 30 to 60% to recover a component soluble in 
that aqueous methanol solution. This makes it possible to obtain a 
fraction having a higher antioxidant activity. 
The aqueous methanol-soluble component recovered from barley as described 
above can be purified by recrystallization from, for example, an aqueous 
methanol solution having a water content of 30 to 70%, preferably 40 to 
60% to obtain an active ingredient of an antioxidant substance as pale 
yellow crystal. The active ingredient of the antioxidant thus isolated has 
been identified by NMR, mass spectrometry or the like 
2'-O-glucosyl-isovitexin having a structure represented by the following 
formula: 
##STR1## 
It is presumed that an antioxidant substance having the aforementioned 
structure or one similar thereto is contained in green leaves of various 
green plants such as cereals and that substance constitutes the active 
ingredient of the antioxidant substance of the present invention. 
As will be apparent from examples hereinbelow, the antioxidant substance of 
the present invention has a high antioxidant activity as potent as or more 
potent than .alpha.-tocopherol, and is useful as an antioxidant in the 
field of foods, medicines and the like. 
For example, the antioxidant substance of the present invention is free of 
various metal elements and substances promoting denaturation of foods 
which are usually contained in green leaves used as raw material, and 
hence it can be blended advantageously in various inorganic or organic 
materials or compositions in the field of foods, medicines and the like 
which requires antioxidizing properties. For example, the antioxidant 
substance of the present invention may be mixed with sugars such as 
fructose, glucose, dextrin, and starch; amino acids; organic acids such as 
citric acid, malic acid, tartaric acid, and succinic acid; various 
vitamins; colorants, perfumes, various viscosity increasing agents, and 
the like, after being included by cyclodextrin, crown ether or the like, 
if desired. In particular, the antioxidant substance of the present 
invention can be sterilized by filtration when it is in the form of 
aqueous compositions since it gives substantially no adverse influence on 
the solubility in water or transparency of the resulting composition. 
The antioxidant substance of the present invention has advantages that it 
can be mixed or blended as powder prepared by spray-drying or vacuum 
drying or the like with a raw material for medicines, paints, cosmetics, 
foaming agents and the like, such as talc, zinc oxide, sodium carbonate, 
sodium hydrogen carbonate, titanium dioxide, kaolin, and calcium phosphate 
to produce novel industrial products and that it causes no change in the 
quality of the products. In addition, the antioxidant substance of the 
present invention, which is soluble in water and also in alcohols, is 
useful for the stabilization of inorganic and organic compositions, and 
makes it possible to produce excellent novel products, for example, 
antioxidants for the preparation of polymers; emulsion paints; cosmetics; 
paper products; foods; medicines; materials for medical therapy and the 
like. 
For example, the antioxidant substance provided according to the present 
invention may be used for maintaining freshness and quality of or 
preserving various foods by blending it with the foods. Here, the term 
"foods" is used in a broader sense and includes not only typical foods but 
also beverages (including beverages), seasonings and the like, for 
example, fruits and their processed foods (e.g., canned fruits, bottled 
fruits, jams, marmalades, etc.), fishes, meats and their processed foods 
(e.g., hams, sausages, corned beef, etc.) breads and noodles (Japanese 
noodles, buckwheat noodle, Chinese noodle, spaghetti, macaronis, etc.) 
fruit juice, various drinkings, cookies, candies, dairy products (e.g., 
butter, cheese, etc.) vegetable plant fat and oils, margarine, plant 
proteins, retort foods, frozen foods, various seasonings (e.g., miso (bean 
paste), soy sauce, sauce, etc.), alcoholic beverages (e.g., fruit liquors, 
sake, etc.) and the like. 
The antioxidant substance of the present invention is pale yellow or 
colorless, soluble in water and alcohols and can be well absorbed by 
living organisms so that it can be blended readily with the aforementioned 
foods without giving any substantial adverse influence on their 
compositions and components or appearance. For example, it can be freely 
blended with various additives which are often used in foods, including 
sweeteners such as fructose, glucose, and millet jelly; organic acid such 
as citric acid, malic acid, tartaric acid, and succinic acid and salts 
thereof; various vitamins, colorants, perfumes, various vegetable 
viscosity increasing agents, and the like, without giving no influence to 
their solubility in water and transparency, but permitting treatments such 
as filtration, sterilization and the like. 
The antioxidant substance of the present invention can be used 
advantageously as an agent for retaining the freshness or quality of such 
foods and uptake of the antioxidant substance of the invention is helpful 
for the maintenance or promotion of health. 
When the antioxidant substance of the present invention is used as an agent 
for preserving the freshness or quality of foods, the amount of the 
antioxidant substance may be varied within a wide range and it is 
difficult to set it to a specific value. However, as a guideline, it can 
be used in an amount of 0.1 to 10% by weight, and preferably 0.5 to 7% by 
weight, in the form of an extract with aqueous ethanol solution having a 
water content of 20%; 0.001 to 5% by weight, and preferably 0.01 to 2% 
weight, in the form of methanol fraction having a water content of 40%; 
and 0.001 to 1% by weigh, and preferably 0.005 to 0.5% by weight, in the 
form of 2"-O-glucosyl-isovitexin. 
