U.S. patent application number 12/222492 was filed with the patent office on 2010-02-25 for drugs, foods or drinks with the use of algae-derived physiologically active substances.
This patent application is currently assigned to TAKARA BIO INC.. Invention is credited to Tatsuji ENOKI, Katsushige IKAI, Ikunoshin KATO, Nobuto KOYAMA, Eiji NISHIYAMA, Hiroaki SAGAWA, Takeshi SAKAI, Takanari TOMINAGA, Fu-Gong YU.
Application Number | 20100048491 12/222492 |
Document ID | / |
Family ID | 27520204 |
Filed Date | 2010-02-25 |
United States Patent
Application |
20100048491 |
Kind Code |
A2 |
ENOKI; Tatsuji ; et
al. |
February 25, 2010 |
DRUGS, FOODS OR DRINKS WITH THE USE OF ALGAE-DERIVED
PHYSIOLOGICALLY ACTIVE SUBSTANCES
Abstract
Medicinal compositions for treating, ameliorating or preventing
diseases with sensitivity to 3,6-anhydrogalactopyranose represented
by formula (1): ##STR1## foods, drinks, cosmetics, etc. containing
as the active ingredient at least one member selected from the
group consisting of the above-mentioned compound, its aldehyde, its
hydrate and 2-O-methylated derivatives thereof and soluble sugar
compounds containing the above compound. This compound also shows,
for example, an apoptosis-inducing activity, a carcinostatic
activity and inhibitory activities on the production of active
oxygen, lipid peroxide radicals and NO, which makes it useful also
as the active ingredient of antioxidants and preservatives.
Inventors: |
ENOKI; Tatsuji; (Otsu-shi,
Shiga, JP) ; SAGAWA; Hiroaki; (Kusatsu-shi, Shiga,
JP) ; TOMINAGA; Takanari; (Otsu-shi, Shiga, JP)
; NISHIYAMA; Eiji; (Moriyama-shi, Shiga, JP) ;
KOYAMA; Nobuto; (Uji-shi, Kyoto, JP) ; SAKAI;
Takeshi; (Hirosaki-shi, Aomori, JP) ; YU;
Fu-Gong; (Hirosaki-shi, Aomori, JP) ; IKAI;
Katsushige; (Koka-gun, Shiga, JP) ; KATO;
Ikunoshin; (Uji-shi, Kyoto, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
UNITED STATES
2026285197
202-737-3528
mail@browdyneimark.com
|
Assignee: |
TAKARA BIO INC.
4-1, Seta 3- chome
Otsu-shi, Shiga
JP
520-2193
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20090099101 A1 |
April 16, 2009 |
|
|
Family ID: |
27520204 |
Appl. No.: |
12/222492 |
Filed: |
August 11, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11/034,243 |
Jan 13, 2005 |
|
|
|
12222492 |
Aug 11, 2008 |
|
|
|
10/228,195 |
Aug 26, 2008 |
7417031 |
|
|
11/034,243 |
Jan 13, 2005 |
|
|
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09/554,235 |
Jun 28, 2005 |
6911432 |
|
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PCT/JP98/05065 |
Nov 11, 1998 |
|
|
|
10/228,195 |
Aug 27, 2002 |
|
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Current U.S.
Class: |
514/23 ;
514/456 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 17/00 20180101; A61P 27/16 20180101; A23L 33/105 20160801;
A61P 25/00 20180101; A61P 37/08 20180101; A01N 43/04 20130101; A61P
1/04 20180101; A61P 9/10 20180101; A61P 1/00 20180101; A61K 31/341
20130101; A23L 3/3562 20130101; A61P 37/00 20180101; A61P 39/06
20180101; A61K 31/7048 20130101; A61P 3/10 20180101; A61P 1/10
20180101; A61P 25/22 20180101; A61P 9/02 20180101; A61P 13/12
20180101; A61P 19/02 20180101; A61P 37/02 20180101; A23L 3/3544
20130101; A23L 3/3472 20130101; A61P 9/00 20180101; A61P 43/00
20180101; A61P 27/02 20180101; A61P 11/06 20180101; A61P 35/00
20180101 |
Class at
Publication: |
514/023 ;
514/456 |
International
Class: |
A61K 31/70 20060101
A61K031/70; A61K 31/335 20060101 A61K031/335 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 11, 1997 |
JP |
323917/1997 |
Jan 19, 1998 |
JP |
20146/1998 |
Apr 27, 1998 |
JP |
130973/1998 |
May 29, 1998 |
JP |
164410/1998 |
Jul 13, 1998 |
JP |
212041/1998 |
Claims
1. A method for treating a disease that requires induction of
apoptosis for its treatment, a carcinomatous disease, a disease
that requires protection against oxidation for its treatment, a
disease that requires inhibition of active oxygen production for
its treatment, a disease that requires inhibition of nitric
monoxide production for its treatment, a disease that requires
inhibition of lipid peroxide radical production for its treatment
or a disease that requires immunoregulation for its treatment, the
method comprising administering a pharmaceutical composition which
comprises as an active ingredient at least one member selected from
the group consisting of: (1) a compound selected from the group
consisting of 3,6-anhydrogalactopyranose represented by the formula
(I): ##STR8## an aldehyde thereof, a hydrate thereof, and a
2-O-methylated derivative of said 3,6-anhydrogalactopyranose, said
aldehyde or said hydrate; and (2) a soluble saccharide containing
the compound at its reducing end.
2. A method according to claim 1, wherein the saccharide is a
product produced by acid decomposition under acidic conditions
below pH 7 and/or enzymatic digestion of a substance containing at
least one compound selected from the group consisting of
3,6-anhydrogalactopyranose represented by said formula I, an
aldehyde or a hydrate thereof, and a 2-O-methylated derivative of
the 3,6-anhydrogalactopyranose, said aldehyde or said hydrate.
3. A method according to claim 2, wherein the substance containing
at least one compound selected from the group consisting of
3,6-anhydrogalactopyranose represented by formula I, an aldehyde
and a hydrate thereof, and 2-O-methylated derivatives of the
3,6-anhydrogalactopyranose, the aldehyde and the hydrate is at
least one substance selected from the group consisting of agar,
agarose and carrageenan.
4. A method according to claim 1, wherein the saccharide is at
least one saccharide selected from the group consisting of
agarobiose, agarotetraose, agarohexaose, agarooctaose,
.kappa.-carabiose, and
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose.
Description
[0001] This is a division of co-pending parent application Ser. No.
10/228,195 filed Aug. 27, 2002, itself a division of grandparent
application Ser. No. 09/554,235, now U.S. Pat. No. 6,475,990,
issued on Nov. 5, 2002.
FIELD OF THE INVENTION
[0002] The present invention relates to use of a physiologically
active substance derived from algae. More specifically, it relates
to a pharmaceutical composition, an antioxidant, a preservative
composition for keeping freshness of foods and drinks, and a
cosmetic composition which comprise the physiologically active
substance as an active ingredient, as well as a functional food or
drink which comprises the physiologically active substance.
Furthermore, it relates to a saccharide for exhibiting the
function.
BACKGROUND OF THE INVENTION
[0003] Recently, a mode of death of cells or tissues called as
apoptosis (self-blasting or self-destruction of cells) has been
noticed.
[0004] The apoptosis is a death which has been originally
programmed in the genome of a cell and is different from necrosis
which is a pathological cell death. Certain external or internal
factors trigger the activation of a gene that programs the
apoptosis to cause the biosynthesis of a programmed death protein.
In some cases, a programmed death protein which has been present in
a cell in its inactive form becomes activated. The active
programmed death protein thus formed decomposes the cell to lead
death.
[0005] Activation of the apoptosis in desired tissues or cells
would make it possible to eliminate cells which are unnecessary or
harmful from a living body in a natural manner, which is of very
importance.
OBJECTS OF THE INVENTION
[0006] Oligosaccharides derived from algae such as agar are
expected to be developed as raw materials for foods (Food Chemical,
1988-2, 40-44; Bessatsu Food Chemical (Extra Number Food
Chemical)-4, 1990, December, 127-131; JP-A-6-38691). However, their
physiological functions such as an apoptosis-inducing activity are
unknown.
[0007] The main object of the present invention is to develop a
highly safe substance having a physiological function such as an
activity of inducing apoptosis derived from a naturally occurring
material, as well as to provide a pharmaceutical composition for
preventing or treating a disease sensitive to the substance, such
as a composition for inducing apoptosis comprising the substance as
an active ingredient, and a functional food or drink comprising the
substance as a constituent component.
SUMMARY OF THE INVENTION
[0008] In brief, the first aspect of the present invention is a
pharmaceutical composition which comprises as an active ingredient
at least one member selected from the group consisting of:
[0009] a compound selected from the group consisting of
3,6-anhydrogalactopyranose represented by formula 1: ##STR2## an
aldehyde and a hydrate thereof, and 2-O-methylated derivatives of
the 3,6-anhydrogalactopyranose, the aldehyde and the hydrate;
and
[0010] a soluble saccharide containing the compound at its reducing
end,
said composition being used for treating or preventing a disease
sensitive to the compound.
[0011] The second aspect of the present invention is a food or
drink comprising at least one member selected from the group
consisting of:
[0012] a compound selected from the group consisting of
3,6-anhydrogalactopyranose represented by formula 1, an aldehyde
and a hydrate thereof, and 2-O-methylated derivatives of the
3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and
[0013] a soluble saccharide containing the compound at its reducing
end,
[0014] said food or drink being used for ameliorating a disease
state of or preventing a disease sensitive to the compound.
[0015] The third aspect of the present invention is an antioxidant
which comprises as an active ingredient at least one member
selected from the group consisting of
[0016] a compound selected from the group consisting of
3,6-anhydrogalactopyranose represented by formula 1, an aldehyde
and a hydrate thereof, and 2-O-methylated derivatives of the
3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and
[0017] a soluble saccharide containing the compound.
[0018] The fourth aspect of the present invention is a food and
drink comprising the antioxidant of the third aspect of the present
invention.
[0019] The fifth aspect of the present invention is a saccharide
for an antioxidant selected from the group consisting of:
[0020] a compound selected from the group consisting of
3,6-anhydrogalactopyranose represented by formula 1, an aldehyde
and a hydrate thereof, and 2-O-methylated derivatives of the
3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and
[0021] a soluble saccharide containing the compound.
[0022] The sixth aspect of the present invention is a preservative
composition for keeping freshness of foods and drinks which
comprises as an active ingredient at least one member selected from
the group consisting of:
[0023] a compound selected from the group consisting of
3,6-anhydrogalactopyranose represented by formula 1, an aldehyde
and a hydrate thereof, and 2-O-methylated derivatives of the
3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and
[0024] a soluble saccharide containing the compound.
[0025] The seventh aspect of the present invention is a cosmetic
composition comprising as an active ingredient at least one
saccharide selected from the group consisting of agarobiose,
agarotetraose, agarohexaose, agarooctaose, .kappa.-carabiose and
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose.
[0026] The eighth aspect of the present invention is an acidic food
or drink comprising at least one member selected from the group
consisting of:
[0027] a compound selected from the group consisting of
3,6-anhydrogalactopyranose represented by formula 1, an aldehyde
and a hydrate thereof, and 2-O-methylated derivatives of the
3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and
[0028] a soluble saccharide containing the compound.
[0029] The further aspect of the present invention is use of at
least one member selected from the group consisting of:
[0030] a compound selected from the group consisting of
3,6-anhydrogalactopyranose represented by formula 1, an aldehyde
and a hydrate thereof, and 2-O-methylated derivatives of the
3,6-anhydrogalactopyranose, the aldehyde and the hydrate; and
[0031] a soluble saccharide containing the compound, in preparation
of a pharmaceutical composition, a food or drink, an antioxidant, a
preservative composition for keeping freshness of foods and drinks
or a cosmetic composition.
[0032] Hereinafter, the present invention will be explained in
detail with reference to the attached drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0033] FIG. 1 illustrates a gel filtration elution pattern of agar
decomposed with 0.12 N HCl.
[0034] FIG. 2 illustrates a gel filtration elution pattern of agar
decomposed with 1 N HCl.
[0035] FIG. 3 illustrates a size-exclusion HPLC chromatogram of
agar decomposed with an acid.
[0036] FIG. 4 illustrates a mass spectrum of an apoptosis-inducing
and carcinostatic substance.
[0037] FIG. 5 illustrates a .sup.1H-NMR spectrum of an
apoptosis-inducing and carcinostatic substance (hydrate form).
[0038] FIG. 6 illustrates a .sup.1H-NMR spectrum of an
apoptosis-inducing and carcinostatic substance (aldehyde form).
[0039] FIG. 7 illustrates an elution pattern of normal phase HPLC
of agarobiose, agarotetraose and agarohexaose.
[0040] FIG. 8 illustrates a mass spectrum of the peak at 66.7
min.
[0041] FIG. 9 illustrates a mass spectrum of the peak at 78.5
min.
[0042] FIG. 10 illustrates a mass spectrum of the peak at 85.5
min.
[0043] FIG. 11 illustrates a mass spectrum of
3,6-anhydro-L-galactose.
[0044] FIG. 12 illustrates a .sup.1H-NMR spectrum of
3,6-anhydro-L-galactose (hydrate).
[0045] FIG. 13 illustrates a .sup.1H-NMR spectrum of
3,6-anhydro-L-galactose (aldehyde).
[0046] FIG. 14 illustrates the relation between the incubation time
and the number of viable cells obtained by incubating HL-60 cells
with addition of one of oligosaccharides at a final concentration
of 250 .mu.M.
[0047] FIG. 15 illustrates the relation between the incubation time
and the number of viable cells obtained by incubating HL-60 cells
with addition of one of oligosaccharides at a final concentration
of 125 .mu.M.
[0048] FIG. 16 illustrates an elution pattern in normal phase HPLC
chromatogram of agar treated by heating in 0.5 M phosphate.
[0049] FIG. 17 illustrates a calibration curve of agarobiose.
[0050] FIG. 18 illustrates the relation between the heating time
and the amount of agarobiose produced in 0.2% agar solution in 0.1
M HCl.
[0051] FIG. 19 illustrates the relation between the heating time
and the amount of agarobiose produced in 0.2% agar solution in 0.1
M citric acid.
[0052] FIG. 20 illustrates the production of agaro-oligosaccharides
in 500 mM citric acid at 80.degree. C.
[0053] FIG. 21 illustrates the production of agaro-oligosaccharides
in 500 mM citric acid at 95.degree. C.
[0054] FIG. 22 illustrates the production of agaro-oligosaccharides
in 1200 mM lactic acid at 80.degree. C.
[0055] FIG. 23 illustrates the production of agaro-oligosaccharides
in 1200 mM lactic acid at 95.degree. C.
[0056] FIG. 24 illustrates the production of agaro-oligosaccharides
in 1000 mM malic acid at 80.degree. C.
[0057] FIG. 25 illustrates the production of agaro-oligosaccharides
in 1000 mM malic acid at 95.degree. C.
[0058] FIG. 26 illustrates the production of agaro-oligosaccharides
in 1000 mM malic acid at 70.degree. C.
[0059] FIG. 27 illustrates a normal phase HPLC chromatogram of
K-carrageenan decomposed with an acid.
