Note: Descriptions are shown in the official language in which they were submitted.
2 ~
-- 1 --
~NTIOXIDATION ACTIVE SUBSTANCE AND UTILIZATION
THEREOF
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 utiliza-
tion of the same as an additive to foods, cosmetics andthe like.
Hitherto, there have been known various anti-
oxidants 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, ~-tocopherol, and ascorbic acid, and
phenol type synthetic antioxidants such as butylhydroxy-
anisole (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 acti-
vity, 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 ~-tocopherol. The present invention
has been achieved based on this discovery.
According to one aspect of the present inven-
tion, 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
.
.
~ E~j
invention, there is provided an antioxidant substance
derived from ~ 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.
~ ereafter, the antioxidant substances of the
present invention will be describea in more detail.
~erein, 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 addi-
tion, 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 papper, green leaves of
carrot, and green leaves of radish, non-cultivated bege-
tables which grow in fields or wastelands or mountainssuch as bamboo grass, and ashitaba ~a kind of Japanese
parsley: Angelica keiskei (~ig.) Koidz.); and further
fresh-water or see-water algae such as Spirulina~
Chlorella, wakame (a Japanese see weed: Undaria pinnati--
fida Suringar), and green laver (Enteromorpha).
As the cereals which can be used favorably inthe 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
2 ~
such as a mixer, a juicer or the like and then removed o~
crude solid contents by siEt~ng, filtration or the like,
if desired, to prepare a sucked juice (hereina~ter,
refer~ed 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 an enough amount of water or n-hexane. This extrac-
tion treatment can be performed usually at room tempera-
ture 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 solidi-
fied 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 solu-
tion 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 afore-
mentioned a~ueous ethanol-soluble component can be
treated with a suitable adsorbent such as Styrene-DVB
. . ,;
. .
2 ~ ~ ~ 2 ~ ~
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 pos-
sible to obtain a fraction having a higher antioxidant
activity.
The aqueous methanol-soluble component
recovered ~rom 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'-0-glucosyl-isovitexin having a structure represented
by the following formula:
HO ~ O OH
o ~
OH
O ~ OH
3() ~C~2 ~ ~
~/
f 0
0~1
(cf. Example 1 below)
2~
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 ~-tocopherol, and is useEul 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 anti-
oxidant substance of the present invention can be steri-
lized 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,
s ~3 7~
-- 6 --
zinc oxide, sodium carbonate, sodium hydrogen carbonate,
titanium dioxide, kaolin, and calcium phosphate to pro-
duce 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 main-
taining freshness and quality of or preserving variousfoods 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 pro-
cessed foods (e.g., hams, sausages, corned beef, etc.)
breads and noodles (Japanese noodles, buckwheat noodle,
Chines 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 inven-
tion 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 substantially no adverse influence
on their compositions and components or appearance. For
C~ A 9 ~
example, it can be freely blended with various additives
which are often used in foods, including sweeteners such
as fructose, glucose, and millet jelly; orgnaic 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 solubi-
lity in water and transparency, but permitting treatments
such as filtration, sterilization and the like.
The antioxidant substance of the present inven-
tion 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 fresh-
ness 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"-0-glucosyl-isovitexin.
The antioxidant substance provided according to
the present invention exhibits excellent effects on the
prevention of spots (chloasma), freckles, chapping,
burning with W rays (sunburnt), etc. and is safe. Hence
it can be utilized for the prevention of spots tchloasma,
freckles, chapping, burning with UV rays (sunburnt) and
protection of hairs and the like by blending it with
cosmetics for skins and hairs.
As described above, the antioxidant substance
2 ~ 5
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 ~or 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 substantially no adverse in-
fluence 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 sub-
stance 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 aforemen-
tioned 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 ~itamins, 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.
Brief description of the drawings referred to in
the examples below are as follows:
Fig. 1 is a chart illustrating results of the
measurement by FAB-MS method of 2'-O-glucosyl-isovitexin
obtained in Example 1:
Fig. 2 is a chart illustrating a UV ray absorp-
tion spectrum of 2'-O-glucosyl-isovitexin in H2O system
~!192~
Fig. 3 is a chart illustrating a UV ray absorp-
tion spectrum of 2'-O-glucosyl isovitexin in MeOH system;
Fig. 4 is a chart illustrating an infrared
absorption spectrum of 2'-O-glucosyl-isovitexin;
13 Fig. 5 is a chart illustrating results of
C-NMR analysis of 2'-O-glucosyl-isovitexin;
Fig. 6 is a graph illustrating results oE
measurements on lipid peroxides in each extract obtained
in Example a and ~-tocopherol by TBA method (results of
measurement on optical density at S35 nm~;
Fig. 7 is a gas chromatograph chart for lipid
peroxides derived from arachidonic acid with Fenton's
reagent, MA and 4HN;
Fig. 8 is a gas chromatograph chart for lipid
peroxides derived from arachidonic acid with Fenton's
reagent, MA and 4HN;
Fig. 9 is a gas chromatograph chart for lipid
peroxides derived from arachidonic acid with Fenton's
reagent, MA and 4HN;
Example 1 (Fractionation and Preparation 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 bout
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 sepa-
rated. Repeating this procedure, n-hexane-insoluble
component was obtained.