The antioxidant substance provided according to the present invention 
exhibits excellent effects on the prevention of spots (chloasma), 
freckles, chapping, burning with UV rays (sunburn), etc. and is safe. 
Hence it can be utilized for the prevention of spots (chloasma, freckles, 
chapping, burning with UV rays (sunburn) and protection of hair and the 
like by blending it with cosmetics for skins and hair. 
As described above, the antioxidant substance of the present invention is 
pale yellow, soluble in water and alcohols and highly absorbable to living 
organisms, and can be readily blended with cosmetics for skin and hair, 
e.g., water, alcohols, aqueous alcohol solutions, lotions, creams, cream 
emulsions, hair tonics, hair growing agents, bath compositions, soaps, 
ointments and the like without giving any substantially adverse effects on 
their composition or appearance. 
The amount of the aforementioned antioxidant substance to be blended may be 
varied within a wide range depending on the kind and utility of the 
cosmetics but generally it is suitable to blend the antioxidant substance 
in an amount of 0.01 to 10% by weight, and preferably 0.1 to 5% by weight, 
and 0.001 to 1% by weight and preferably 0.005 to 0.5% by weight as 
2'-O-glucosyl-isovitexin, active ingredient, based on the weight of the 
matrix of the cosmetics. 
The matrix which can be used in the aforementioned cosmetics is not limited 
particularly and there can be various matrices conventionally used in skin 
and hair cosmetics such as water, alcohols, propylene glycol, stearic 
acid, glycerol, cetyl alcohol, liquid paraffin and the like. As usual, the 
cosmetics may, if desired, contain one or more of vitamins, extracts from 
galenics, hormones and medicines for external applications, and the like. 
Hereafter, the present invention will be explained more concretely by way 
of examples and with reference to the attached drawings.

EXAMPLE 1: FRACTIONATION AND PREATION OF ACTIVE INGREDIENT 
To lyophilized powder (2.0 g) of green juice of barley before ripening was 
added 500 ml of n-hexane, and the mixture was stirred well at room 
temperature for about 5 minutes. Thereafter, insoluble matter was 
separated by centrifugation (8,000 rpm, 10 minutes), and 500 ml of 
n-hexane was added to the insoluble matter thus separated. Repeating this 
procedure, n-hexane-insoluble component was obtained. 
The n-hexane-insoluble component was added to 500 ml of an aqueous ethanol 
solution having a water content of 20 v/v %. After stirring the mixture 
well at room temperature for about 5 minutes, insoluble matter was 
filtered. The insoluble matter filtered was treated again with an aqueous 
ethanol having a water content of 20 v/v %, and the filtrate obtained was 
combined with the previous filtrate, followed by evaporation of the 
solvent under reduced pressure. This gave 13.0 g of ethanol extract. 
The ethanol-soluble content was adsorbed on Amberlite XAD-2 column, and 
eluted serially with deionized water, aqueous methanol solutions having 
water contents of 80 v/v %, 60 v/v %, 40 v/v %, 20 v/v %, and 0 v/v %, 
respectively, and acetone to obtain various eluates. 
The respective eluates were distilled under reduced pressure to evaporate 
the solvents and as a result 4.77 g of water-extract, 180 mg of 20% 
methanol-extract, 131 mg of 40% methanol-extract, 199 mg of 60% 
methanol-extract, 32 mg of 80% methanol-extract, 165 mg of 100% 
methanol-extract, 0.87 mg of acetone-extract were obtained (here, % of 
methanol indicates the concentration of methanol (v/v %) in each aqueous 
methanol solution). 
The 60% methanol-extract thus obtained was recrystallized from 60% methanol 
to obtain 180 mg of pale yellow crystals. The structure of the crystal was 
performed by mass spectrometry and NMR. 
Mass spectrometry was performed by using an FAB-MS: VG ZAB-2F (Xenon Gun) 
(Jon Tech) type mass spectrometer, and results as shown in FIG. 1 were 
obtained. The mass spectrum thus obtained showed a peak of [M+H.sup.+ ] at 
m/z=595, and the molecular weight was determined to be 594. Taking this 
together with the results of elemental analysis, the molecular formula of 
the present substance was judged to be C.sub.27 H.sub.30 O.sub.15. 
Upon UV absorption spectrum of the present substance was measured in 
H.sub.2 O and in methanol, absorptions by flavonoid glucoside were 
observed as shown in FIGS. 2 and 3, respectively. 
Infrared absorption spectrum was measured by KBr method using JASCO 
FT/IR-7000S and the results obtained are shown in FIG. 4. Absorption at 
3422 cm.sup.-1 indicates the presence of OH group. 