[0060] FIG. 28 illustrates a mass spectrum of an apoptosis-inducing
and carcinostatic substance.
[0061] FIG. 29 illustrates a .sup.1H-NMR spectrum of an
apoptosis-inducing and carcinostatic substance.
[0062] FIG. 30 illustrates the results of gel filtration with
Cellulofine GCL-25.
[0063] FIG. 31 illustrates the results of gel filtration with a
Sephadex LH-20 column.
[0064] FIG. 32 illustrates a mass spectrum of
.beta.-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose.
[0065] FIG. 33 illustrates a .sup.1H-NMR spectrum of
.beta.-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose.
[0066] FIG. 34 illustrates the relation between the concentration
of agarobiose and the level of .sup.3H-thymidine uptake in
lymphocyte blastgenesis induced by ConA.
[0067] FIG. 35 illustrates the relation between the concentration
of agarobiose and the level of .sup.3H-thymidine uptake in a mixed
lymphocyte reaction.
[0068] FIG. 36 illustrates NO.sub.2.sup.- concentrations in culture
media in the presence of various concentrations of agarobiose.
[0069] FIG. 37 illustrates NO.sub.2.sup.- concentrations in culture
media in the presence of various concentrations of
neoagarobiose.
[0070] FIG. 38 illustrates NO.sub.2.sup.- concentrations in culture
media in the presence of a solution of agar digested by
hydrochloric acid or citric acid.
[0071] FIG. 39 illustrates NO.sub.2.sup.- concentrations in culture
media in the presence of 3,6-anhydro-D-galactose or galactose.
[0072] FIG. 40 illustrates NO.sub.2.sup.- concentrations in culture
media under various conditions.
[0073] FIG. 41 illustrates carcinostatic activity of the
oligosaccharide of the present invention.
[0074] FIG. 42 illustrates inhibition of PCA reaction by the
oligosaccharide of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0075] An aldehyde of 3,6-anhydrogalactopyranose of formula 1
(hereinafter simply referred to as "3,6-anhydro-galactopyranose")
of the present invention is a compound of formula 2: ##STR3##
[0076] A hydrate thereof is a compound of formula 3: ##STR4##
[0077] A 2-O-methylated derivative of the
3,6-anhydrogalactopyranose is a compound of formula 4: ##STR5##
[0078] An aldehyde of the methylated derivative is a compound of
formula 5: ##STR6##
[0079] A hydrate of the methylated derivative is a compound of
formula 6: ##STR7##
[0080] The structures of formulas 1 to 6 used herein may be
represented by different expression forms. It is intended that such
different expression forms and their possible tautomers are
included in formulas 1 to 6. In addition, the configuration of
formulas 1 to 6 is not limited to specific one as far as the
desired activities are exerted, and may be in the D-form or L-form,
or a mixture thereof.
[0081] The soluble saccharide of the present invention is, without
limitation, a soluble saccharide containing at least one compound
selected from 3,6-anhydrogalactopyranose, an aldehyde and a hydrate
thereof, and 2-O-methylated derivatives of the
3,6-anhydrogalactopyranose, the aldehyde and the hydrate
(hereinafter collectively referred to as "the compounds of formulas
1 to 6"), and can be obtained by decomposition of a substance
containing at least one compound selected from the compounds of
formulas 1 to 6 (hereinafter simply referred to as "a raw
substance") under acidic conditions below pH 7 with an acid and/or
enzyme, or by chemical synthesis. The soluble saccharide of the
present invention is not limited to specific one in so far as it
dose not solidify or semi-solidify (gelate) when used. Therefore,
any saccharides containing at least one compound selected from the
compounds of formulas 1 to 6 which become solated when used are
included in the soluble saccharides of the present invention.
Examples of the soluble saccharides suitably used in the present
invention include a saccharide whose non-reducing end is a sugar
other than L-galactose-6-sulfate, for example, agarobiose,
agarotetraose, agarohexaose, agarooctaose, .kappa.-carabiose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like.
[0082] The raw substances used for obtaining the soluble
saccharides are not limited to specific one and include, for
example, viscous polysaccharides from red algae such as agarose,
agaropectin, funoran, porphyran, carrageenan, furcellaran, and
hypnean [Kyoritsu-shuppan Inc., "Tatouseikagaku
1--Kagakuhen--(Biochemistry of Polysaccharides 1--Chemistry--), pp.
314 (1969)].
[0083] The raw substances also include materials that contain these
polysaccharides. For example, as raw materials for agarose and
agaropectin, red algae belonging to Gelidiaceae such as Gelidium
amansii, Gelidium japonicum, Gelidium pacificum, Gelidium
subcostatum, Pterocladia tenuis, Acanthopeltis japonica and the
like, red algae belonging to Gracilariaceae such as Gracilaria
verrucosa, Gracilaria gigas and the like, red algae belonging to
Ceramiaceae such as Ceramium kondoi, Campylaephora hypnaeoides and
the like, and other red algae are used. Usually, several kinds of
algae are used in combination as the raw materials. Although algae
dried in the sun are usually used as the raw materials, both fresh
and dried algae can be used in the present invention. Algae which
are bleached while spraying water during drying, i.e., bleached raw
algae, can also be used.
[0084] The raw material algae are extracted with hot water and then
cooled to obtain "gelidium jelly". Water is removed from this
"gelidium jelly" by freeze-dehydration or compress-dehydration,
followed by drying to obtain agar. Agar in various forms such as
bar, belt, board, thread, powder and the like can be used
regardless of the source algae. Usually, agar contains about 70% of
agarose and about 30% of agaropectin. The agar can be further
purified to prepare agarose with high purity. Purified agarose with
high purity or law purity having various agarose contents can be
used.
[0085] The raw substances include the above-mentioned raw material
algae, gelidium jelly, agar, purified agarose, purified agaropectin
and intermediate products or side products obtained during
preparation of these substances.
[0086] Agarose is a polysaccharide whose main structure is
alternately linked D-galactose and 3,6-anhydro-L-galactose. In the
structure, 1-position of D-galactose and 4-position of
3,6-anhydro-L-galactose are linked to each other through
.beta.-glycoside bond and 1-position of 3,6-anhydro-L-galactose and
3-position of D-galactose are linked to each other through
.alpha.-glycoside bond. The .alpha.-1,3-bond is hydrolyzed by mild
hydrolysis with a dilute acid or .alpha.-agarase [Carbohydr. Res.,
Vol. 66, p. 207 (1978)], and the .beta.-1,4-bond is hydrolyzed by
.beta.-agarase selectively.
[0087] Carrageenan is a polysaccharide which is contained in red
algae such as Gigartinaceae, Solieriaceae, Hypneaceae and the like.
.kappa.-Carrageenan, .lamda.-carrageenan and .eta.-carrageenan are
known.
[0088] .kappa.-Carrageenan has a fundamental structure in which
1-position of D-galactose-4-sulfate is linked to 4-position of
3,6-anhydro-D-galactose through .beta.-glycoside bond, 1-position
of 3,6-anhydro-D-garactose is linked to 3-position of
D-galactose-4-sulfate through .alpha.-glycoside bond, and they are
repeated alternately. .lamda.-Carrageenan has a fundamental
structure in which 1-position of D-galactose is linked to
4-position of D-galactose-2,6-disulfate through .beta.-glycoside
bond, 1-position of D-galactose-2,6-disulfate is linked to
3-position of D-galactose through .alpha.-glycoside bond, and they
are repeated alternately. Carrageenan is utilized as a gelatinizing
agent of foods.
[0089] The raw substances of the present invention also include
partially decomposed products of the above-mentioned raw substances
using a chemical, physical and/or enzymatic method.
[0090] Examples of chemical decomposition include hydrolysis under
acidic to neutral conditions. Examples of physical decomposition
include radiation of electromagnetic waves or ultrasonic waves.
Examples of enzymatic digestion include hydrolysis with a hydrolase
such as agarase, carrageenase and the like.
[0091] Decomposition of the raw substances under acidic to neutral
conditions are not limited to specific one in so far as the
decomposition produces the compounds of formulas 1 to 6 and the
soluble saccharides containing at least one of these compounds
which have an apoptosis-inducing activity; a carcinostatic
activity; antioxidant activities such as an activity of inhibiting
active oxygen production, an activity of inhibiting nitrogen
monoxide (hereinafter referred to as NO) production; an
immunoregulatory activity; or the like. Examples of the saccharides
include agarobiose, agarotetraose, agarohexaose, agarooctaose,
.kappa.-carabiose (hereinafter simply referred to "carabiose")
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose, and
the like; and the saccharides containing the compounds selected
from the compounds of formulas 1 to 6 at their reducing ends whose
non-reducing ends are saccharides other than
L-galactose-6-sulfate.
[0092] For example, the raw substance is dissolved or suspended in
an acid and reacted to produce the compound selected from the
compounds of formulas 1 to 6 and the soluble saccharides containing
at least one of these compounds to be used in the present
invention. The reaction time required for the production of the
compound selected from the compounds of formulas 1 to 6 and the
soluble saccharides containing at least one of these compounds can
be reduced by heating upon reaction.
[0093] The kind of the acid to be used for dissolution or
suspension of the raw substances (for example, a substance that
contains agarose or an agarose) is not limited to a specific one
and may be inorganic acids such as hydrochloric acid, sulphuric
acid, nitric acid and the like, organic acids such as citric acid,
formic acid, acetic acid, lactic acid, ascorbic acid and the like,
solid acids such as cation exchange resins, cation exchange fibers,
cation exchange membranes and the like.
[0094] The concentration of the acid is not limited, but the acid
can be used at a concentration of 0.0001 to 5 N, preferably 0.01 to
1 N. In addition, the reaction temperature is not limited, but the
reaction may be carried out at 0 to 200.degree. C., preferably 20
to 130.degree. C. Furthermore, the reaction time is not limited,
but the reaction may be carried out for a few seconds to a few
days. The kind and the concentration of the acid, the reaction
temperature and the reaction time may be suitable selected
depending on the particular kind of the raw substance containing at
least one compound selected from the compounds of formulas 1 to 6,
such as agarose or carrageenan, as well as the compound of interest
selected from the compounds of formula 1 to 6, the yield of the
saccharide containing the compound, and the degree of
polymerization of the soluble saccharide of interest containing the
compound selected from the compounds of formulas 1 to 6 at its
reducing end. In general, the acid decomposition reaction proceeds
more rapidly by selecting a strong acid rather than a weak acid, a
high acid concentration rather than a low acid concentration, and a
high temperature rather than a low temperature.
[0095] Furthermore, in general, when a solid acid is used, a strong
cationic exchange resin gives better decomposition reaction
efficiency than a weak cationic exchange resin does. In addition,
when the amount of the solid acid relative to the amount of the raw
substance is more and the reaction temperature is higher, the acid
decomposition reaction proceeds more rapidly.
[0096] For example, a solution of the saccharide used in the
present invention which is obtained by suspending agar in 0.1 N
hydrochloric acid in an amount of 10% by weight, dissolving the
agar by heating at 100.degree. C. for 13 minutes and removing
insoluble materials does not gelate any longer even when the
solution is cooled to its freezing point. When the saccharide
contained in this solution is analyzed by gel filtration HPLC,
normal phase HPLC and the like, saccharides with high molecular
weight are scarcely observed and almost all of the saccharides are
decomposed to soluble saccharides composed of 10 or less sugars.
Likewise, in case of a solid acid, a solution of the saccharide of
the present invention obtained by converting 1 part by weight of a
Na-type commercially available strong cationic exchange resin to
its H type with 1 N hydrochloric acid, placed in 79 parts by weight
of deionized water, adding and suspending 10 parts by weight of
agar and heating the mixture at 95.degree. C. for 180 minutes dose
not gelate any longer, even when the solution is cooled to its
freezing point. When the saccharide contained in this solution is
analyzed by gel filtration HPLC, normal phase HPLC and the like,
saccharides with high molecular weight are scarcely observed and
almost all of the saccharides are decomposed to soluble saccharides
composed of 10 or less sugars.
[0097] Furthermore, for producing the soluble saccharide used in
the present invention which has the compound selected from the
compounds of formulas 1 to 6 at its reducing end, a large amount of
the physiologically active oligosaccharide, such as a saccharide
for an antioxidant, can be produced by using an organic acid such
as citric acid, lactic acid or malic acid, suitably selecting the
acid concentration ranging from several 10 mM to several M, the
heating temperature ranging from 70 to 95.degree. C., and the
heating time ranging from several 10 minutes to 24 hours. In
addition, the physiologically active oligosaccharide produced has
long-term storage stability if it is maintained under acidic
conditions while preventing them from becoming alkaline after
hydrolysis.
[0098] The decomposed raw substances may be used directly or after
being neutralized as the compounds to be used in the present
invention, i.e., the compound selected from the compounds of
formulas 1 to 6 and the soluble saccharides containing at least one
of these compounds, for example, saccharides such as agarobiose,
agarotetraose, agarohexaose, agarooctaose, .kappa.-carabiose and
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like. However, they may be further purified. The compound
selected from the compounds of formulas 1 to 6 and the soluble
saccharides containing these compounds at their reducing ends, for
example, an oligosaccharide such as agarobiose, agarotetraose,
agarohexaose, agarooctaose, .kappa.-carabiose and
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose can be
purified by using, for example, its apoptosis-inducing activity or
carcinostatic activity as an index. As the means for purification,
a known method such as a chemical method, a physical method or the
like can be used. The compound selected from the compounds of
formulas 1 to 6 or the soluble saccharide containing at least one
of the compounds, which is an apoptosis-inducing substance,
produced in the acid decomposition products can be purified by
combining known purification methods such as gel filtration,
fractionation using a molecular weight fractionating membrane,
solvent extraction and chromatography using ion exchange resins or
the like.
[0099] The structures of the resultant compounds can be analyzed by
the known methods such as mass spectrometry, nuclear magnetic
resonance, measurement of ultraviolet absorption spectrum or
infrared absorption spectrum and the like.
[0100] Agarobiose, one example of the active ingredient of the
present invention, is a disaccharide in which 1-position of
D-galactose and 4-position of 3,6-anhydro-L-galactose are linked to
each other through .beta.-glycoside bond. An .alpha.-isomer and a
.beta.-isomer exist because an anomer carbon is present at
1-position of 3,6-anhydro-L-galactose, and agarobioses to be used
in the present invention include both of the isomers.