The n-hexane-insoluble component was added 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
.:
9 2 ~ ~
-- 10 --
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 distil~ed 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, % o~ methanol indicates the con-
centration 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 spec-
trometer, and results as shown in Fig. 1 were obtained.The mass spectrum thus obtained showed a peak of [M +
H ] 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 C27H30O15.
Upon UV absorption spectrum of the present
substance was measured in H2O and in methanol, absorp-
tions by flavonoid glucoside were observed as shown in
Figs. 2 and 3, respectively.
Infrared absorption spectrum was measurRd by
KBr method using JASCO FT/IR-7000S and the results
2 ~ ~
obtained are shown in Fig. 4. Absorption at 3422 cm 1
indicates the presence of OH group.
Upon hydrolysis with hydrochloric acid-methanol
by a conventional ~ethod, the present substance released
one molecule of glucose to produce isovitexin.
Further, 13C NMR spectrum ~500 MHz) of the
present substance was measured by GE OMEGA 300 type
nuclear magnetic resonance spectrum absorption spectro-
meter using 25 mg of purified antioxidant substance and
tetramethylsilane (TMS, (CH3)4Si) as an internal
standard, and results shown in Fig. 5 were obtained. In
Fig. 5, ehemieal shift was indieated by "~". The puri-
fied antioxidant substance gave signal eorresponding to
27 earbon atom in MeOH-d4, whieh suggested the follow-
ing structural formula:
~0
~ ZH~O~
HO ~ o
- HO
O HO ~
OH
~} ~loJ~OH
based on standard value of 13C-NMR for isovitexin
(Remarathnam, N., Osawa, T., Namiki, M. and Kawakishi,
S.: J. Agrie. Food Chem., 37 316-319 (1989)).
- 12 - -
From the above formula, the present substance
is named 2'-Oglucosyl-isovitexin.
Example 2 (Fractionation and Preparation 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 Preparation of Active
Ingredient)
Lyophilized powder t20 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
2~ that obtained in Example 1.
Example 4 tMeasurement of lipid peroxide by TBA method)
To 7.5 mg of linoleic acid was added 0.22 mg of
~-tocopherol or 0.22 mg of water-extract obtained in
Example 1. Then 200 ~ of Fenton's reagent (FeC12,
2 2)
was added to each of the resulting mixture, and the
mixtures thus obtained were incubated at 37C for 16
hours (total amount: 5 ml).
To 0.2 ml of each solution were added 0.2 ml of
aqueous 8 ~ SDSl) solution, 1.5 ml of acetate buffer
(pH 3.5) and 1.5 ml of an aqueous 0.67 % TBA2 ) olu
tion, and the mixture was heated for 1 hour in a boiling
water bath (95 to 100C).
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,
J ~ ~
- 13 -
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, MAD3) and 4NH (4-Hydroxynonenal))
To microsomes and 7.5 mg of arachidonic acid
was added 0.22 mg of -tocopherol or 0.22 mg of anti-
oxidant substance obtained in Example 1. To the mixture
was added 5 ml of Tris hydrochloric acid buffer solution
(05 M Trizma ~Cl, pH 7.4; 0.15 M KCl; 0.2 ~ SDS). The
resulting mixture was shaken mildly to make a suspension,
to which was added 200 ~1 of Fenton's reagent (FeC12,
H2O2). The mixture was reacted at 37C for 16 hours.
After the reaction was stopped by addition of 50 1 of 4
~ BHT4), 4O~L1 of N-methylhydrazine and the reaction
mixture was left to stand at room temperature ~or 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 dichloro-
methane 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 chromatogrpahic analysis, which
was subjected to gas chromatography under the following
conditions:
Capillary column: DB-WAX
25 m X 0.25 mm
Column temperature: 35C (retention time:
1.0 minutes) - 190C (retention
time: 20 minutes) temperature
elevation rate: 40C/minute
Inlet temperature: 250C
Detector temperature: 3~0~C
,
t ~ 2 l~ ~
- 14 -
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 corresonding to isovitexin to which one mole-
cule of glucose is connected strongly inhibits the produc-
tion of not only ~AD but also 4HN (4-hydroxynonenal~ and
has an antioxidant activity more patent than
-tocopherol.