Upon hydrolysis with hydrochloric acid-methanol by a conventional method, 
the present substance released one molecule of glucose to produce 
isovitexin. 
Further, .sup.13 C NMR spectrum (500 MHz) of the present substance was 
measured by GE OMEGA 300 type nuclear magnetic resonance spectrum 
absorption spectrometer using 25 mg of purified antioxidant substance and 
tetramethylsilane (TMS, (CH.sub.3).sub.4 Si) as an internal standard, and 
results shown in FIG. 5 were obtained. In FIG. 5, chemical shift was 
indicated by ".delta.". The purified antioxidant substance gave signal 
corresponding to 27 carbon atom in MeOH-d.sub.4, which suggested the 
following structural formula: 
##STR2## 
based on standard value of .sup.13 C-NMR for isovitexin (Remarathnam, N., 
Osawa, T., Namiki, M. and Kawakishi, S.: J. Agric. Food Chem., 37 316-319 
(1989)). 
From the above formula, the present substance is named 
2'-O-glucosyl-isovitexin. 
EXAMPLE 2: FRACTIONATION AND PREATION OF ACTIVE INGREDIENT 
Lyophilized powder (20 g) of green juice of wheat before ripening as 
treated in the same manner as in Example 1 to obtain 118 mg of 60% 
methanol-extract. Further, the extract was repeatedly recrystallized from 
60% methanol to obtain 106 mg of pale yellow crystals. The present 
substance was found to be the same substance as that obtained in Example 
1. 
EXAMPLE 3: FRACTIONATION AND PREATION OF ACTIVE INGREDIENT 
Lyophilized powder (20 g) of green juice of comfrey before ripening was 
treated in the same manner as in Example 1 to obtain 40 mg of 60% 
methanol-extract. Further, the extract was repeatedly recrystallized from 
60% methanol to obtain 37 mg of white crystals. The present substance was 
found to be the same substance as that obtained in Example 1. 
EXAMPLE 4: MEASUREMENT OF LIPID PEROXIDE BY TBA METHOD 
To 7.5 mg of linoleic acid was added 0.22 mg of .alpha.-tocopherol or 0.22 
mg of water-extract obtained in Example 1. Then 200 .mu.l of Fenton's 
reagent (FeCl.sub.2, H.sub.2 O.sub.2) was added to each of the resulting 
mixture, and the mixtures thus obtained were incubated at 37.degree. C. 
for 16 hours (total amount: 5 ml). 
To 0.2 ml of each solution were added 0.2 ml of aqueous 8% SDS.sup.1 
solution, 1.5 ml of acetate buffer (pH 3.5) and 1.5 ml of an aqueous 0.67% 
TBA.sup.2H) solution, and the mixture was heated for 1 hour in a boiling 
water bath (95.degree. to 100.degree. C.). 
After cooling, 5 ml of butanol was added. The resulting mixture was stirred 
vigorously and then the butanol layer was separated by centrifugation 
(2,000 rpm, 10 minutes), and optical density of the butanol layer was 
measured at 535 nm. Results obtained are shown in FIG. 6. 
Notes: 1) SDS=sodium dodecylsulfate 
2) TBA=thiobarbituric acid 
EXAMPLE 5: GAS CHROMATOGRAPHIC ANALYSIS OF PEROXIDE PRODUCT OF LIPID, 
MAD.sup.3) AND 4NH (4-HYDROXYNONENAL) 
To microsomes and 7.5 mg of arachidonic acid was added 0.22 mg of 
.alpha.-tocopherol or 0.22 mg of antioxidant substance obtained in Example 
1. To the mixture was added 5 ml of Tris hydrochloric acid buffer solution 
(05M Trizma HCl, pH 7.4; 0.15M KCl; 0.2% SDS). The resulting mixture was 
shaken mildly to make a suspension, to which was added 200 .mu.l of 
Fenton's reagent (FeC.sub.12, H.sub.2 O.sub.2). The mixture was reacted at 
37.degree. C. for 16 hours. After the reaction was stopped by addition of 
50 l of 4% BHT.sup.4), 40 .mu.l of N-methylhydrazine and the reaction 
mixture was left to stand at room temperature for 1 hour to allow to 
produce an N-methylhydrazine derivative. After adding thereto 15 ml of 
saturated saline, the reaction mixture was extracted with 5 ml of 
dichloromethane for 3 hours. 
The dichloromethane layer was separated, a predetermined amount of an 
internal standard solution for gas chromatography (I. S.) was added 
thereto, and dichloromethane was added to make exactly 10 ml to prepare a 
sample for gas chromatographic analysis, which was subjected to gas 
chromatography under the following conditions: 
______________________________________ 
Capillary column: 
DB-WAX 
25 m .times. 0.25 mm 
Column temperature: 
35.degree. C. (retention time: 
1.0 minutes) - 190.degree. C. (retention 
time: 20 minutes) temperature 
elevation rate: 40.degree. C./minute 
Inlet temperature: 
250.degree. C. 