[0101] The saccharide containing the compound selected from the
compounds of formulas 1 to 6 at its reducing end used as the active
ingredient in the present invention is one in which one or more
sugars are bound to one or more hydroxide groups other than that at
1-position of the compound selected from the compounds of formulas
1 to 6, and is not limited to a specific one in so far as it has an
apoptosis-inducing activity, a carcinostatic activity, antioxidant
activities such as an activity of inhibiting active oxygen
production, an activity of inhibiting NO production, etc., and/or
an immunoregulatory activity. Examples thereof include
decomposition products of the raw substances such as products from
agarose obtained by decomposition with acid or digestion with
.alpha.-agarase such as agarobiose, agarotetraose, agarohexaose,
agarooctaose, agarodecaose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like. Furthermore, products from carrageenan obtained by
decomposition with acid or digestion with carrageenase such as
carabiose can also be exemplified. Furthermore, the saccharides of
the present invention which contain the compounds selected from the
compounds of formulas 1 to 6 at their reducing ends include those
in which one or more sugars selected from hexoses such as glucose,
mannose, galactose, etc., pentoses such as xylose, arabinose,
ribose, etc., uronic acids such as glucuronic acid, galacturonic
acid, mannuronic acid, gluronic acid, etc., amino sugars such as
glucosamine, galactosamine, etc., sialic acids such as
N-acetylneuraminic acid, etc., deoxy sugars such as fucose, etc.,
as well as esters, amides and lactones thereof are bound to hydroxy
groups other than that at 1-position of the compounds selected from
the compounds of formulas 1 to 6. Furthermore, the saccharides of
the present invention which contain the compounds selected from the
compounds of formulas 1 to 6 at their reducing ends include those
in which pyruvate and/or sulfate groups are bound to the
saccharides containing the compounds selected from the compounds of
formulas 1 to 6 at their reducing ends, for example, the
saccharides such as agarobiose, agarotetraose, agarohexaose,
agarooctaose, .kappa.-carabiose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like as well as the saccharides whose hydroxy groups are
methylated. As described above, preferably, the saccharides of the
present invention which contain the compounds selected from the
compounds of formulas 1 to 6 at their reducing ends are those whose
non-reducing ends are sugars other than L-galactose-6-sulfate.
[0102] Since an anomer carbon is present at 1-position of the
compound at the reducing end of the saccharide containing
3,6-anhydrogalactopyranose or its 2-O-methylated derivative at its
reducing end, an .alpha.-isomer and a .beta.-isomer exist for such
a compound. Both can be used as the saccharides of the present
invention which contain 3,6-anhydrogalactopyranose or its
2-O-methylated derivative at their reducing ends.
[0103] The molecular weight is not specifically limited in so far
as the compound has a physiological activity such as an
apoptosis-inducing activity, a carcinostatic activity, antioxidant
activities such as an activity of inhibiting active oxygen
production, an activity of inhibiting NO production and/or an
immunoregulatory activity.
[0104] Of course, a mixture of an .alpha.-isomer, a .beta.-isomer,
an aldehyde and a hydrated, and a mixture of a D-isomer and a
L-isomer can be used in the present invention as the compound
selected from the compounds of formulas 1 to 6 or the saccharide
containing the compound at its reducing end.
[0105] Thus, the compound selected from the compounds of formulas 1
to 6 or the saccharide containing the compound at its reducing end
used in the present invention has an apoptosis-inducing activity, a
carcinostatic activity, antioxidant activities such as an activity
of inhibiting active oxygen production, an activity of inhibiting
lipid peroxide radical production, an activity of inhibiting NO
production, an immunoregulatory activity and an anti-allergic
activity. Then, according to the present invention, first, there is
provided a pharmaceutical composition comprising as an active
ingredient at least one of the compounds selected from the
compounds of formulas 1 to 6 and the soluble saccharides containing
these compounds at their reducing ends for treating or preventing a
disease sensitive to at least one of these compounds, for example,
a therapeutic or prophylactic composition for the disease.
[0106] Examples of diseases sensitive to these compounds include a
diseases that requires induction of apoptosis for its treatment or
prevention, a carcinomatous disease, a diseases that requires
inhibition of active oxygen production for its treatment or
prevention, a disease that requires inhibition of lipid peroxide
radical production for its treatment or prevention, a disease that
requires inhibition of NO production for its treatment or
prevention or a disease that requires immunoregulation for its
treatment or prevention such as an allergic disease. The
pharmaceutical composition for treating or preventing these
diseases of the present invention can be used as a composition for
inducing apoptosis, a carcinostatic composition, antioxidants such
as an inhibitor of active oxygen production, an inhibitor of lipid
peroxide radical production, an inhibitor of nitrogen monoxide
production, an anti-inflammatory composition, an immunoregulator,
an anti-allergic composition and the like.
[0107] For example, the composition for inducing apoptosis of the
present invention is useful for eliminating auto-reactive
lymphocytes from patients suffered from autoimmune diseases, tumor
cells, cells infected with a virus and the like. It can be used to
eliminate unnecessary or harmful cells from a living body in a
natural manner by causing apoptosis in the desired tissues or
cells. Examples of diseases for which the composition for inducing
apoptosis of the present invention is effective include autoimmune
diseases such as systemic lupus erythematosus, immune mediated
glomerulonephritis, multiple sclerosis, collagen disease, etc.,
rheumatism, and the like.
[0108] The composition for inducing apoptosis of the present
invention can be used in a method for inducing apoptosis and the
method is useful for elucidation of mechanism of induction of
apoptosis, as well as screening for apoptosis-inducing compounds
and inhibitors of apoptosis induction.
[0109] Since the activity of inducing apoptosis by the composition
for inducing apoptosis of the present invention is inhibited by
Caspase inhibitor, for example, IL-LS converting enzyme inhibitor V
[Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone: manufactured by Takara
Shuzo]. Thus, the apoptosis induced by the composition is
considered to be a cell death due to apoptosis depending on
Caspase.
[0110] Caspase has been shown that it functions as an important
mediator of apoptosis because it increases prior to various cell
death; its overexpression induces cell death; the apoptosis is
inhibited by a peptide inhibitor or an inhibitory protein such as
CrmA and p35; and, in a knockout mouse for Caspase-1 or Caspase-3,
a part of apoptosis normally observed is inhibited [Seikagaku
(Biochemistry), vol. 70, p. 14-21 (1998)]. That is, during
apoptosis process, Caspase which is a cysteine protease is
activated to decompose nuclear or cytoplasmic proteins. Caspase is
first synthesized as a precursor and then activated by processing.
Regulation of this Caspase activation decides the life or death of
cells. The mammals have 10 or more types of Caspases. An upstream
Caspase processes a downstream Caspase to amplify the activity of
decomposing intracellular proteins in a cascade mode [Saibo Kogaku
(Cell Technology), Vol. 17, p. 875-880 (1998)]. On the contrary,
the processing activity can be inhibited by inhibitor of the
cysteine protease, Caspase, to stop cell death by Caspase dependent
apoptosis.
[0111] The compound used in the present invention is useful for
inhibition of production of oxidizing materials such as active
oxygen. Then, an antioxidant such as an inhibitor of active oxygen
production which comprises the compound as its active ingredient is
useful for treating or preventing diseases caused by production
and/or excess of active oxygen.
[0112] In general, active oxygen can be classified into radical
active oxygen and non-radical active oxygen. The radical active
oxygen includes hydroxy radical, hydroxyperoxy radical, peroxy
radical, alkoxy radical, nitrogen dioxide (NO.sub.2), NO,
thylradical and superoxide. On the other hand, the non-radical
active oxygen includes singlet oxygen, hydrogen peroxide, lipid
hydroperoxide, hypochlorous acid, ozone and peroxonitrite. All of
them are related to various pathological states such as
inflammatory diseases, diabetes, cancers, arteriosclerosis,
neurosis, ischemic re-perfusion disorder and the like.
[0113] In a living body, active oxygen is always produced at a low
concentration in some pathways. These are superoxide
physiologically leaking out from an electron transport system such
as mitochondria, hydrogen peroxide, hydroxy radical catalyzed with
a transition metal such as copper and iron, hypochlorous acid
formed by neutrophils or monocytes for protecting against
infections, NO produced by decomposition of arginine and the like,
and they are inevitable. A living body has a system eliminating
active oxygen including enzymes and low molecular weight compounds
against the production of active oxygen to maintain the balance
between the production and the elimination. However, a living body
is damaged oxidatively when the system for producing active oxygen
becomes predominant over the eliminating system due to the
activation of the above-mentioned pathways for some reasons or, to
the contrast, due to the inactivation of the eliminating system.
Such conditions are called as oxidative stress. Furthermore, in
addition to the imbalance inside the body, the living body is
always exposed to oxidative stress by materials outside the body
such as the atmosphere, foods and the like. Therefore, oxidative
stress is inevitable in everyone's daily life.
[0114] That is, as described above, the oxidative stress is related
to various diseases and a living body is always exposed to
circumstances in which diseases are caused by or disease conditions
become more serious due to oxidative stress. Therefore, the
antioxidant such as the inhibitor of active oxygen production of
the present invention is useful for preventing and treating the
diseases caused by oxidative stress or preventing the worsening of
the disease conditions due to such oxidative stress.
[0115] Furthermore, a lipid peroxidation reaction is always
associated with oxidative stress and proceeds at once upon
production of a lipid peroxide radical. 4-Hydroxy-2-nonenal (HNE)
produced therein is a toxic aldehyde specifically targeting
glutathione or a protein. Reaction products of HNE and protein are
detected in various disease tissues and considered to be inducing
factors of disease conditions associated with oxidative stress.
Then, the antioxidant which comprises the antioxidant substance
used in the present invention which can inhibit production of lipid
peroxide radicals is useful for preventing and treating age-related
diseases caused by oxidative stress.
[0116] NO is the main component of an endothelium-derived relaxing
factor (EDRF) [Nature, Vol. 327, p. 524-526 (1987)]. According to
the present invention, there is provided a pharmaceutical
composition for treating or preventing a diseases that requires
inhibition of NO production for its treatment or prevention.
[0117] In the present invention, diseases that require inhibition
of NO production are not limited to specific one and include for
example, systematic hypotension caused by toxic shock, treatment
with some cytokines and the like, blood pressure response
reduction, autoimmune diseases, inflammation, arthritis, rheumatoid
arthritis, diabetes, inflammatory bowel diseases, vascular function
failure, pathogenic angiectasis, tissue injury, cardiovascular
ischemia, hyperalgesia, cerebral ischemia, diseases associated with
vascularization, cancers and the like, inclusive the diseases
described in JP-A 9-504524, JP-A 9-505288, JP-A 8-501069, JP-A
8-512318 and JP-A 6-508849.
[0118] For NO synthases (NOS) which produces NO and L-citrulline
from L-arginine and oxygen, a cNOS type which are constitutively
expressed, and an iNOS which is a inducible type are known. In
macrophages and the like, iNOS is induced by stimulation of
cytotoxin or cytokines (for example, LPS, INF-.gamma.) to produce
NO. iNOS itself is essential to maintain a living body system.
However, on the other hand, it has been shown that iNOS causes
various diseases when it is expressed excessively by various
factors to produce excess NO.
[0119] The present inventors have confirmed that the compounds
selected from the compounds of formulas 1 to 6 and the soluble
saccharides containing these compounds at their reducing ends such
as agarobiose, agarotetraose and agarohexaose inhibit this iNOS
expression. The confirmation was carried out at protein level by
western blotting and at messenger RNA level by RT-PCR. That is, the
compounds used in the present invention are useful for treating and
preventing diseases that require inhibition of NO production by
inhibiting expression of iNOS which is overexpressed by various
factors to produce excess NO.
[0120] The compounds of the present invention inhibit NO production
in macrophages and are useful for treating and preventing diseases
caused by NO production in macrophages, inflammation, cancers and
the like. In addition, inhibition of NO production by the
saccharides used in the present invention is not antagonistic
inhibition of NO production inducing substances such as LPS or
INF-.gamma.. Increase in inhibitory effect on NO production is
observed by addition of the saccharides used in the present
invention in advance. Therefore, the compounds of the present
invention are very useful as those for preventing antioxidant
production.
[0121] The inhibitor of NO production of the present invention is
useful for studying the mechanism of NO production, and the mode of
action of NO and can be used for screening of materials involved in
the mechanism of NO production.
[0122] Vascularization is necessary for growth of a solid cancer,
and vascular endothelial growth factor/vascular permeability factor
(VEGF) play important roles in this process. In various tumor
cells, VEGF is induced by NO. The inhibitor of NO production of the
present invention also inhibits VEGF production of tumor cells by
inhibiting NO production, thereby inhibiting vascularization around
cancer tissues. When the inhibitor of NO production of the present
invention is administered to a mouse in which tumor cells have been
transplanted subcutaneously to form a solid cancer, vascularization
around the cancer tissue becomes insufficient and the cancer falls
out.
[0123] Nitrosoamines are a series of compounds in which nitro group
is attached to a secondary amine and several hundred types are
known. Many of them show carcinogenic activity to animals by
damaging DNA. Nitrosoamines are considered to have a high relation
to carcinogenesis of a human being and usually produced by a
reaction of a nitrite and an amine in a stomach. NO also produces a
nitrosoamine by reaction with an amine under physiological
conditions at a neutral pH range. NO production is accelerated in a
patient suffered from clonorchiasis or cirrhosis that have a high
relation to a cancer epidemiologically. Therefore, in particular,
carcinogenesis of a high-risk group can be prevented by
administration of the inhibitor of NO production of the present
invention to prevent acceleration of NO production. As described
hereinabove, the inhibitor of NO production of the present
invention shows its carcinostatic activity in two step, that is,
suppression of carcinogenesis and inhibition of vascularization in
cancer tissues.
[0124] NO also induces edema which is specifically recognized in
inflammatory lesions, i.e., vascular permeability accelerating
activity [Maeda et al., Japanese Journal of Cancer Research, Vol.
85, p. 331-334 (1994)] and accelerates biosynthesis of
prostaglandins which are inflammatory mediators [Salvemini et al.,
Proceedings of National Academy of Sciences, USA, Vol. 90, p.
7240-7244 (1993)]. On the other hand, NO reacts with a superoxide
radical quickly to produce peroxonitrite ion and this peroxonitrite
ion also considered to cause inflammatory damages of cells and
tissues.
[0125] NO production is induced when activated immune cells enter
in an organ and release cytokines. Insulin-dependent diabetes is
induced by specific destruction of islet .beta. cells and this
destruction is considered to be caused by NO. Synovial fluid in the
lesion of a patient suffered from rheumatoid arthritis,
osteoarthrosis, gouty arthritis and arthritis associated with
Behcet disease contains NO at a concentration higher than that in
the normal joint of the same patient or joints of healthy people.
When the inhibitor of NO production of the present invention is
administered to such patients, NO production in the lesion is
inhibited to improve disease conditions.
[0126] NO production is increased during cerebral ischemia and
after re-perfusion, which causes damages in cerebral tissues.
Administration of the inhibitor of NO production of the present
invention to a patient during cerebral ischemia relieves the damage
of cerebral tissue and improves the prognosis.
[0127] The immunoregulator of the present invention has
immunoregulatory activities such as an activity of suppressing
lymphocyte blastogenesis and an activity of suppressing mixed
lymphocyte reaction. Thus, the immunoregulator of the present
invention is useful as a pharmaceutical composition for treating or
preventing diseases caused by abnormality of these immune systems
or immune factors.
[0128] Lymphocyte blastogenesis is a reaction in which mitogen
binds to a receptor on the surface of a lymphocyte to activate the
lymphocyte and promotes its division and proliferation. Mixed
lymphocyte reaction is a reaction in which lymphocytes obtained
from allogeneic animals are mixed and cultured, thereby inducing
activation of lymphocytes due to incompatibility of major
histocompatibility antigens to promote the division and
proliferation of lymphocytes. The immunoregulator of the present
invention suppress these reactions and is useful as a
pharmaceutical composition for treating and preventing chronic
diseases caused by abnormal acceleration of lymphocytes, for
example, autoimmune diseases such as chronic nephritis, ulcerative
colitis, type I diabetes and rheumatoid arthritis and is also
useful for suppression of graft rejection.