Notes: 3) MAD = malondialdehyde
4) sHT = butylhydroxytoluene
Example 6
To the powder (100 g) of green ~uice 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-hexaneinsoluble 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 adsor~ed 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 %, respec-
tively, 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 %
2 ~ ~
- 15 -
methanol-extract, 1.5 g of 60 % methanol-extract, 170 mg
of 80 % methanol-ex-tract, 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 eontaining ~-carotene having
compositions as shown in Tables 1 and 2 were prepared,
and antioxidant aetivities of 60 % methanol-extract and
2'-O-glueosyl-isovitexin, respectively, at pH 3 or 5 on
~-carotene were measured using water and vitamin C as
control.
Determination of B-carotene was performed based
on the method preseribed in Hygienic Examination Methods,
Commentary, ed. Japan Pharmaeeutical Association,
P-347~349 ~1990) published by Rinbara Shuppan Co., Ltd.).
Table 1 Composition of Model Juice
ComponentComposition
_
Inverted sugar20.0 g
Citrie aeid, anhydrous 1.0 g
B-earotene* 0.65 g
H2O to make 100 ml
pH adjusted to 3.0
Note: * earotene base: produced by Sanei
Kagaku Kogyo Co., Ltd.
A J~
-- 16 --
Table 2 Composition of Model Juice
.
Component Composition
Inverted sugar 20.0 g
Citric acid tdihydrate) 0O43 g
Sodium citrate (dihydrate) 1.23 g
~-Carotene*0. 656 mg
H2O to make100 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 ~-carotene. The reaction
temperature was 18C.
.
h~A~2~j
Table 3 Antioxidant Activity at pH 3
Residual Ratio ~%)
. of ~-Caro tene
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*
~ote: * Expressed as amount of 2'-O-glucosyl-
isovitexin.
Table 4 Antioxidant Activity at pH S
Residual Ratio (%)
. of ~-Carc tene
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 2'-O-glucosyl
isovitexin.
2 ~ 5
- 18 -
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 anti-
oxidant showed considerable color fading at pH 3 andbecame col~rless at pH 5.
_xample 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 180C and an air
exhaustion temperature of 120C 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
190C and an air exhaustion temperature of 120C to
obtain 430 g of powdery raw material.
Example 9
f~
-- 19 --
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 170C
and an air exhaution temperature of 110C to obtain 127 g
of powdery raw material as a cyclodextrin inclusion
compound.
EXample 10
Kaolin (200 g) was mixed with 100 g of sub-
stance A obtained in Example 7 to prepare a 30 % suspen-
sion, which was then spray-dried at an air absorption
temperature of 170C and an air exhaustion temperature of
110C to obtain 270 g of powdery raw material.
Example 11
After being desalted, 100 ml of a 4 ~ sodium
silicate solution was adjusted to p~ 9 with 1 % potassium
hydroxide, and 15 ml aliquot thereof was heated at 95C
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 90C 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
. 9 2 .~ ~
- 20 -
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 lO0
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 reten-
tion 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 7Day l4Day 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
~otes: * 500 mg/liter
** E mix P-20, 500 mg/liter as vitamin E
~ 3~3
- 21 -
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 0Day 7Day 14 Day 20
Water A B C C
20 Vitamin C* A B B C
Vitamin E** A B B C
Extract A A A B
Notes: * 500 mg/liter
** E mix P-120, 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 85C for
30 minutes.
Granulated sugar 20 g
Fructose 35 g
~a~92~
- 22 -
Citric acid 3 g
Succinic acid 0.1 g
Component soluble in an aqueous
ethanol solution having a water
content of 20 ~ 10 g
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
~uice was sterilized with heating at 85C for 30 minutes.
Granulated sugar 20 g
Fructose 35 g
Citric acid 3 g
Tartaric acid 0.1 g
~-Carotene (carotene base
No. 31256 produced by Sanei
Kagaku Rogyo Co., Ltd.) 10 mg
Fraction eluted with an aqueous
methanol solution having a water
content of 40 % 0.5 g
Perfume suitable amount
To make 1 liter
After leaving this formulation to stand under
natural light for 15 days, ~-carotene remained in an
amount of 83 %. On the other hand, ~-carotene disap-
, . :
? 1~ r~
pear~d completely in a formulation in which the fractioneluted with an aqueous methanol solution having a water-
content of 40 ~ was absent. Determination of ~-carotene
was performed based on the method prescribed in Hygienic
Examination Methods, Commentary, ed. Japan Pharmaceutical
Association, p.347-349 tl990) 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, ~-carotene
remained in an amount of 73.2 % while in a juice in which
2'-O-glucosyl-isovitexin was absent ~-carotene disap-
peared 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, andadding 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 con-
siderably 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/l,000 g of margarin, and the margarin obtained was
preserved at room temperature for 3 months. The prepara-
- 24 -
tion containing no such fraction suffered denaturation
and its fragrance changed resulting in that it wasun-
suitable for use for food while the margarin of the
present invention containing the fraction suffered sub-
stantially no such change in fragrance.