Detector temperature: 
300.degree. C. 
Detector: NPD (nitrogen-phosphorus detector) 
Carrier Gas: Helium 
______________________________________ 
The results obtained are shown in FIGS. 7 to 9. From the chromatographic 
charts, it revealed that the antioxidant substance of the present 
invention having a structure corresponding to isovitexin to which one 
molecule of glucose is connected strongly inhibits the production of not 
only MAD but also 4HN (4-hydroxynonenal) and has an antioxidant activity 
more potent than -tocopherol. 
Notes: 3) MAD=malondialdehyde 
4) BHT=butylhydroxytoluene 
EXAMPLE 6 
To the powder (100 g) of green juice obtained in Example 1 was added 2.5 
liters of n-hexane, and the mixture was stirred at room temperature for 
about 5 minutes. Insoluble matter as separated by centrifugation (8,000 
rpm, 10 minutes) and 2.5 liters of n-hexane was added thereto. This 
procedure was repeated to obtain n-hexane insoluble component. 
To the insoluble component was added 2.5 liters of aqueous ethanol solution 
having a water content of 20 v/v %, and extraction was repeated similarly 
to the above-described procedure to obtain 20 v/v % (water content) 
ethanol-soluble component. After distilling off ethanol under reduced 
pressure, 72 g of a fraction which was able to be extracted with aqueous 
ethanol solution having a water content of 20 v/v % ethanol. 
This fraction (60 g) was adsorbed by Amberlite XAD-2 column and then 
serially eluted with distilled water, aqueous methanol solution having 
water contents of 80 v/v %, 60 v/v %, 40 v/v % 20 v/v % and 0 v/v %, 
respectively, and acetone. 
The solvents were distilled off from the eluates under reduced pressure to 
obtain 27 g of water-extract, 1.1 g of 20% methanol-extract, 680 mg of 40% 
methanol-extract, 1.5 g of 60% methanol-extract, 170 mg of 80% 
methanol-extract, and 5.3 mg of acetone-extract. Separately, 1.5 g of 60% 
methanol-extract which was prepared similarly was recrystallized to obtain 
1.2 g of 2'-O-glucosyl-isovitexin. 
Model juices containing .beta.-carotene having compositions as shown in 
Tables 1 and 2 were prepared, and antioxidant activities of 60% 
methanol-extract and 2'-O-glucosyl-isovitexin, respectively, at pH 3 or 5 
on .beta.-carotene were measured using water and vitamin C as control. 
Determination of .beta.-carotene was performed based on the method 
prescribed in Hygienic Examination Methods, Commentary, ed. Japan 
Pharmaceutical Association, p. 347-349 (1990) published by Kinbara Shuppan 
Co., Ltd.). 
TABLE 1 
______________________________________ 
Composition of Model Juice 
Component Composition 
______________________________________ 
Inverted sugar 20.0 g 
Citric acid, anhydrous 
1.0 g 
.beta.-Carotene* 0.65 g 
H.sub.2 O to make 100 ml 
pH adjusted to 3.0 
______________________________________ 
Note: 
*carotene base: produced by Sanei Kagaku Kogyo Co., Ltd. 
TABLE 2 
______________________________________ 
Composition of Model Juice 
Component Composition 
______________________________________ 
Inverted sugar 20.0 g 
Citric acid (dihydrate) 
0.43 g 
Sodium citrate (dihydrate) 
1.23 g 
.beta.-Carotene* 0.656 mg 
H.sub.2 O to make 100 ml 
pH adjusted to 5.05 
______________________________________ 
Note: 
*carotene base: produced by Sanei Kagaku Kogyo Co., Ltd. 
Tables 3 and 4 show antioxidant activities of the respective fractions on 
.beta.-carotene. The reaction temperature was 18.degree. C. 
TABLE 3 
______________________________________ 
Antioxidant Activity at pH 3 
Residual Ratio (%) 
of .beta.-Carotene 
Antioxidant Day 0 Day 7 
______________________________________ 
Water 100 18.0 
L-Ascorbic acid: 0.17 mM 
100 44.8 
2'-O-Glucosyl- 100 55.0 
isovitexin: 0.17 mM* 
60% Methanol-fraction: 
100 52.0 
0.17 mM* 
______________________________________ 
Note: 
*Expressed as amount of 2O-glucosyl-isovitexin. 
TABLE 4 
______________________________________ 
Antioxidant Acitivity at pH 5 
Residual Ratio (%) 
of .beta.-Carotene 
Antioxidant Day 0 Day 7 
______________________________________ 
Water 100 0 
L-Ascorbic Acid: 0.17 mM 
100 30.0 
2'-O-Glucosyl- 100 41.0 
isovitexin: 0.17 mM* 
60% Methanol-fraction: 
100 44.5 
0.17 mM* 
______________________________________ 
Note: 
*Expressed as amount of 2O-glucosyl isovitexin. 