[0129] In mast cells sensitized with IgE antibody, degranulation is
induced by binding of an antigen and a chemical mediator is
released. This type I, i.e. the immediate-type allergic reaction
plays an important role in allergy diseases whose representative
examples are asthma and atopic dermatitis, and substances which
suppress release of chemical mediators from mast cells are
considered to be very effective for treating and preventing these
allergic diseases.
[0130] Passive cutaneous anaphylaxis (PCA) of a rat which is a
model of the type I allergic reaction is initiated with
degranulation of mast cells, followed by release of chemical
mediators contained in granules such as histamine and serotonin to
cause increase in vascular permeability and finally to cause
pigment leakage in a local skin. This model is used as a model for
estimating anti-allergic compounds in vivo most frequently.
[0131] The compounds selected from the compounds of formulas 1 to 6
and the soluble saccharides containing these compounds at their
reducing ends has an activity of inhibiting PCA and the present
invention also provides an anti-allergic composition comprising at
least one member selected from the group consisting of the
compounds selected from the compounds of formulas 1 to 6 and the
soluble saccharides containing these compounds at their reducing
ends as its active ingredient.
[0132] The anti-allergic composition is very useful for treating
and preventing diseases which can be treated by inhibition of the
type I allergic reaction, such as bronchial asthma, atopic
dermatitis, allergic rhinitis, pollinosis, hives, contact
dermatitis, allergic conjunctivitis and the like.
[0133] The above-mentioned pharmaceutical composition for treating
or preventing diseases of the present invention, for example, the
composition for inducing apoptosis, can be prepared by using at
least one member selected from the group consisting of the
compounds selected from the compounds of formula 1 to 6 and the
soluble saccharides containing these compounds at their reducing
ends as its active ingredient, and formulate it with a known
pharmaceutically acceptable carrier.
[0134] In general, the compound is combined with a pharmaceutically
acceptable liquid or solid carrier and, if necessary, to this is
added solvent, dispersing agent, emulsifier, buffering agent,
stabilizer, excipient, binder, disintegrant, lubricant and the like
to obtain a preparation in the form of a solid preparation such as
tablet, granule, powder, epipastic, capsule and the like, and a
liquid preparation such as normal solution, suspension, emulsion
and the like. In addition, a dried preparation which can be
reconstituted as a liquid preparation by addition of a suitable
carrier before use can be obtained.
[0135] The composition for inducing apoptosis of the present
invention can be administrated as either an oral preparation or a
parenteral preparation such as injectable preparation, drips or the
like.
[0136] The pharmaceutical carrier can be selected according to the
above-mentioned particular administration route and dosage form.
For an oral preparation, for example, starch, lactose, sucrose,
mannit, carboxymethylcellulose, cornstarch, inorganic salts and the
like are used. For preparing the oral preparation, binder,
disintegrant, surfactant, lubricant, fluidity promoting agent,
tasting agent, coloring agent, flavoring agent and the like can
also be added.
[0137] A parenteral preparation can be prepared according to
conventional methods by dissolving or suspending the active
ingredient of the present invention, that is the saccharide having
the activity of inducing apoptosis, in a diluent such as injectable
distilled water, physiological saline, aqueous glucose solution,
injectable vegetable oil, sesame oil, peanut oil, soybean oil, corn
oil, propylene glycol, polyethylene glycol or the like, and, if
necessary, adding sterilizer, stabilizer, osmotic regulator,
smoothing agent and the like to the resultant solution or
suspension.
[0138] The composition for inducing apoptosis of the present
invention can be administrated through a suitable route for the
dosage form of the composition. The administration method is not
limited and the composition can be used internally or externally
(or topically) or by injection and the like. The injectable
preparation can be administrated intravenously, intramuscularly,
subcutaneously, intradermally and the like. External preparations
include a suppository and the like.
[0139] A dosage of the composition for inducing apoptosis of the
present invention can be appropriately determined and varies
depending on the particular dosage form, administration route and
purpose as well as age, weight and conditions of a patient to be
treated. In general, a daily dosage for an adult person is 10 .mu.g
to 200 mg/kg in terms of the amount of the active ingredient
contained in the composition. As the dosage, of course, can vary
dependent on various factors, in some cases, a less dosage than the
above may be sufficient but, in other cases, a dosage more than the
above may be required. The pharmaceutical composition of the
present invention can be administrated orally as it is, or it can
be administered daily by admixing with appropriate foods and
drinks.
[0140] The carcinostatic composition of the present invention can
be prepared by using at least one member selected from the group
consisting of the compounds selected from the compounds of formulas
1 to 6 and the soluble saccharides containing these compounds at
their reducing ends as its active ingredient and formulating it
with a known pharmaceutical carrier. The carcinostatic composition
can prepared according to the same manner as that described above
with respect to the composition for inducing apoptosis.
[0141] The carcinostatic composition can be administrated through a
suitable route for the dosage form of the composition. A method for
administration is not limited and the composition can be
administrated internally or externally (or topically) or by
injection and the like. An injectable preparation can be
administrated, for example, intravenously, intramuscularly,
subcutaneously, intradermally and the like. External preparations
include a suppository and the like.
[0142] A dosage of the carcinostatic composition of the present
invention can be determined and varies depending on the particular
dosage form, administration route and purpose as well as age,
weight and conditions of a patient to be treated. In general, a
daily dosage for an adult person is 10 .mu.g to 200 mg/kg in terms
of the amount of the active ingredient contained in the
composition. As the dosage, of course, can vary dependent on
various factors, in some cases, a less dosage than the above may be
sufficient, but, in other cases, a dosage more than the above may
be required. The pharmaceutical composition of the present
invention can be administrated orally as it is, or it can be
administrated daily by admixing with appropriate foods and
drinks.
[0143] The antioxidant, the inhibitor of active oxygen production,
the inhibitor of lipid peroxide radical production, the inhibitor
of NO production, the immuno-regulator and the anti-allergic
composition of the present invention can be prepared according to
the same manner as that described above with respect to the
composition for inducing apoptosis. The same dosage and
administration route as those described above with respect to the
composition for inducing apoptosis can be used.
[0144] That is, the antioxidant, the inhibitor of active oxygen
production, the inhibitor of lipid peroxide radical production, the
inhibitor of NO production, the immuno-regulator and the
anti-allergic composition of the present invention are
administrated through a suitable route for the particular dosage
form of the composition. A method for administration is not limited
and the composition can be administrated internally or externally,
or by injection and the like. An injectable preparation can be
administrated intravenously, intramuscularly, subcutaneously,
intradermally and the like. External preparations include a
suppository and the like.
[0145] A dosage of the antioxidant, the inhibitor of active oxygen
production, the inhibitor of lipid peroxide radical production, the
inhibitor of NO production, the immuno-regulator and the
anti-allergic composition of the present invention can be
determined and varies depending on the particular dosage form,
administration route and purpose as well as age, weight and
conditions of the patient to be treated. In general, a daily dosage
for an adult person is 10 .mu.g to 200 mg/kg in terms of the amount
of the active ingredient contained in the composition. As the
dosage, of course, can vary dependent on various factors, in some
cases, a less dosage than the above may be sufficient, but, in
other cases, a dosage more than the above may be required. The
pharmaceutical composition of the present invention can be
administrated orally as it is, or it can be administrated daily by
admixing with appropriate foods and drinks.
[0146] The foods or drinks of the present invention are those
comprising, produced by adding thereto and/or produced by diluting
at least one member selected from the group consisting of the
compounds selected from the compounds of formulas 1 to 6 and the
soluble saccharides containing these compounds, for example,
saccharides prepared by acid decomposition under acidic conditions
below pH 7 and/or enzymatic digestion of the raw substances, such
as agarobiose, agarotetraose, agarohexaose, agarooctaose,
.kappa.-carabiose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like. Since the food or drink has an activity of inducing
apoptosis, a carcinostatic activity, an antioxidant activity, an
immunoregulatory activity and the like. Thus, it is very useful for
ameliorating disease states of and preventing diseases sensitive to
at least one member selected from the group consisting of the
compounds selected from the compounds of formulas 1 to 6 and the
soluble saccharides containing these compounds at their reducing
ends, such as a disease that requires induction of apoptosis for
its treatment or prevention, a carcinomatous disease, a disease
that requires inhibition of active oxygen production for its
treatment or prevention, a disease that requires inhibition of NO
production for its treatment or prevention or a disease that
requires immunoregulation for its treatment or prevention, an
allergic disease and the like.
[0147] A process for producing the foods or drinks of the present
invention is not limited to a specific one, and cooking, processing
and other generally employed processes for producing foods and
drinks can be used in so far as the resultant foods or drinks
contain as their active ingredients at least one member selected
from the group consisting of the compounds selected from the
compounds of formulas 1 to 6 and the soluble saccharides containing
those compounds at their reducing ends prepared, for example, by
acid decomposition under acidic conditions below pH 7 and/or
enzymatic digestion of the raw substances, such as agarobiose,
agarotetraose, agarohexaose, agarooctaose, .kappa.-carabiose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like.
[0148] The foods or drinks of the present invention are not limited
to a specific one and examples thereof include cereal processed
products (e.g., wheat flour products, starch processed products,
premixed products, noodles, macaroni, breads, bean jams, buckwheat
noodles, fu (wheat gluten bread), rice noodle, gelatin noodles, and
packed rice cake, etc.), fat and oil processed products (e.g.,
plastic fat and oil, tempura oil, salad oil, mayonnaise, dressings,
etc.), soybean processed products (e.g., tofu, miso, fermented
soybeans, etc.), meet processed products (e.g., hams, bacon,
pressed ham, sausage, etc.), processed marine products (e.g.,
frozen ground fish meat, boiled fish paste, tubular roll of boiled
fish paste, cake of ground fish, deep-fried patty of fish paste,
fish ball, sinew, fish meat ham, sausage, dried bonito, processed
fish egg products, canned marine food, fish boiled in sweetened soy
sauce, etc.), dairy products (e.g., raw milk, cream, yogurt,
butter, cheese, condensed milk, powdered milk, ice cream, etc.),
processed vegetables and fruit products (e.g., pastes, jams,
pickles, fruit juices, vegetable drinks, mixed drinks, etc.),
confectioneries (e.g., chocolates, biscuits, sweet buns, cakes,
rice-cake sweets, rice sweets, etc.), alcohol drinks (e.g., sake,
Chinese liquors, wines, whiskies, shochu, vodkas, brandies, gins,
rums, beer, soft alcohol drinks, fruit liquors, liqueurs, etc.),
luxury drinks (e.g., green tea, tea, oolong tea, coffee, soft
drinks, lactic acid drinks, etc.), seasonings (e.g., soy sauce,
sauce, vinegar, sweet sake, etc.), canned food, bottled food and
bagged food (e.g., various cooked food such as rice topped with
cooked beef and vegetables, rice boiled together with meat and
vegetables in a small pot, steamed rice with red beans, curry,
etc.), semi-dried or condensed food (e.g., liver paste, the other
spread, soup of buckwheat noodles or "udon", condensed soups,
etc.), dried food (e.g., instant noodles, instant curry, instant
coffee, powdered juice, powdered soup, instant miso soup, cooked
food, cooked drinks, cooked soup, etc.), frozen food (e.g.,
sukiyaki, chawan-mushi, grilled eel, hamburger steak, shao-mai,
Chinese meat dumpling, various stick, fruit cocktail, etc.), solid
food, liquid food (e.g., soup, etc.), processed agricultural
products and forest products such as spices, processed livestock
products, processed marine products and the like.
[0149] In so far as the food or drink of the present invention
comprises, is produced by adding thereto and/or produced by
diluting at least one member selected from the group consisting of
the compounds selected from the compounds of formulas 1 to 6 and
the soluble saccharides containing these compounds at their
reducing ends, for example, saccharides prepared by acid
decomposition under acidic conditions below pH 7 and/or enzymatic
digestion of the raw substances, such as agarobiose, agarotetraose,
agarohexaose, agarooctaose, .kappa.-carabiose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like, in an amount necessary for exhibiting the physiological
functions, their forms are not limited to a specific one and may be
any edible forms including tablets, granule, capsule and the
like.
[0150] 3,6-Anhydrogalactopyranose, a 2-O-methylated derivative
thereof and the saccharides containing these compounds at their
reducing ends tend to open at their hemi-acetal rings to form
aldehyde groups at the ends. These aldehyde groups as well as the
aldehyde group of the aldehyde of the 3,6-anhydrogalactopyranose
tend to react with compounds which are reactive with aldehyde
group, for example, nucleophiles such as amino acids. The compounds
of formulas 1 to 6 or the saccharides, for example, the
oligosaccharides thus reacted are in such a state that they lose
the compounds selected from the compounds of formulas 1 to 6 at
their reducing ends. Therefore, they lose various physiological
activities of the member selected from the compounds selected from
the compounds of formulas 1 to 6 and the oligosaccharides
containing these compounds at their reducing ends. That is, in
order to maintain the member selected from the compounds selected
from the compounds of formulas 1 to 6 and the saccharides
containing these compounds at their reducing ends in the foods or
drinks stably, a molar concentration of a compound reactive with
the aldehyde should be kept lower than that of the aldehyde.
[0151] In the production of the food or drink of the present
invention, it is possible to provide the food or drink that
contains the member selected from the group consisting of the
compounds selected from the compounds of formulas 1 to 6 and the
oligosaccharides containing these compounds at their reducing ends
in a high content without substantial reduction of the amount
thereof by controlling the amount of a compound that is reactive
with the aldehyde. Such control has not been considered heretofore
in the prior art.
[0152] It is also found that the member selected from the group
consisting of the compounds selected from the compounds of formula
1 to 6 and the saccharides containing these compounds at their
reducing ends is stable under acidic conditions. Then, an acidic
food or acidic drink which contains at least one member selected
from the group consisting of the compounds selected from the
compounds of formulas 1 to 6 and soluble saccharide containing
these compounds at their reducing ends in a high content can be
provided by carrying out all of the steps of producing the food or
drink of the present invention under acidic conditions to prepare
the acidic food or acidic drink.
[0153] In the production of the acidic food or drink of the present
invention, the kind of the acid to be used for acid decomposition
of the raw substances is not limited to a specific one, and both
organic and inorganic acids can be used. However, a better taste of
the resultant acid decomposition product of agar is obtained when
an organic acid are used. Then, organic acids are preferably used
to obtain an acid decomposition product having a novel flavor. The
organic acid can be selected depending on the particular purpose.
It can be used alone, or two or more of them can be used in
combination. Preferred examples of the organic acids include acetic
acid, citric acid, malic acid, lactic acid, tartaric acid, succinic
acid, fumaric acid and the like. Decomposition conditions are not
specifically limited. For example, when citric acid is used as the
organic acid, acid decomposition of agar as a raw substance is
carried out at 60 to 130.degree. C., preferably 90 to 105.degree.
C., for 3 to 300 minutes, preferably 30 to 200 minutes, thereby
modifying an acidic taste of the organic acid to obtain the
composition having a good balanced taste, mild texture and a smooth
acidic taste. An acidulant having the desired acidic taste can be
obtained by heat treatment of a composition containing 0.05 to 30%
by weight, preferably 0.2 to 10% by weight of an organic acid, and
1 to 20% by weight, preferably 5 to 15% by weight of at least one
member selected from the group consisting of the compounds selected
from the compounds of formulas 1 to 6 and the soluble saccharides
containing these compounds at their reducing ends. The acidulant
thus obtained is very useful for the production of soft drinks,
acidic seasonings, acidic foods and the like.