Example 19
During preparing solid yoghurt by a conven-
tional method, 0.2 % of the Eraction extracted with an
aqueous ethanol solution hav:ing a water content of 20 %
was added. The yoghurt was iEilled 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 ~-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 40Cr 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
;
9 ~
- 25 -
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 hexaneinsoluble
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 after 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
30 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
2 ~ r~;3
- 26 -
everyday after washing ~ace .in the morning and before
sleeping t~ examine effects on improvement of spots and
freckles. Resul.ts 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 quiet: Effective
Boundary between spot or freckle portion and other
portion:
Boundary apparent: No effect
Boundary unclear: Slightly effective
Boundary almost: Effective
undiscernible
Table 7
effective Effective No
Color 15 7 8
Boundary 17 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'-0-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.
h ~ 2 ~ ~
Example 25
Formulation of cream:
Stearic acid 10 g
Isopropyl myristate 5 g
5 Cetyl alco~ol 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 result-
ing mixture was emulsified and when the temperature of
the emulsion reached 60C, 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 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.
2~92~i
- 28 -
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 _
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 la 10 7 5
Slightly 7 10 10 14 15 15
effective
Effective _ _ 26 a 1 o
Example 25
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.
. 9 2 ~ ~
- 29 -
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 everyday application of the
hair growing agent were judged according to the following
criteria:
Dandruff Thin ~air State
Class (State) RatingClass (State) Rating
Very much -3 Very much -3 i
Much -2 Much -2
15 Medium -1 Medium -1
Normal 0 Normal 0
Slight +3 Slight +3
.
Color, Gloss
Class (State) Rating
25 Bad ~3
Normal 0
: Good ~3
Based on the aforementioned criteria the states
before and after the application are shown in Table 10.
2 ~ .~S ~
- 30 -
Table 10
.. _ _ ....
sefore Application After Application
ex Age Dund- Thin Color, Dund- Thin Color,
ruff Hair Gloss ruff Hair Gloss
30 -1.5 -1 -3 O -1 O
27 -2 -1 O -1 O +3
23 -2 -1 O -1 O +3
35 -1.5 -1 O O O ~1.5
40 -1 -2 O O -1 +1.5
53 -1 -2 -3 O -1.5 O
43 O -3 -3 O -205 O
43 -1 -2 O O -1.5 O
48 -2 -2 -3 -1 -1 +1.5
55 -1 -3 -3 O -2.5+1.5
52 -1 -3 -3 +3 _~ +1.5
38 -1 -2 -3 O -1 O
35 -1.5 -1 O -1 -1 O
37 -1.5 -2 O O -1
40 -1 -3 -3 ~O -2 +1.5
45 -1 -2 -3 O -lo 5+1.5
43 -1 -2 -3 O -1 +1.5
56 -1 -2 -3 O -1 +1.5
5~ -2 -1 O -1 -1 +1.5
55 -2 -2 -3 -1 -1.5 O
30 -2 O +3 O O +3
3.S -2 O O -1 O +2
~ r
- 31 -
Table 10 (Continued)
_ Before Applicat:ion After Applicatlon
. _ . __
Sex Age Dund-Thin Co;Lor, Dund- Thin Color,
ruffHair Glossruff Hair Gloss
_ . ..
~ 37 -3 O O -1 O +3
$ 90 -1 --3 ;
40 -2 O --1.5 O O O ~.
~ 43 -2 O -1.5 -1 O O .
T 45 -1 -1.5 O O +3
T 47 -2 -1 -1 -2 +1.5 +3
47 -2 O -2 O O +l
47 -2 -1 -2 O O O
50 -3 O -3 O O O
-2 -1 -2 O O O
~ 50 -1 -1 -3 O -1 +1.5
T 51 ~3 -1 -1.5 -l.S O O
T 52 -2 -1 -2
$ 52 -3 -1 -3 O O +1.5
. 53 -2 -2 -3 -1 -1 O
54 -1 -1 -3 O O +1.5
55 -1 -1 -3 O O +1.5
56 -2 -2 -3 O O O
, ' -