Further, juice containing 2'-O-glucosyl-isovitexin and 60% methanol-extract 
fraction showed no color change both at pH 3 and at pH 5 and retained 
always fresh pale red color while juice containing no antioxidant showed 
considerable color fading at pH 3 and became colorless at pH 5. 
EXAMPLE 7 
After being washed, your green leaves of barley were sucked to obtain green 
juice, which was then powderized by a suitable drying method such as 
spray-drying, lyophilization or the like. The green juice powder thus 
obtained (10 kg) was extracted twice each with 200 liters of hexane. Water 
(100 liters) was added to hexane-insoluble portion and water-soluble 
component was spray-dried to obtain 3.8 kg of spray-dried product. Then, 
100 liters of aqueous ethanol solution having a water content of 20 v/v % 
was added thereto obtain 2.7 kg of 20% (water content) ethanol-soluble 
component, from which ethanol was distilled off. To this were added 70 
liters of an aqueous methanol solution having a water content of 40 v/v % 
to extract 40% (water content) methanol-soluble component, and then 
methanol was distilled off to obtain 2 kg of 40% (water content) 
methanol-soluble component. This component was named substance A. To 100 g 
of substance A was added 400 g of talc to prepare a suspension, which was 
then spray-dried at an air absorption temperature of 180.degree. C. and an 
air exhaustion temperature of 120.degree. C. to produce 470 g of powdery 
raw material. 
EXAMPLE 8 
A solution containing 100 g of substance A obtained in Example 7 and 400 g 
of dextrin was prepared, which was spray-dried at an air absorption 
temperature of 190.degree. C. and an air exhaustion temperature of 
120.degree. C. to obtain 430 g of powdery raw material. 
EXAMPLE 9 
Water (300 ml) was added to 100 g of Lintex-P (Sanraku Co., Ltd.) and the 
mixture was kneaded to form a slurry. Methanol fractionation as in Example 
6 was exactly performed stepwise to separate a substance which as a 
fraction extracted with an aqueous methanol having a water content of 40%. 
This substance was recrystallized from an aqueous methanol having a water 
content of 40% to obtain 2'-O-glucosyl-isovitexin. The 
2'-O-glucosyl-isovitexin (40 g) thus obtained was added to the slurry and 
stirred at room temperature for 90 minutes, followed by spray-drying at an 
air absorption temperature of 170.degree. C. and an air exhaustion 
temperature of 110.degree. C. to obtain 127 g of powdery raw material as a 
cyclodextrin inclusion compound. 
EXAMPLE 10 
Kaolin (200 g) was mixed with 100 g of substance A obtained in Example 7 to 
prepare a 30% suspension, which was then spray-dried at an air absorption 
temperature of 170.degree. C. and an air exhaustion temperature of 
110.degree. C. to obtain 270 g of powdery raw material. 
EXAMPLE 11 
After being desalted, 100 ml of a 4% sodium silicate solution was adjusted 
to pH 9 with 1% potassium hydroxide, and 15 ml aliquot thereof was heated 
at 95.degree. C. for 15 minutes. Then, 10 g of 2'-O-glucosyl-isovitexin 
obtained by the method of Example 1 was portionwise added to 85 ml of the 
sodium silicate solution which remained, and then concentrated at 
90.degree. C. for 8 hours to produce spherical silica containing the 
antioxidant substance. 
EXAMPLE 12 
After being washed with water, sterilized and washed again with water, 100 
kg of young leaves of barley were crushed with a crusher and sucked with a 
sucker to obtain about 95 liters of young barley leaf extract. This young 
barley leaf extract was spray-dried to obtain 4 kg of green juice powder, 
which was then extracted with 20 liters of an aqueous ethanol solution 
having a water content of 20%, followed by removal of the solvent to 
obtain 700 g of a liquid extract. 
As a result of analysis, the extract was found to contain 
2'-O-glucosyl-isovitexin in a concentration of 0.7% by weight. This 
extract was diluted with water to prepare an aqueous solution having a 
concentration of 100 mg/liter expressed in terms of 
2'-O-glucosyl-isovitexin. 
In this aqueous solution was immersed a Kabosu fruit (a kind of citrus 
fruit, Citrus sphaerocarpa hort. ex Tanaka) for 30 minutes and then taken 
out and left to stand at room temperature. Therefore, freshness retention 
tests were performed using water, vitamin C and vitamin E as controls by 
visual evaluation of the color of the kabosu fruit after lapse of a 
predetermined time, judging green as A, partially yellowing as B, and 
wholly yellowing as C. The results obtained are shown in Table 5. 