[0154] The acidic food or acidic drink of the present invention
contains a large amount of at least one member, which has a
physiological activity, selected from the group consisting of the
compounds selected from the compounds of formulas 1 to 6 and the
soluble saccharides containing these compounds, for example,
saccharides prepared by acid decomposition under acidic conditions
below pH 7 and/or enzymatic digestion of the raw substances, such
as agarobiose, agarotetraose, agarohexaose, agarooctaose,
.kappa.-carabiose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like. The physiological functions of the compounds such as an
activity of inducing apoptosis, a carcinostatic activity and the
like provide an effect of preventing carcinogenesis, an effect of
suppressing cancer or the like upon eating the food or drink. That
is, the acidic food or acidic drink of the present invention is a
healthy food or drink which has effects of ameliorating the disease
states of or preventing the diseases sensitive to at least one
member selected from the group consisting of the compounds selected
from the compounds of formulas 1 to 6 and the soluble saccharides
containing these compounds, and is particularly useful for keeping
gastrointestinal health.
[0155] The compounds selected from the compounds of formulas 1 to 6
and the soluble saccharides containing said compounds of the
present invention, for example, saccharides prepared by acid
decomposition under acidic conditions below pH 7 and/or enzymatic
digestion of the raw substances, such as agarobiose, agarotetraose,
agarohexaose, agarooctaose, .kappa.-carabiose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like have antioxidant activities such as an activity of
inhibiting active oxygen production, an activity of inhibiting
lipid peroxide radical production and the like, and can be used in
the production antioxidant foods or antioxidant drinks as an
antioxidant such as an inhibitor of active oxygen production, an
inhibitor of lipid peroxide radical production, an inhibitor of NO
production and the like.
[0156] That is, according to the present invention, there is
provided an antioxidant, in particular, an antioxidant for foods
and drinks, which comprises at least one member selected from the
group consisting of the compounds selected from the compounds of
formulas 1 to 6 and the soluble saccharides containing these
compounds as its active ingredient.
[0157] The form of the antioxidant of the present invention is not
limited to a specific one, and can be suitably selected according
to the foods and drinks to be applied, for example, powder, paste,
emulsion and the like. The antioxidant food or drink which
comprises the member selected from the compounds and the
saccharides used in the present invention as its active ingredient
can be readily and simply produced by using the antioxidant of the
present invention.
[0158] According to the present invention, there is also provided a
saccharide for an antioxidant selected from the group consisting of
the compounds selected from the compounds of formulas 1 to 6 and
the soluble saccharide containing these compounds. For example, the
saccharide for an antioxidant can be obtained as a product produced
by acid decomposed under acidic conditions below pH 7 and/or
enzymatic digestion of the raw substance. In addition, its purified
or partial purified product can also be used. Examples of the raw
substances include those derived from red algae such as agar,
agarose, carrageenan and the like. They can be used alone or two or
more of them can be used in combination. The examples of
representative saccharides for an antioxidant are, not limited
specifically, soluble polysaccharides containing the compounds
selected from the compound of formulas 1 to 6, for example,
agarobiose, agarotetraose, agarohexaose, agarooctaose,
.kappa.-carabiose and
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like.
[0159] The saccharide for an antioxidant of the present invention
is useful for eliminating or suppressing the production of oxidants
in a living body, such as active oxygen. Then, the saccharide for
an antioxidant is useful for ameliorating disease states of or
preventing diseases caused by production or excess of active
oxygen.
[0160] As described above, oxidative stress, which is generated
from oxidative damage of a living body in case where the system for
producing active oxygen is predominant over an elimination system,
is involved in various diseases. Thus, a living body is always
exposed to circumstances which lead to diseases caused by oxidative
stress or worsening of the diseases conditions. Therefore, it is
desirable to take a suitable amount of an antioxidant everyday for
preventing, treating or preventing worsening of diseases caused by
oxidative stress. For daily intake of suitable amount of an
antioxidant, it is desirable to take it from foods and drinks. The
foods and drinks of the present invention which comprise, produced
by adding thereto, and/or produced by adding the saccharide for an
antioxidant are very useful for antioxidant foods or drinks or
anti-oxidative stress foods or drinks.
[0161] The member selected from the compounds and the saccharides
used in the present invention also have ability of retaining water
and at least one of them can be used as an active ingredient for
the production of an anti-constipation composition, an
anti-constipation food and an anti-constipation drink.
[0162] Furthermore, according to the present invention, there is
provided a cosmetic composition which comprises as its active
ingredient the soluble saccharide containing the compound selected
from the compounds of formulas 1 to 6 in its reducing end, for
example, an oligosaccharide such as agarobiose, agarotetraose,
agarohexaose, agarooctaose, .kappa.-carabiose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose or the
like. The saccharide can be obtained as a product produced by acid
decomposition under acidic conditions below pH 7 and/or enzymatic
digestion of the raw substance. The purified or partially purified
decomposition product can also be used. As the raw substance, that
derived from red algae, for example, agar, agarose, carrageenan or
the like can be used alone or two or more of them can be used in
combination.
[0163] The above-mentioned compound can be used as an active
ingredient for the production of cosmetic compositions including
fundamental cosmetic compositions such as cream, milky lotion,
lotion, facial cleansing and puck, makeup cosmetics such as
lipstick and foundation, body soap, soap and the like. The compound
is also effective to the hair and the cosmetic composition of the
present invention can be produced in the form of hair care
products, for example, hair products such as hair tonic, hair
liquid, hair set lotion, hair blow agent, hair cream, hair coat,
and the like and hair toiletry products such as shampoo, hair
rinse, hair treatment, and the like. The amount of the compound
mixed in the cosmetic composition can be determined appropriately
according to its skin beautifying/whitening activity, humectant or
moisturizing activity, antioxidant activity and the like. As other
cosmetic components, those mixed in conventional cosmetic
compositions can be used. Skin beautifying/whitening activity and
humectant or moisturizing activity can be measured by conventional
methods, for example the method described in JP-A 8-310937.
[0164] The cosmetic composition of the present invention has
excellent properties based on a skin beautifying/whitening
activity, a humectant or moisturizing activity, an antioxidant
activity, an activity of inhibiting active oxygen production and an
anti-oxidative stress activity to the skin; a humectant or
moisturizing activity, an antioxidant activity, an activity of
inhibiting active oxygen production and an anti-oxidative stress
activity to the hair; and the like.
[0165] The present invention also provides a preservative
composition for keeping freshness of foods and drinks which
comprises as its active ingredient at least one member selected
from the group consisting of the compounds selected from the
compounds of formulas 1 to 6 and the soluble saccharides containing
these compounds at their reducing ends, for example,
oligosaccharides such as agarobiose, agarotetraose, agarohexaose,
agarooctaose, .kappa.-carabiose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like. The saccharide can be obtained as a product produced by
acid decomposition under acidic conditions below pH 7 and/or
enzymatic digestion of the substance. The purified or partially
purified decomposition product also can be used. As the raw
substance, that derived from red algae which comprises the compound
selected from the compounds of formulas 1 to 6, for example, agar,
agarose, carrageenan and the like can be used alone or two or more
can be used in combination.
[0166] At least one member selected from the group consisting of
the compounds selected from the compounds of formulas 1 to 6 and
the soluble saccharides containing the compounds at their reducing
ends, for example, a saccharide such as agarobiose, agarotetraose,
agarohexaose, agarooctaose, .kappa.-carabiose,
.beta.-D-galactopyranosyl-3,6-anhydro-2-O-methyl-L-galactose and
the like has an antioxidant activity, a freshness keeping activity
and a tyrosinase inhibitory activity. The preservative composition
for keeping freshness of foods and drinks of the present invention
which prevents effectively color change, decay, oxidation and the
like of foods can be produced by using the compound as its active
ingredient according to a known formulation process. The
preservative composition of the present invention is very useful
for keeping a flavor and freshness of various foods, perishable
foods, and processed foods.
[0167] No acute toxicity is observed when administering either of
the member selected from the group consisting of the compounds
selected from the compounds of formula 1 to 6 and the soluble
saccharides containing these compounds used in the present
invention to a mouse at a dosage of 1 g/kg orally or
intraperitoneally.
[0168] The following examples further illustrate the present
invention in detail but are not to be construed to limit the scope
thereof.
Example 1
[0169] (1) A suspension of 400 mg of commercially available agar
powder (manufactured by Wako Pure Chemical Industries, Ltd.) in 20
ml of 1 N HCl was heated with a microwave oven to obtain a
solution. The resulting solution was cooled and was adjusted to pH
4 with sodium hydroxide. To the solution was added 384 mg of citric
acid and the solution was adjusted to pH 3 with sodium hydroxide.
Water was added thereto to make the total volume up to 40 ml and
the solution was heated at 120.degree. C. for 4 hours. The
resulting acid decomposition solution was adjusted to pH 6.5 with
sodium hydroxide and filtrated with a 0.2 .mu.m filter
(manufactured by Corning).
[0170] Human promyelocytic leukemia cell HL-60 (ATCC CCL-240) was
incubated at 37.degree. C. in RPMI 1640 medium (manufactured by
Gibco) supplemented with 10% of fetal bovine serum (JRH Bioscience)
which had been treated at 56.degree. C. for 30 minutes, and
suspended in the same medium at a concentration of 500 cells/90
.mu.l. Each 90 .mu.l portion of the suspension was distributed into
each well of a 96 well plate (manufactured by Falcon). To the
suspension in each well was added 10 .mu.l of the above-mentioned
acid decomposition solution, a 10-fold dilution of the solution or
water, and incubated with 5% CO.sub.2 at 37.degree. C. After 24
hour and 48 hour from the initiation of the incubation, the cell
morphology was observed under an optical microscope. Then,
according to the MTT method described in "Apoptosis Jikken
Protocol" (Syuzyun-sha, Tanuma, Seiichi ed., pp. 156 (1994)), 5
mg/ml of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide (manufactured by Sigma) and 10 .mu.l of phosphate buffered
saline solution were added to the culture and the incubation was
continued for additional 4 hours. Then, 100 .mu.l of 2-propanol
containing 0.04 N hydrochloric acid was added to the culture, and
the mixture was thoroughly stirred. An absorbance at 590 nm was
measured and the number of viable cells was calculated from the
absorbance measured for each of the wells, which was compared each
other.
Example 2
[0171] (1) A suspension of 1 g of commercially available agar (Agar
Noble, manufactured by Difco) in 100 ml of 0.1 N hydrochloric acid
was heated with a microwave oven until boiling to prepare a
solution. After cooling to room temperature and adjusting to pH 6,
the solution was filtered through Cosmonice filter (manufactured by
Nacalai Tesque) and 2 ml of the filtrate was separated with reverse
phase HPLC under the following conditions. [0172] Column: TSK-gel
ODS 80Ts (20 mm.times.250 mm, manufactured by Toso) [0173] TSK
guard column ODS-80Ts .quadrature.20 mm.times.50 mm, manufactured
by Toso) [0174] Mobile phase: aqueous 0.1% trifluoroacetic acid
(TFA) solution [0175] Flow rate: 9 ml/min [0176] Detection:
absorbance at 215 nm
[0177] Each elution peak was fractionated, collected, evaporated to
dryness under reduced pressure and then dissolved in 300 .mu.l of
water. Each fraction was sterilized by filtration and its 10 .mu.l
portion was placed in a well of a 96 well microtiter plate. Then,
90 .mu.l of RPMI 1640 medium (manufactured by Nissui) containing
10% fetal bovine serum (manufactured by Gibco) and 5,000 HL-60
cells (ATCC CCL-240) was added thereto, followed by incubation at
37.degree. C. for 48 hours with 5% CO.sub.2. The cell morphology
was observed under an optical microscope. Then, 5 mg/ml
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide and
10 .mu.l of phosphate buffered saline solution were added thereto
and the incubation was continued for additional 4 hours. To the
culture was added 100 .mu.l of 2-propanol containing 0.04 N
hydrochloric acid and the resultant mixture was thoroughly stirred.
The absorbance at 590 nm was measured to determine a cell
proliferation rate.
[0178] As a result, apoptosis corpuscles were observed in the group
to which the fraction from the peak at 8.26 min. was added. And, as
compared with the control group to which water was added, the
absorbance at 590 nm was lower and the cell proliferation was
inhibited.
[0179] (2) A 100 .mu.l of the fraction from the peak at 8.26 min.
described in Example 2-(1) was separated with size exclusion HPLC
chromatography as follows. [0180] Column: TSK-gel .alpha.-2500 (7.8
mm.times.300 mm, manufactured by Toso) [0181] TSK guard column a (6
mm.times.40 mm, manufactured by Toso) [0182] Mobile phase: aqueous
0.01% TFA solution [0183] Flow rate: 0.8 ml/min [0184] Detection:
differential refractometer
[0185] The separation pattern of the size exclusion HPLC
chromatography is illustrated in FIG. 3. That is, FIG. 3
illustrates the size exclusion HPLC chromatogram of the acid
decomposition product of agar. The horizontal axis represents the
elution time (min.) and the vertical axis represents the output
from the differential refractometer.
[0186] Each separated peak was fractionated, collected and
evaporated to dryness under reduced pressure. Each fraction was
dissolved in water at a concentration of 10 mg/ml, sterilized by
filtration and then, according to the same manner as that described
above in Example 2-(1), an apoptosis-inducing activity and an
antiproliferation activity against tumor cell were measured. As a
result, the peaks at the elution time 8.87 min. and 9.40 min. had
both activities.
[0187] The substances at the elution time 8.87 min. and 9.40 min.
were separately dissolved in a phosphate buffered saline solution,
and allowed to stand at 37.degree. C. for 1 hour. Then, according
to the same manner as that described above, they were analyzed by
the size exclusion HPLC chromatography. As a result, the peaks at
8.87 min. and 9.40 min. were observed in both samples, and the
ratio of the peak area was almost identical among the samples. This
revealed that the substances at the elution time 8.87 min. and 9.40
min. were in an equilibrium state when they were dissolved in the
aqueous phosphate buffer.
[0188] The fractions from the peaks at the elution time 8.87 min.
and 9.40 min. were combined and evaporated to dryness under reduced
pressure to obtain an apoptosis-inducing and carcinostatic
substance.
[0189] (3) The apoptosis-inducing and carcinostatic substance
described in Example 2-(2) was subjected to mass spectrometry with
DX302 mass spectrometer (manufactured by Nippon Denshi). The
measurement was carried out by using glycerol as a matrix with
negative ion mode.
[0190] FAB-MS
[0191] m/z 323 [M--H].sup.-
[0192] 415 [M+glycerol-H].sup.-
[0193] 507 [M+2glycerol-H].sup.-
[0194] The results are shown in FIG. 4. That is, FIG. 4 illustrates
the mass spectrum of the apoptosis-inducing and carcinostatic
substance. The horizontal axis represents m/z value and the
vertical axis represents the relative intensity (%).