TABLE 5 
______________________________________ 
Freshness Retention Test on Kabosu Fruit 
Day Number 
Day 0 Day 7 Day 14 Day 20 
______________________________________ 
Water A B C C 
Vitamin C* A A B B-C 
Vitamin E** A A B B-C 
Extract A A A A 
______________________________________ 
Notes: 
*500 mg/liter 
**E mix P20, 500 mg/liter as vitamin E 
EXAMPLE 13 
The extract (100 g) obtained in Example 12 was extracted with an aqueous 
methanol solution having a water content of 40 and dried to obtain 65 g of 
an extract. Using this extract, freshness retention tests were performed 
on sudachi fruit (a kind of a citrus fruit, Citrus sudachi hort. ex 
Shirai) similarly to Example 12. The results obtained are shown in Table 
6. 
TABLE 6 
______________________________________ 
Freshness Retention Test on Sudachi Fruit 
Day Number 
Day 0 Day 7 Day 14 Day 20 
______________________________________ 
Water A B C C 
Vitamin C* A B B C 
Vitamin E** A B B C 
Extract A A A B 
______________________________________ 
Notes: 
*500 mg/liter 
**E mix P120, 500 mg/liter as vitamin E. 
EXAMPLE 14 
A component soluble in an aqueous ethanol solution having a water content 
of 20% prepared in the same manner as in Example 7 was formulated as set 
forth below to obtain a beverage. After preparation, the resulting juice 
was sterilized with heating at 85.degree. C. for 30 minutes. 
______________________________________ 
Granulated sugar 20 g 
Fructose 35 g 
Citric acid 3 g 
Succinic acid 0.1 g 
Component soluble in an aqueous 
10 g 
ethanol solution having a water 
content of 20% 
Perfume suitable amount 
To make 1 liter 
______________________________________ 
This preparation was a beverage which had a favorable fragrance and was 
effective for the suppression of foul breath. 
EXAMPLE 15 
The component soluble in an aqueous ethanol solution having a water content 
of 20% prepared in Example 7 was adsorbed on Amberlite XAD-2 column and 
eluted with an aqueous methanol solution having a water content of 40%. 
The fraction obtained was dried and blended with a juice having the 
following composition to obtain a beverage. After preparation, the 
resulting juice was sterilized with heating at 85.degree. C. for 30 
minutes. 
______________________________________ 
Granulated sugar 20 g 
Fructose 35 g 
Citric acid 3 g 
Tartaric acid 0.1 g 
.beta.-Carotene (carotene base 
10 mg 
No. 31256 produced by Sanei 
Kagaku Kogyo Co., Ltd.) 
Fraction eluted with an aqueous 
0.5 g 
methanol solution having a water 
content of 40% 
Perfume suitable amount 
To make 1 liter 
______________________________________ 
After leaving this formulation to stand under natural light for 15 days, 
.beta.-carotene remained in an amount of 83%. On the other hand, 
.beta.-carotene disappeared completely in a formulation in which the 
fraction eluted with an aqueous methanol solution having a water-content 
of 40% was absent. Determination of .beta.-carotene was performed based on 
the method prescribed in Hygienic Examination Methods, Commentary, ed. 
Japan Pharmaceutical Association, p. 347-349 (1990) published by Kinbara 
Shuppan Co., Ltd.). 
EXAMPLE 16 
A juice was prepared by repeating the procedure of Example 15 except that 
0.01% 2'-O-glucosyl-isovitexin was added instead of the aqueous methanol 
solution having a water content of 40%. After leaving this formulation to 
stand under natural light for 15 days, .beta.-carotene remained in an 
amount of 73% while in a juice in which 2'-O-glucosyl-isovitexin was 
absent .beta.-carotene disappeared completely. 
EXAMPLE 17 
Corned beef was prepared by providing beef meat, removing fat therefrom, 
adding salt thereto, pre-serving for 5 days, cooking to loosen meat 
fibers, and adding the fraction eluted with an aqueous methanol solution 
having a water content of 40% in an amount of 0.1% together with table 
salt, spices, fats, seasonings. The corned beef thus prepared was stored 
in a refrigerator for 10 days. As a result, the preparation in which the 
fraction eluted with an aqueous methanol solution having a water content 
of 40% was absent changed color considerably due to oxidation while the 
preparation of the invention retained fresh pink color immediately after 
production. 
EXAMPLE 18 
Upon preparation of margarin by a conventional method, the extract with an 
aqueous ethanol solution having a water content of 20% was added in a 
proportion of 10 g/1,000 g of margarin, and the margarin obtained was 
preserved at room temperature for 3 months. The preparation containing no 
such fraction suffered denaturation and its fragrance changed resulting in 
that it was unsuitable for use for food while the margarin of the present 
invention containing the fraction suffered substantially no such change in 
fragrance. 
EXAMPLE 19 
During preparing solid yoghurt by a conventional method, 0.2% of the 
fraction extracted with an aqueous ethanol solution having a water content 
of 20% was added. The yoghurt was filled in a small vessel and incubated 
therein to prepare a yoghurt preparation. This preparation had a unique 
favorable fragrance and was stable after preservation for a long time. 
EXAMPLE 20 
A liquid yoghurt was prepared by preparing and incubating a yoghurt by a 
conventional method, and then adding 0.01% of .beta.-carotene and 0.02% of 
2"-O-glucosyl-isovitexin. After superimposing the preparations thus 
obtained one on another and exposing them to the direct rays of the sun 
for 3 days, no decoloration occurred. 
EXAMPLE 21 
To durum wheat flour (7 kg) was mixed with 3 kg of a high protein content 
wheat flour, and the mixture was charged in a mixer, and 0.025 g of the 
extract with an aqueous ethanol solution having a water content of 20% 
obtained in Example 7 was added. After adding thereto 2.5 kg of water at 
about 40.degree. C., the mixture was kneaded and extruded through an 
extruder while being stirred, and dried and cut to a constant length to 
produce spaghetti containing an antioxidant substance. 
EXAMPLE 22 
To a mixture of high protein content wheat flour (350 g), 20 g of 
granulated sugar, 3.4 g of yeast, 5 g of water, and 25 g of butter was 
added 0.7 g of the extract with an aqueous ethanol solution having a water 
content of 20% obtained in Example 7, and the resulting formulation was 
treated under conventional conditions for baking bread to obtain bread 
containing an antioxidant substance. 
EXAMPLE 23 
After being washed, young green leaves of barley were sucked to obtain 
green juice, which was then powderized by spray-drying to obtain 1 kg of 
green juice powder. This was extracted twice each with 20 liters of 
hexane. Water (10 liters) was added to hexane insoluble portion and 
water-soluble component was spray-dried to obtain 380 g of spray-dried 
product. Then, 10 liters of an aqueous ethanol solution having a water 
content of 20% was added thereto to obtain 300 g of component soluble in 
an aqueous ethanol solution having a water content of 20%, from which 
ethanol was distilled off. To this were added 10 liters of an aqueous 
methanol solution having a water content of 40% to extract a component 
soluble in an aqueous methanol solution having a water content of 40%, and 
then methanol was distilled off to obtain 250 g of a component soluble in 
an aqueous methanol solution having a water content of 40%. This component 
was named substance A. Using substance A which was soluble in water and in 
aqueous alcohol solutions, a lotion having the following composition was 
prepared: 
______________________________________ 
Substance A 5 g 
95% Ethanol 100 g 
Methyl p-hydroxybenzoate 
0.05 g 
Perfume 0.5 g 
Colorant 0.005 g 
Purified water suitable amount 
Total to make 1,000 g 
______________________________________ 
APPLICATION EXAMPLE 1 
The lotion prepared in Example 23 was used everyday after washing face in 
the morning and before sleeping to examine effects on improvement of spots 
and freckles. Results obtained on a panel of 30 women are shown in Table 
7. The tests were performed in a period of 6 months. Evaluation was made 
on the following criteria. 
Color of spots and freckles: 
Brown: No effect 
Pale brown: Slightly effective 
Almost gone: Effective 
Boundary between spot or freckle portion and other portion: 
Boundary apparent: No effect 
Boundary unclear: Slightly effective 
Boundary almost: Effective undiscernible 
TABLE 7 
______________________________________ 
Slightly No 
effective Effective 
effect 
______________________________________ 
Color 15 7 8 
Boundary 17 8 5 
______________________________________ 
EXAMPLE 24 
The methanol fractionation as in Example 23 was exactly performed stepwise 
to separate substance A which was a fraction eluted with an aqueous 
methanol solution having a water content of 40%. The present substance was 
recrystallized from an aqueous methanol solution having a water content of 
40% to obtain 2'-O-glucosyl-isovitexin, which was added to the formulation 
for lotion described in Example 23 instead of substance A in a 
concentration of 0.1% to prepare a lotion. 
EXAMPLE 25 
______________________________________ 
Formulation of Cream: 
______________________________________ 
Stearic acid 10 g 
Isopropyl myristate 5 g 
Cetyl alcohol 5 g 
Liquid paraffin 7 g 
Glycerol monostearate 
2 g 
Polyoxyethylene stearate 
5 g 
Methyl p-hydroxybenzoate 
0.1 g 
______________________________________ 
were mixed and to the mixture were added 3 g of glycerol, 1 g of propylene 
glycol, and 50 g of water. The resulting mixture was emulsified and when 
the temperature of the emulsion reached 60.degree. C., 0.3 g of 
2'-O-glucosyl-isovitexin obtained in Example 1 was added, following by 
addition of a small amount of perfume while stirring to prepare a cream. 
APPLICATION EXAMPLE 2 
The cream prepared in Example 25 was used for 6 months everyday after 
washing face in the morning and before sleeping to examine effects on 
improvement of spots and freckles as well as chapping. Results obtained on 
a panel of 30 men an 30 women are shown in Table 7. Judgement on spots and 
freckles was made in the same manner as in Application Example 1 of 
example 23 while chapping was judged based on slipping of cream on skin by 
touching on the following criteria. 