[0195] Nuclear magnetic resonance spectrum of the
apoptosis-inducing and carcinostatic substance described in Example
2-(2) was measured with JNM-A500 nuclear magnetic resonance
apparatus (manufactured by Nippon Denshi).
[0196] .sup.1H-NMR: .delta. 3.36 (1H, dd, J=8.0, 10.0 Hz), 3.51
(1H, dd, J=3.0, 10.0 Hz), 3.56 (1H, m), 3.58 (1H, m), 3.63 (1H, m),
3.67 (1H, m), 3.70 (1H, dd, J=3.0, 10.0 Hz), 3.77 (1H, d, J=3.0
Hz.quadrature., 3.83 (1H, dd, J=4.5, 10.0 Hz), 3.93 (1H, dd, J=5.0,
3.5 Hz), 4.23 (1H, m), 4.25 (1H, m), 4.41 (1H, d, J=8.0 Hz), 4.85
(1H, d, J=6.0 Hz)
[0197] The sample was dissolved in heavy water and the chemical
shift value of HOD was shown as 4.65 ppm.
[0198] .sup.13C-NMR: .delta. 61.9, 69.4, 71.5, 73.37, 73.42, 73.8,
76.0, 76.1, 83.7, 86.5, 90.7, 103.3
[0199] The sample was dissolved in heavy water and the chemical
shift value of dioxane was shown as 67.4 ppm.
[0200] .sup.1H-NMR spectrum of the apoptosis-inducing and
carcinostatic substance is shown in FIG. 5. In FIG. 5, the
horizontal axis represents the chemical shift value (ppm) and the
vertical axis represents the signal intensity. The sample was also
dissolved in heavy dimethyl sulfoxide and .sup.1H-NMR spectrum was
measured.
[0201] .sup.1H-NMR: .delta. 9.60 (1H, H of aldehyde)
[0202] .sup.1H-NMR spectrum of the apoptosis-inducing and
carcinostatic substance in heavy dimethyl sulfoxide solvent is
shown in FIG. 6. In FIG. 6, the horizontal axis represents the
chemical shift value (ppm) and the vertical axis represents the
signal intensity.
[0203] The apoptosis-inducing and carcinostatic substance described
in Example 2-(2) was identified as agarobiose on the basis of the
analytical results of mass spectrometry, .sup.1H-NMR and
.sup.13C-NMR. And, the .sup.1H-NMR in heavy dimethyl sulfoxide
solvent demonstrated that 3,6-anhydrogactose at the reducing end of
agarobiose was mainly present as an aldehyde whose ring was opened
in a non-aqueous solvent. In addition, .sup.1H-NMR in heavy water
solvent demonstrated that it was present as a hydrated of the
aldehyde in an aqueous solution.
[0204] The results as described above revealed that the
apoptosis-inducing and carcinostatic substance obtained in Example
2-(2) was agarobiose.
[0205] (4) The apoptosis-inducing and carcinostatic substance
obtained in Example 2-(2), i.e., agarobiose, was dissolved in water
at a concentration of 0.78 mg/ml, and its 10 .mu.l portion was
placed in the well of a 96 well microtiter plate to measure an
apoptosis-inducing activity and an antiproliferation activity
against cell according to the same manner as described in Example
2-(1). As a result, apoptosis corpuscles were observed under an
optical microscope and, as compared with the control group to which
water as added, cell proliferation was suppressed by about 86% in
the group to which agarobiose was added. Namely, agarobiose at a
concentration of 78 .mu.g/ml induced apoptosis in HL-60 cells and
inhibited cell proliferation.
Example 3
[0206] (1) A suspension of 2.5 g of commercially available agar
(Agar Noble) in 50 ml of 0.1 N HCl was heated at 100.degree. C. for
13 minutes to prepare a solution. After cooling to room temperature
and neutralizing to about neutral pH with NaOH, the solution was
filtered through Cosmonice filter and separated with normal phase
HPLC as follows. [0207] Column: TSk-gel Amide-80 (21.5 mm.times.300
mm, manufactured by Toso) [0208] Solvent A: aqueous 90%
acetonitrile solution [0209] Solvent B: aqueous 50% acetonitrile
solution [0210] Flow rate: 5 ml/min [0211] Elution: linear gradient
from solvent A to solvent B (80 min.)>Solvent B (20 min.) [0212]
Detection: absorbance at 195 nm [0213] Amount of sample applied: 2
ml
[0214] The peaks at the retention time 66.7 min., 78.5 min. and
85.5 min. were fractionated and collected and they were subjected
to mass spectrometry. As a result, these substances were
agarobiose, agarotetraose and agarohexaose, respectively. The
separation with HPLC as described above was repeated 8 times and
the fractions thus separated were evaporated to dryness under
reduced pressure to obtain 122 mg of agarobiose, 111 mg of
agarotetraose, and 55 mg of agarohexaose, respectively.
[0215] The results are shown in FIGS. 7 to 10. That is, FIG. 7
illustrates the elution pattern of agarobiose, agarotetraose and
agarohexaose in the normal phase HPLC. The horizontal axis
represents the retention time (min.) and the vertical axis
represents the absorbance at 195 nm. FIG. 8 illustrates the mass
spectrum of the peak at 66.7 min. The horizontal axis represents
the m/z value and the vertical axis represents the relative
intensity (%). FIG. 9 illustrates the mass spectrum of the peak at
78.5 min. The horizontal axis represents the m/z value and the
vertical axis represents the relative intensity (%). FIG. 10
illustrates the mass spectrum of the peak at 88.5 min. The
horizontal axis represents the m/z value and the vertical axis
represents the relative intensity (%).
[0216] (2) To 450 .mu.l of 100 mM aqueous agarobiose solution
obtained in Example 3-(1) were added 50 .mu.l of 10-fold
concentrated phosphate buffered saline (T900, manufactured by
Takara Shuzo) and 50 .mu.l of 10 units/.mu.l of
.beta.-galactosidase (G5635, manufactured by Sigma) in phosphate
buffered saline. The resultant mixture was incubated at 37.degree.
C. for 1 hours.
[0217] To the reaction mixture was added 5 ml of a mixture of
1-butanol:ethanol=1:1 and then insoluble materials were removed by
centrifugation. The resultant solution was applied on silica gel
BW-300SP for column chromatography (3.times.50 cm, manufactured by
Fuji Silysia Chemical Ltd.) and separated using
1-butanol:ethanol:water=5:5:1 as the eluent with pressurizing at
0.3 kg/cm.sup.2 with a compressor.
[0218] Fractionation was carried out to collect 7 ml fractions, and
a portion of each fraction was taken up and analyzed with thin
layer chromatography. As a result, Fraction Nos. 14 to 17 contained
3,6-anhydro-L-galactose with high purity. These fractions were
combined and evaporated to dryness under reduced pressure to obtain
3.8 mg of 3,6-anhydro-L-galactose. The structure of this substance
was confirmed by mass spectrometry and nuclear magnetic
resonance.
[0219] The results are shown in FIGS. 11 to 13. That is, FIG. 11
illustrates the mass spectrum of 3,6-anhydro-L-galactose. The
horizontal axis represents the m/z value and the vertical axis
represents the relative intensity (%). FIG. 12 illustrates the
.sup.1H-NMR spectrum of 3,6-anhydro-L-galactose in heavy water and
FIG. 13 illustrates the .sup.1H-NMR spectrum of
3,6-anhydro-L-galactose in heavy dimethyl sulfoxide solvent. In the
figures, the horizontal axes represent the chemical shift value,
and the vertical axes represent the signal intensity.
[0220] For 3,6-anhydro-L-galactose, .sup.1H-NMR spectrum in heavy
dimethyl sulfoxide solvent also showed the proton signal of
aldehyde at 9.60 ppm. This demonstrated that it was present as an
aldehyde whose ring was opened in a non-aqueous solvent.
Furthermore, from the .sup.1H-NMR spectrum in heavy water, it was
present as a hydrate of the aldehyde in an aqueous solution.
Example 4
[0221] (1) A 20 mM solution of 3,6-anhydro-L-galactose obtained in
Example 3 was diluted 2-, 4- and 8-folds with sterilized water and,
according to the same manner as that described in Example 2-(1), an
apoptosis-inducing activity and an anti-proliferation activity
against tumor cells of respective dilutions were measured. As a
result, in the group to which the 2-fold dilution of
3,6-anhydro-L-galactose was added (at the final concentration of 1
mM), apoptosis corpuscles were observed and the absorbance at 590
nm became less than one-half of that of the control group to which
water was added.
[0222] (2) A 50 mM solution of agarobiose, agarotetraose or
agarohexaose obtained in Example 3-(1) was sterilized by filtration
and diluted 2-, 4-, 8-, 16-, 32-, 64- and 128-folds with sterilized
water. According to the same manner as that described in Example
2-(1), an anti-proliferation activity against various cells of the
resultant dilutions was measured. The cells and culture media used
are shown in Tables 1 and 2. TABLE-US-00001 TABLE 1 Cells Medium
Human promyelocyctic leukemia RPMI 1640 medium (Nissui) HL-60 (ATCC
CCL 240) supplemented with 10% fetal bovine serum (Gibco) Human
peripheral lymphocyte the same as the above RPMI 1778 (ATCC CCL
156) Mouse monocyte DMEM medium supplemented RAW 264.7 (ATCC TIB
71) with 10% fetal bovine serum (Nissui) Human gastric cancer cell
RPMI 1640 medium MKN 45 (Riken gene bank, RCB supplemented with 10%
fetal 1001) bovine serum Human hepatoma cancer cell DMEM medium
supplemented HepG2 (ATCC HB 8065) with 10% fetal bovine serum Human
colonic adenocarcinoma McCoy's medium supplemented HT-29 (ATCC HTB
38) with 10% fetal bovine serum (BioWhittaker) Human colonic
adenocarcinoma the same as the above HCT116 (ATCC CCL-247)
Fibrosarcoma DMEM medium supplemented HT-1080 (ATCC CCL-121) with
10% fetal bovine serum (BioWhittaker)
[0223] TABLE-US-00002 TABLE 2 Cells Medium Glial blast cell DMEM
medium supplemented A-172 (ATCC CRL1620) with 10% fetal bovine
serum Human breast cancer DMEM medium supplemented MCF7 (ATCC
HTB-22) with 10% fetal bovine serum Human breast cancer RPMI 1640
medium T-47D (ATCC HTB-133) supplemented with 10% fetal bovine
serum Human bladder carcinoma McCoy's medium supplemented T24 (ATCC
HTB-4) with 10% fetal bovine serum Human cancer of the uterine DMEM
medium supplemented cervix cell with 10% fetal bovine serum HeLa S3
(ATCC CCL-22) Human lung cancer the same as the above A549 (ATCC
CCL-185) Human colonic adenocarcinoma RPMI 1640 medium WiDr (ATCC
CCL-218) supplemented with 10% fetal bovine serum
[0224] As a result, agarobiose, agarotetraose and agarohexaose
exhibited an antiproliferation activity against these cells. The
results are shown in Table 3.
[0225] The number in Table 3 represents the dilution rate of the
dilution added to the group whose absorbance at 590 nm was less
than one-half of that of the control group to which water was
added. The dilution rate 1 corresponds to the concentration of 5 mM
in the cell culture medium. Then, for agarobiose, agarotetraose and
agarohexaose, the concentrations required for 50% proliferation
inhibitory rate (IC.sub.50) are calculated based on the change in
the absorbance at 590 nm, and are shown in Table 4. TABLE-US-00003
TABLE 3 Cells Agarobiose Agarotetraose Agarohexaose HL-60 32 64 64
RPMI1788 64 128 128 RAW264.7 16 32 32 MKN45 8 16 16 HepG2 8 8 16
HT-29 8 16 32 HCT116 16 32 32 HT-1080 8 16 16 A-172 16 16 16 MCF7
16 16 16 T-47D 16 16 16 T24 8 8 8 HeLa S3 8 8 16 A549 8 8 16 WiDr 8
8 16
[0226] TABLE-US-00004 TABLE 4 Cells Agarobiose Agarotetraose
Agarohexaose HL-60 170 97 78 RPMI1788 44 28 22 RAW264.7 179 133 109
MKN45 344 196 166 HepG2 652 413 430 HT-29 622 208 144 HCT116 244
158 136 HT-1080 317 216 185 A-172 289 185 151 MCF7 274 276 238
T-47D 210 183 158 T24 352 399 365 HeLa S3 353 400 334 A549 570 494
279 WiDr 405 399 334
Example 5
[0227] A suspension of 5 g of commercially available agar in 50 ml
of 0.1 N HCl was heated at 100.degree. C. for 13 minutes. After
cooling to room temperature, the solution was neutralized to about
neutral pH with NaOH and 2 ml of the solution was applied to a
column (10.times.255 mm) packed with activated carbon (60-150 mesh,
079-21, manufactured by Nacalai Tesque) washed with water. The
column was washed with 200 ml of water and then eluted with each
200 ml of 2.5%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%,
27.5%, 30%, 35%, 40%, 45% and 50% aqueous ethanol in this
order.
[0228] Each eluted fraction was concentrated 10-folds under reduced
pressure and spotted on a silica gel sheet 60F.sub.254
(manufactured by Merck) and developed with
1-butanol:ethanol:water=5:5:1. Orcinol reagent [prepared by
dissolving 400 mg of orcinol monohydrate (manufactured by Nacalai
Tesque) in 22.8 ml of sulfuric acid and adding water thereto to
make the final volume up to 200 ml] was sprayed to observe the
resultant spots.
[0229] As a result, agarobiose with high purity was contained in
the fractions eluted with 5% and 7.5% aqueous ethanol;
agarotetraose with high purity was contained in the fractions
eluted with 15% and 17.5% aqueous ethanol; agarohexaose with high
purity was contained in the fractions eluted with 22.5% and 25%
aqueous ethanol; and agarooctaose with high purity was contained in
the fractions eluted with 27.5% and 30% aqueous ethanol.
Example 6
[0230] Agarobiose, agarotetraose, agarohexaose and agarooctaose
(hereinafter, sometimes, these oligosaccharaides are referred to as
agarooligosaccharides) obtained in Example 5 were dissolved in
water at a concentration of 2.5 mM or 1.25 mM separately, and were
sterilized by filtration. HL-60 cells were suspended in RPMI 1640
medium containing 10% fetal bovine serum at a concentration of
2.5.times.10.sup.5 cells/4.5 ml and to this suspension was added
0.5 ml of each of the oligosaccharide solutions. The resultant
mixture were incubated with 5% CO.sub.2 at 37.degree. C. for 24
hours or 48 hours. A part of the cell culture was taken up,
followed by addition of Trypan Blue thereto and observing under a
microscope to count the number of viable cells. As a result, the
number of viable cells in each group was decreased as compared with
the group to which water was added (control) and apoptosis
corpuscles were observed.
[0231] The results are shown in FIGS. 14 and 15. That is, FIG. 14
illustrates the relation between the incubation time and the number
of viable cells when HL-60 cells were cultured with addition of one
of the oligosaccharides at the final concentration of 250 .mu.M.
FIG. 15 illustrates the relation between the incubation time and
the number of viable cells when HL-60 cells were cultured with
addition of one of the oligosaccharides at the final concentration
of 125 .mu.M. In FIGS. 14 and 15, the horizontal axes represent the
incubation time (hrs.) and the vertical axes represent the number
of viable cells (.times.10.sup.5/5 ml). The closed circle
(.circle-solid.) represents the addition of water (control), the
open diamond (.diamond.) represents the addition of agarobiose, the
open circle (.largecircle.) represents the addition of
agarotetraose, the open triangle (.DELTA.) represents the addition
of agarohexaose and the open square (.quadrature.) represents the
addition of agarooctaose.
Example 7
[0232] (1) A suspension of 0.2 g of .kappa.-carrageenan
(manufactured by Sigma, C-1263) or .lamda.-carrageenan
(manufactured by Wako Pure Chemical Industries, Ltd., 038-14252) in
20 ml of 0.1 N HCl was heated at 95.degree. C. for 10 minutes.
After neutralizing with 1N NaOH, the resultant mixture was diluted
1.5-, 2.25-, 3.38- and 5.06-folds with water and an
anti-proliferation activity against HL-60 cells was measured
according to the same manner as that described in Example 2-(1). As
a result, in the groups to which the 1.5-, 2.25- and 3.38-fold
dilutions of heated .kappa.-carrageenan and the 1.5- and 2.25-fold
dilutions of heated .lamda.-carrageenan were added, the absorbance
at 590 nm was less than one-half of that of the control group to
which water was added, and apoptosis corpuscles were observed.
[0233] (2) Commercially available agar (Agar Noble), agarose L03
(manufactured by Takara Shuzo) and commercially available
bar-shaped agar were suspended in 1N HCl at a concentration of 1%,
respectively, and heated at 100.degree. C. for 15 minutes. After
cooling, the heated mixtures were neutralized with 1N NaOH and
diluted 2-, 4-, 8- and 16-folds with water, respectively. An
anti-proliferation activity against HL-60 cells of the resultant
dilutions were measured according to the same manner as that
described in Example 2-(1). As a result, in the groups to which the
2- to 8-fold dilutions of the acid decomposition products of agar
and agarose and the 2- and 4-fold dilutions of the acid
decomposition product of bar-shaped agar were added, the absorbance
at 590 nm was less than one-half of that of the control group to
which water was added, and apoptosis corpuscles were observed.
[0234] When each of the acid decomposition products was analyzed
with normal phase HPLC, agarooligosaccharaides such as agarobiose,
etc. were detected for all the decomposition products.
Example 8
[0235] (1) Commercially available agar was suspended in each of the
following aqueous acid solutions at a concentration of 1%.
[0236] 0.5 M, 1M or 2M citric acid; 0.1 M, 0.5 M, 1 M or 2 M nitric
acid; 0.1 M or 0.5 M sulfuric acid; 0.1 M, 0.5 M or 1 M phosphoric
acid; 0.1 M hydrochloric acid.
[0237] The resultant agar suspensions were heated with a microwave
oven until the agar was dissolved, followed by neutralization with
NaOH. The solutions were diluted with 2-, 4-, 8-, 16- or 32-folds
with distilled water. Then, an apoptosis-inducing activity and an
antiproliferation activity against HL-60 cells were measured
according to the same manner as that described in Example
2-(1).
[0238] As a result, agar heated in the above-listed various acids
induced apoptosis in HL-60 cells and inhibited cell proliferation.
The results are shown in Table 5. The number in Table 5 represents
the dilution rate of the dilution added to the group whose
absorbance at 590 nm was less than one half of that of the control
group to which water was added. In addition, the number in the
parentheses represents the dilution rate of a solution (prepared,
without adding agar, by neutralizing the highest concentration of
the acid used in this Example with NaOH and diluting the resulting
solution with distilled water) added to the group whose absorbance
at 590 nm was less than one-half of that of the control group to
which water was added. TABLE-US-00005 TABLE 5 0.1 M 0.5 M 1 M 2 M
Citric acid 4 8 16(8) Nitric acid 8 16 16(2) Sulfuric acid 8 16(2)
Phosphoric acid 4 8 16(1) Hydrochloric acid 16
[0239] (2) The substances obtained by heating agar in the acids in
Example 8-(1) were analyzed with normal phase HPLC as follows.
[0240] Column: PALPAK type S (4.6.times.250 mm, manufactured by
Takara Shuzo, CA8300) [0241] Solvent A: aqueous 90% acetonitrile
solution [0242] Solvent B: aqueous 50% acetonitrile solution [0243]
Flow rate: 1 ml/min. [0244] Elution: solvent A (10 min.)>linear
gradient from solvent A to solvent B (40 min.)>solvent B (10
min.) [0245] Detection: absorbance at 195 nm [0246] Column
temperature: 40.degree. C.
[0247] As a result, the samples heated in 0.5 M, 1 M and 2 M citric
acid, 0.1 M, 0.5 M, 1 M and 2 M nitric acid, 0.1 M and 0.5 M
sulfuric acid, 0.1 M, 0.5 M and 1 M phosphoric acid, and 0.1 M
hydrochloride acid contained agarooligosaccharides such as
agarobiose, etc.
[0248] The representative result is shown in FIG. 16. That is, FIG.
16 illustrates the normal phase HPLC elution pattern of agar heated
in 0.5 M phosphoric acid. In FIG. 16, the horizontal axis
represents the retention time (min.) and the vertical axis
represents the absorbance at 195 nm.
Example 9
[0249] A suspension of 5 g of commercially available agar (Ina agar
type S-7, manufactured by Ina Shokuhin Kogyo) in 45 ml of 20, 50 or
100 mM citric acid was heated at 95.degree. C. Samples were
obtained after heating for a period of time as described below.
[0250] For 20 mM citric acid, 310 min., 350 min., 380 min., 440
min. and 530 min.
[0251] For 50 mM citric acid, 100 min., 120 min., 140 min., 160
min., 180 min., 200 min., 220 min., 240 min., 260 min., 290 min.
and 320 min.
[0252] For 100 mM citric acid, 60 min., 70 min., 80 min., 90 min.,
100 min., 120 min., 140 min., 160 min., 180 min., 200 min., 220
min. and 240 min.
[0253] 1 .mu.l of 10-fold dilution of each sample was spotted on a
silica gel 60 sheet F.sub.254 (manufactured by Merck), developed
with 1-butanol:ethanol:water=5:5:1 and detected by orcinol-sulfurlc
acid method.
[0254] As a result, each sample contained agarooligosaccharides
such as agarobiose, agarotetraose and agarohexaose, etc.
[0255] For the samples treated with 20 mM citric acid, the
agarooligosaccharide content was increased by heating for as long
as 350 minutes and, thereafter, remained almost constant.
[0256] For the samples treated with 50 mM citric acid, the
agarooligosaccharide content was increased by heating for as long
as 200 minutes and, thereafter, remained almost constant.
[0257] For the samples treated with 100 mM citric acid, the
agarooligosaccharide content was increased by heating for as long
as 160 minutes and, thereafter, remained almost constant.
[0258] The final agarooligosaccharide content increased with the
increase in the concentration of citric acid.
[0259] Each sample was analyzed with the normal phase HPLC
according to the same manner as that described in Example 8-(2). As
a result, results consistent with those obtained by thin layer
chromatography were obtained. However, the sample treated with 100
mM citric acid contained more impurities than that treated with 50
mM citric acid, and the impurities increased with the increase in
the heating time.
Example 10
[0260] (1) Agarobiose prepared in Example 3-(1) was dissolved at a
concentration of 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM or
1 mM in water. One microliter of each sample was spotted on a
silica gel 60 sheet F.sub.254, developed three times with
chloroform:methanol:acetic acid=7:2:2 and color-developed by
orcinol-sulfuric acid method. Image data of the color-developed
sheet was obtained using FOTODYNE FOTO/Analyst Archiver Ecripse
(sold by Central Kagaku Bouekisha). The image data was
image-processed using an image analysis software 1-D Basic
(manufactured by Advanced American Biotechnology) and the intensity
of the agarobiose spot at each concentration was converted to a
numerical value to prepare a calibration curve.
[0261] A graph of the calibration curve is shown in FIG. 17. That
is, FIG. 17 illustrates the calibration curve for agarobiose, and
the graph was prepared by plotting each agarobiose concentration
versus the intensity of each spot. In FIG. 17, the horizontal axis
represents the agarobiose concentration (mM) and the vertical axis
represents the intensity of spot. The equation in FIG. 17
represents the relation of the intensity of spot (y) and the
agarobiose concentration (x). For a sample whose agarobiose
concentration is unknown, the agarobiose concentration can be
calculate from the equation by determining the intensity of
spot.
[0262] Likewise, calibration curves for agarotetraose, agarohexaose
and agarooctaose obtained in Example 5 were prepared.
[0263] (2) A suspension of 0.2 g of commercially available agar
(Agar Noble) in 90 ml of water was heated with a microwave oven and
cooled to about room temperature. To this resultant solution was
added 10 ml of 1 M HCl or 1 M citric acid to prepare 0.2% agar
solution in 0.1 M HCl or 0.2% agar solution in 0.1 M citric acid.
The solution was heated at 90.degree. C. and samples were obtained
at 5 min., 10 min., 20 min., 30 min., 1 hour, 2 hours, 4 hours, 8
hours and 21 hours after initiation of heating. Each sample was
subjected to the thin layer chromatography according to the same
manner as that described in Example 10-(1) and the intensity of
spot was determined to calculate the agarobiose concentration. Each
sample was appropriately diluted to make the agarobiose
concentration within a range between 0.05 mM and 1 mM.
[0264] In FIGS. 18 and 19, the relation between the heating time
and the amount of agarobiose formed in 0.2% agar solution in 0.1 M
HCl and 0.2% agar solution in 0.1 M citric acid. That is, FIG. 18
illustrates the relation between the heating time and the amount of
agarobiose formed in 0.2% agar solution in 0.1 M HCl. FIG. 19
illustrates the relation between the heating time and the amount of
agarobiose formed in 0.2% agar solution in 0.1 M citric acid. In
FIG. 18 and FIG. 19, each horizontal axis represents the heating
time (hrs.) and the vertical axis represents the agarobiose
concentration (mM).
[0265] As shown in FIG. 18, in 0.2% agar solution in 0.1 M HCl, the
agarobiose concentration reached the maximum by heating for one
hour and reduced thereafter. And, as shown in FIG. 19, in 0.2% agar
solution in 0.1 M citric acid, the agarobiose concentration
increased gradually by heating as long as 8 hours and reduction was
observed at 21 hours. The agarobiose concentration in a sample
obtained by heating 0.2% agar solution in 0.1 M HCl for 5 minutes,
or by heating 0.2% agar solution in 0.1 M citric acid for 5, 10, or
20 minutes was below the detectable limitation.
[0266] (3) Agar Noble was suspended in 5, 50 and 500 mM citric acid
at a concentration of 10%, heated at 65, 80 or 95.degree. C.
Samples were obtained at 30 min., 1, 2, 4, 8 or 24 hours after the
initiation of heating and, according to the same manner as that
described in Example 10-(1), the amount of agarooligosaccharides
formed was measured.
[0267] As a result, when agar was dissolved in 5 mM citric acid,
although small amounts of agarooligosaccharides were formed at
80.degree. C., they were scarcely formed at 65.degree. C. At
95.degree. C., a large amount of agarobiose was formed by heating
for 8 to 24 hours, and a large amount of agarotetraose was also
formed by heating for 8 to 24 hours. When agar was dissolved in 50
mM citric acid, agarooligosaccharides were scarcely formed at
65.degree. C. At 80.degree. C., a large amount of agarobiose was
formed by heating for 24 hours, and large amounts of agarotetraose,
agarohexaose and agarooctaose were formed by heating for 4 to 8
hours. At 95.degree. C., a large amount of agarobiose was formed by
heating for 24 hours, and large amounts of agarotetraose,
agarohexaose and agarooctaose were formed by heating for 4 to 8
hours. When agar was dissolved in 500 mM citric acid, small amounts
of agarooligosaccharides were formed by heating at 65.degree. C.
for 4 to 24 hours. At 80.degree. C., a large amount of agarobiose
was formed by heating for 2 to 24 hours, and large amounts of
agarotetraose and agarohexaose were formed by heating for 1 to 6
hours. A large amount of agarooctaose was formed by heating for 1
to 2 hours. At 95.degree. C., a large amount of agarobiose was
formed by heating for 1 to 24 hours, and a large amount of
agarotetraose was formed by heating for 1 to 2 hours. Large amounts
of agarohexaose and agarooctaose were formed by heating for 30
minutes to 1 hour.
[0268] Examples of hydrolysis by 500 mM citric acid are shown in
FIGS. 20 and 21. That is, FIG. 20 illustrates agarooligosaccharide
formation in 500 mM citric acid by heating at 80.degree. C. In FIG.
20, the vertical axis represent the amounts of
agarooligosaccharides formed (open circle: agarobiose, open
triangle: agarotetraose, open square: agarohexaose, symbol x:
agarooctaose) and the horizontal axis represents the time. FIG. 21
illustrates the amounts of agarooligosaccharide formation in 500 mM
citric acid by heating at 95.degree. C. In FIG. 21, the vertical
axis represents the amounts of agarooligosaccharides formed (open
circle: agarobiose, open triangle: agarotetraose, open square:
agarohexaose, symbol x: agarooctaose) and the horizontal axis
represents the time.
[0269] (4) According to the same manner as that described in
Example 10-(3), agarooligosaccharide formation in 50, 500 or 1000
mM acetic acid was measured.
[0270] As a result, when agar was dissolved in 50 mM acetic acid,
small amounts of agarooligosaccharides were formed at 80.degree.
C., while agarooligosaccharides were scarcely formed at 65.degree.
C. When agar was dissolved in 500 mM acetic acid,
agarooligosaccharides were scarcely formed at 65.degree. C. At
80.degree. C. and 95.degree. C., small amounts of
agarooligoshaccharides were formed. When agar was dissolved in 1000
mM acetic acid, agarooligoshaccharides were scarcely formed at
65.degree. C. At 80.degree. C., a large amount of agarobiose was
formed by heating for 24 hours, and small amounts of agarotetraose,
agarohexaose and agarooctaose were formed by heating for 8 hours.
At 95.degree. C., a large amount of agarobiose formed by heating
for 8 hors, and large amounts agarotetraose, agarohexaose and
agarooctaose were also formed by heating for 8 hours.
[0271] (5) According to the same manner as that described in
Example 10-(3), agarooligosaccharide formation in 60, 600 or 1200
mM lactic acid was measured.
[0272] As a result, when agar was dissolved in 60 mM lactic acid,
small amounts of agarooligosaccharides were formed at 95.degree.
C., while agrooligosaccharides were scarcely formed at 65 and
80.degree. C. When agar was dissolved in 600 mM lactic acid, large
amounts of agarobiose were formed by heating at 80.degree. C. for 8
to 24 hours, and large amounts of agarotetraose and agarohexaose
were formed by heating for 4 to 8 hours. A large amount of
agarooctaose was formed by heating for 4 hours. At 95.degree. C., a
large amount of agarobiose was formed by heating for 4 to 8 hours,
and large amounts of agarotetoraose and agarohexaose were formed by
heating for 2 to 6 hours. When agar was dissolved in 1200 mM lactic
acid, a large amount of agarobiose was formed by heating at
80.degree. C. for 4 to 24 hours, and large amounts of
agarotetoraose and agarohexaose were formed by heating for 2 to 6
hours. A large amount of agarooctose was formed by heating for 2
hours. At 95.degree. C., a large amount of agarobiose was formed by
heating for 2 to 8 hours, and large amounts of agarotetraose and
agarohexaose were formed by heating for 1 to 2 hours.