No effect: Rough touch on the skin surface 
Slightly: Rough touch remains slightly, effective but the cream spreads 
well. 
Effective: The cream spreads well on the skin surface. 
The results obtained are shown in Tables 8 and 9. 
TABLE 8 
______________________________________ 
Men 
Number of Months for which 
Cream Was Applied 
1 M 2 M 3 M 4 M 5 M 6 M 
______________________________________ 
No effect 
20 20 17 11 11 10 
Slightly 
10 8 9 15 12 11 
effective 
Effective 
-- 2 4 4 7 9 
______________________________________ 
TABLE 9 
______________________________________ 
Women 
Number of Months for which 
Cream Was Applied 
1 M 2 M 3 M 4 M 5 M 6 M 
______________________________________ 
No effect 
23 20 18 10 7 5 
Slightly 
7 10 10 14 15 15 
effective 
Effective 
-- -- 2 6 8 10 
______________________________________ 
EXAMPLE 26 
Using 2'-O-glucosyl-isovitexin obtained by the method of Example 1, a hair 
growing agent of the following formulation was prepared: 
2-O-Glucosyl-isovitexin (0.1 g) was dissolved in 95 ml of an aqueous 66% 
ethanol solution, and 5 ml of propylene glycol, 0.1 g of perfume, and 0.1 
g of colorant were added to the resulting solution to prepare a hair 
growing agent. 
APPLICATION EXAMPLE 3 
Effects of the hair growing agent obtained in Example 26 were examined on a 
panel of 20 men and 20 women. Effects after 6 month every day application 
of the hair growing agent were judged according to the following criteria: 
______________________________________ 
Class (State) 
Rating 
______________________________________ 
Dandruff 
Very much 
-3 
Much -2 
Medium -1 
Normal 0 
Slight +3 
Thin Hair State 
Very Much 
-3 
Much -2 
Medium -1 
Normal 0 
Slight +3 
Color, Gloss 
Bad -3 
Normal 0 
Good +3 
______________________________________ 
Based on the aforementioned criteria the states before and after the 
application are shown in Table 10. 
TABLE 10 
______________________________________ 
Before Application After Application 
Thin Color, Thin Color, 
Sex Age Dandruff Hair Gloss Dandruff 
Hair Gloss 
______________________________________ 
.male. 
30 -1.5 -1 -3 0 -1 0 
.male. 
27 -2 -1 0 -1 0 +3 
.male. 
23 -2 -1 0 -1 0 +3 
.male. 
35 -1.5 -1 0 0 0 +1.5 
.male. 
40 -1 -2 0 0 -1 +1.5 
.male. 
53 -1 -2 -3 0 -1.5 0 
.male. 
43 0 -3 -3 0 -2.5 0 
.male. 
43 -1 -2 0 0 -1.5 0 
.male. 
48 -2 -2 -3 -1 -1 +1.5 
.male. 
55 -1 -3 -3 0 -2.5 +1.5 
.male. 
52 -1 - 3 -3 +3 -2 +1.5 
.male. 
38 -1 -2 -3 0 -1 0 
.male. 
35 -1.5 -1 0 -1 -1 0 
.male. 
37 -1.5 -2 0 0 -1 0 
.male. 
40 -1 -3 -3 0 -2 +1.5 
.male. 
45 -1 -2 -3 0 -1.5 +1.5 
.male. 
43 -1 -2 -3 0 -1 +1.5 
.male. 
56 -1 -2 -3 0 -1 +1.5 
.male. 
54 -2 -1 0 -1 -1 +1.5 
.male. 
55 -2 -2 -3 -1 -1.5 0 
.female. 
30 -2 0 +3 0 0 +3 
.female. 
35 -2 0 0 -1 0 +2 
.female. 
37 -3 0 0 -1 0 +3 
.female. 
40 -1 0 -3 0 0 0 
.female. 
40 -2 0 -1.5 0 0 0 
.female. 
43 -2 0 -1.5 -1 0 0 
.female. 
45 -1 0 -1.5 0 0 +3 
.female. 
47 -2 -1 -1 -2 +1.5 +3 
.female. 
47 -2 0 -2 0 0 +1 
.female. 
47 -2 -1 -2 0 0 0 
.female. 
50 -3 0 -3 0 0 0 
.female. 
50 -2 -1 -2 0 0 0 
.female. 
50 -1 -1 -3 0 -1 +1.5 
.female. 
51 -3 -1 -1.5 -1.5 0 0 
.female. 
52 -2 -1 -2 0 0 0 
.female. 
52 -3 -1 -3 0 0 +1.5 
.female. 
53 -2 -2 -3 -1 -1 0 
.female. 
54 -1 -1 -3 0 0 +1.5 
.female. 
55 -1 -1 -3 0 0 +1.5 
.female. 
56 -2 -2 -3 0 0 0 
______________________________________