[0273] Examples of hydrolysis by 1200 mM lactic acid are shown in
FIGS. 22 and 23. That is, FIG. 22 illustrates agarooligosaccharide
formation in 1200 mM lactic acid by heating at 80.degree. C. In
FIG. 22, the vertical axis represents the amounts of
agarooligosaccharides formed (open circle: agarobiose, open
triangle: agarotetraose, open square: agarohexaose, symbol x:
agarooctaose) and the horizontal axis represents the time. FIG. 23
illustrates agarooligosaccharide formation in 1200 mM citric acid
by heating at 95.degree. C. In FIG. 23, the vertical axis
represents the amounts of agarooligosaccharides formed (open
circle: agarobiose, open triangle: agarotetraose, open square:
agarohexaose, symbol x: agarooctaose) and the horizontal axis
represents the time.
[0274] (6) According to the same manner as that described in
Example 10-(3), agarooligosaccharide formation in 20, 200, or 1000
mM malic acid was measured.
[0275] As a result, when agar was dissolved in 20 mM malic acid,
small amounts of agarooligosaccharides were formed at 95.degree.
C., but agarooligosaccharides were scarcely formed at 65.degree. C.
and 80.degree. C. When agar was dissolved in 200 mM malic acid,
small amounts of agarooligosaccharides were formed by heating at
65.degree. C. for 24 hours. At 80.degree. C., a large amount of
agarobiose was formed by heating for 8 to 24 hours, and a large
amount of agarotetoraose was formed by heating for 4 to 8 hours. A
large amount of agarohexaose was formed by heating for 4 hours. A
large amount of agarooctaose was formed by heating for 4 hours. At
95.degree. C., a large amount of agarobiose was formed by heating
for 4 to 8 hours, and a large amount of agarotetraose was formed by
heating for 4 hours. When agar was dissolved in 1000 mM malic acid,
at 65.degree. C., small amounts of agarooligosaccharides were
formed by heating for 24 hours. At 80.degree. C., a large amount of
agarobiose was formed by heating for 2 to 24 hours, and a large
amount of agarotetraose was formed by heating for 2 to 6 hours.
Large amounts of agarohexaose and agarooctaose were formed by
heating for 2 hours. At 95.degree. C., a large amount of agarobiose
was formed by heating for 1 to 8 hours, and a large amount of
agarotetoraose was formed by heating for 1 to 2 hours. Large
amounts of agarohexaose and agarooctaose were formed by heating for
at 1 hour.
[0276] Examples of hydrolysis by 1000 mM malic acid are shown in
FIGS. 24 and 25. That is, FIG. 24 illustrates agarooligosaccharides
formation in 1000 mM malic acid by heating at 80.degree. C. In FIG.
24, the vertical axis represents the amounts of
agarooligosaccharides formed (open circle: agarobiose, open
triangle: agarotetraose, open square: agarohexaose, symbol x:
agarooctaose) and the horizontal axis represents the time. FIG. 25
illustrates agarooligosaccharide formation in 1000 mM malic acid by
heating at 95.degree. C. In FIG. 25, the vertical axis represents
the amounts of agarooligosaccharides formed (open circle:
agarobiose, open triangle: agarotetraose, open square:
agarohexaose, symbol x: agarooctaose) and the horizontal axis
represents time.
[0277] (7) Noble agar was suspended in 100, 200, 300, 400, 500,
600, 700, 800, 900 or 1000 mM malic acid at a concentration of 10%
and the suspensions were heated at 70, 80 or 90.degree. C. Samples
were obtained at 30 min., 1, 2, 3, 4, 8, or 24 hours after
initiation of heating and, according to the same manner as that
described in Example 10-(3), agarooligosaccharide formation was
measured.
[0278] At the malic acid concentration of 300 mM or more, large
amounts of agarooligosaccharides were formed even by heating at
70.degree. C. for 8 hours or longer. Examples of hydrolysis in 1000
mM malic acid at 70.degree. C. are shown in FIG. 26. That is, FIG.
26 illustrates agarooligosaccharides formation in 1000 mM malic
acid by heating at 70.degree. C. In FIG. 26, the vertical axis
represents the amounts of agarooligosaccharides formed (open
circle: agarobiose, open triangle: agarotetraose) and the
horizontal axis represents the time.
[0279] Based on the results of Example 10-(3) to (7) as described
above, agarooligosaccharides are preferably produced by using an
acid such as citric acid, lactic acid or malic acid at a
concentration of several ten mM to several M and heating at 70 to
95.degree. C. for several ten minutes to 24 hours.
[0280] (8) Agarobiose was determined using F-kit lactose/galactose
(manufactured by Boehringer Mannheim, code 176303). In the
above-mentioned method, agarobiose was determined by measuring the
concentration of galactose generated from agarobiose by the action
of .beta.-galactosidase in F-kit.
[0281] The determination was carried out according to the
instructions attached to the kit except that .beta.-galactosidase
was reacted at 37.degree. C. for 1 hours. A calibration curve was
prepared using lactose. A molar concentration (mM) was calculated
in terms of lactose, which was then converted to agarobiose
concentration (mg/ml).
[0282] According to the above method, the determination of
agarobiose, agarotetraose, agarohexaose and agarooctaose prepared
in the above-mentioned Example was tried. As a result, for
agarobiose, the calculated value agreed with the actually
determined value. On the other hand, agarotetraose, agarohexaose
and agarooctaose were not substantially detected by the
above-mentioned method. Namely, in practice, it was found that
agarooligosaccharides except agarobiose were not detected by the
above-mentioned method and that the agarobiose concentration in
agarooligosaccharides can be measured using the above-mentioned
method.
[0283] (9) A mixture of 100 g of commercially available agar (Ina
agar type S-7: manufactured by Ina Shokuhin Kogyo) and 10 g of
H-type strong cation exchange resin (Diaion SK104H: manufactured by
Mitsubishi Chemical) was prepared by mixing them in 900 g of
desalted water at 95.degree. C. The mixture was stirred at
95.degree. C. for 180 minutes to carry out acid decomposition of
agar. Then, the resulting mixture was cooled to room temperature,
filtrated by body feed of 1% w/w of activated carbon and 0.5% w/w
of Celite 545 (manufactured by Celite) to obtain a filtrate.
[0284] According to the same manner as that described in Example
8-(2), the filtrate obtained was analyzed by normal phase HPLC to
confirm that agarobiose, agarotetraose, agarohexaose and
agarooctaose were mainly formed as agrooligosaccharides.
[0285] The filtrate was at pH 2.4, and it had the acidity of 1.7,
the brix of 9.4% and the agarobiose content of 7.4% as measured by
using F-kit lactose/galactose described in Example 10-(8).
[0286] (10) To 100 g of desalted water was added 18.5 g of H-type
strong cation exchange resin (Daiyaion SK104H) and the mixture was
stirred at 95.degree. C. At 10 minutes intervals, 10 g of agar (Ina
agar type S-7) was added 5 times, then 15 g of agar was added 2
times, 20 g of agar was added 3 times, and finally 30 g of agar was
added. After adding a total of 185 g of agar, the mixture was
stirred at 95.degree. C. for 150 minutes and then cooled to room
temperature. The resultant mixture was decanted to separate the
resin from the liquid phase. Then, the separated liquid phase was
filtrated by body feed of 3% w/w of activated carbon and 0.5% w/w
of Celite 545 (manufactured by Celite) to obtain a filtrate.
[0287] According to the same manner as that described in Example
8-(2), the filtrate was analyzed by normal phase HPLC to confirm
that agarobiose, agarotetraose, agarohexaose and agarooctaose were
mainly formed as agarooligosaccharides.
[0288] The filtrate was at pH 1.2 and it had the acidity of 11.9,
the brix of 64% and the agarobiose content of 24.4% as measured by
using F-kit lactose/galactose described in Example 10-(8).
[0289] (11) Liquefaction of agar was carried out by preparing a
suspension containing 100 g of commercially available agar (Ina
agar type S-7) in deionized water having various phosphoric acid
concentrations added to a volume of 1 liter, and stirring the
resultant suspension at 95.degree. C.
[0290] According to the same manner as that described with respect
to the suspension containing phosphoric acid, liquefaction of agar
was carried our by preparing a suspension containing agar in
deionized water containing 1% w/v citric acid added to a volume of
1 liter. The term "liquefaction" used herein means a state in which
gelation does not take place even at a freezing point. Time
required for achieving such a state (liquefaction time) was
measured. In addition, agarobiose contents upon liquefaction and
thereafter were measured by using F-kit lactose/galactose described
in Example 10-(8). The results are shown in Tables 6 and 7.
TABLE-US-00006 TABLE 6 Liquefac- Phosphate tion Time held conc.
time at 95.degree. C. Agarobiose (% w/v) (min.) (min.) (g/l) 0.2
150 150 2.57 180 3.80 300 7.97 0.3 120 120 4.88 180 7.07 300 13.90
0.5 110 110 6.09 180 8.48 300 21.40 1.0 90 90 7.58 120 18.80
[0291] TABLE-US-00007 TABLE 7 Liquefac- Citric acid tion Time held
concentration time at 95.degree. C. Agarobiose (% W/V) (min.)
(min.) (g/liter) 1.0 90 90 0.95 120 3.40 150 4.09 300 5.70 360
14.80
Example 11
[0292] (1) A mixture of 150 g of commercially available agar (Ina
agar type S-7, manufactured by Ina Shokuhin Kogyo) and 15 g of
citric acid (anhydrate) for food additives (manufactured by San-Ei
Gen F.F.I. was made up to 1.5 liter with deionized water. The
mixture was warmed to 92.degree. C. and then held at 92-95.degree.
C. for 130 minutes with stirring. Then, the mixture was cooled to
room temperature and filtrate by body feed of 0.5% of Celite 545
(manufactured by Celite) to obtain a filtrate (agar decomposition
oligosaccharide solution). According to the same manner as that
described in Example 8-(2), the filtrate obtained was analyzed by
normal phase HPLC to confirm that agarobiose, agarotetraose,
agarohexaose and agarooctaose were mainly formed as saccharaide
compounds.
[0293] The filtrate was at about pH 2.6 and it had the acidity of
0.92, the brix of 9.2% and the agarobiose content of 43.1 mM as
measured by the method described in Example 10-(8).
[0294] (2) The filtrate (agar decomposition oligosaccharide
solution) prepared in Example 11-(1) was diluted 20-folds and to
this were added acidulant, sweetener and flavor to prepare soft
drinks containing 2.25 mM agarobiose.
[0295] The formulations are shown in Tables 8 and 9. Table 8 shows
the formulation of a grapefruit soft drink and Table 9 shows the
formulation of perilla flavored soft drink.
[0296] The components shown in each table were added to water and
dissolved to prepare the soft drink, and the soft drink was
distributed in 200 ml cans. The soft drink shown in Table 8 was
carbonated to prepare a carbonated drink whose gas pressure was 0.8
kg/cm.sup.2 (20.degree. C.).
[0297] The analytical values for each drink are shown in the lower
columns of Tables 8 and 9. TABLE-US-00008 TABLE 8 Agar
decomposition oligo- 50 ml saccharide solution 1/7 grapefruit 20 g
Vitamin C 0.2 g Citric acid 0.2 g Maltose 1.25 g Grapefruit flavor
1 g Desalted water rest Total 1000 ml pH 3.2 Acidity* 0.23 Brix 2.2
Acidity*: 0.1 N NaOH ml/10 ml (hereinafter the same)
[0298] TABLE-US-00009 TABLE 9 Agar decomposition oligo- 50 ml
saccharide solution Perilla extract 20 g Vitamin C 0.2 g Citric
acid 0.2 g Perilla flavor 0.5 g Desalted water rest Total 1000 ml
pH 2.9 Acidity* 0.10 Brix 0.6
[0299] Each carbonated drink of the present invention was assessed
by 10 panelists in a sensory test which scores in five grades (5:
good, 1: bad). As a control, a soft drink prepared by using an
aqueous citric acid solution having the same acidity instead of the
agar decomposition oligosaccharide solution was used.
[0300] The average scores obtained in the sensory test for the
grapefruit tasted ones and those for the perilla flavored ones are
shown in Tables 10 and 11. TABLE-US-00010 TABLE 10 Product of the
present invention Control Texture mildness 4.6 3.1 smoothness 4.8
3.0 Flavor balance 4.5 2.9 General 4.6 3.1 assessment
[0301] TABLE-US-00011 TABLE 11 Product of the present invention
Control Texture mildness 4.5 2.5 smoothness 4.3 2.4 Flavor balance
4.2 2.5 General 4.3 2.4 assessment
[0302] As compared with the control, the products of the present
invention were assessed to have better flavor balance as well as
milder and smoother texture. Thus, the products are drinks having
novel tastes. Likewise, the soft drinks without carbonation of the
present invention had novel tastes.
[0303] (3) Ethyl alcohol was added to each of the soft drinks
described in Tables 8 and 9 at ethyl alcohol concentrations of 6%
v/v or 8% v/v and the resulting mixtures were distributed in 200 ml
cans. The alcohol drinks were carbonated to prepare the carbonated
alcohol drinks of the present invention in which gas pressure was
0.8 kg/cm.sup.2 (20.degree. C.).
[0304] As compared with the control which did not contain the agar
decomposition oligosaccharide solution, the carbonated alcohol
drinks of the present invention were to have better flavor balance
as well as milder and smoother texture. Thus, the carbonated
alcohol drinks of the present invention had novel tastes.
Example 12
[0305] (1) A drink containing an agar decomposition product
decomposed by citric acid was prepared as follows. The formulation
is shown in Table 12. Namely, for Product 1 of the present
invention in Table 12, 0.1% w/v of the filtrate prepared in Example
11-(1) (agar oligosaccharide solution), 0.25% w/v of agar (Ultra
Agar AX-30: manufactured by Ina Shokuhin Kogyo) and 0.07% w/v of
citric acid were used. For Product 2 of the present invention,
0.25% w/v of agar and 0.08% w/v of citric acid were used without
addition of the agar oligosaccharide solution. In either case of
Products 1 and 2 of the present invention, the drinks containing
the agar decomposition products decomposed by citric acid were
prepared by dissolving agar with hot water, mixing it with the
other components and heating under acidic conditions at 93.degree.
C. for 10 seconds, at 93-80.degree. C. for 20 minutes and at
80-75.degree. C. for 15 minutes. On the other hand, a control was
prepared according to the same formulation as that of Product 2 of
the present invention except that heating was carried out at
93.degree. C. for 10 seconds.
[0306] Each drink with thickness containing the agar decomposition
product decomposed by citric acid was assessed by 10 panelists in a
sensory test which scores in five grades (5: good, 1: bad). The
mean scores obtained in the sensory test are shown in Table 13.
TABLE-US-00012 TABLE 12 Product 1 Product 2 Agar (g) 2.5 2.5 Agar
oligosaccharide solution 1.0 0 (ml) 1.5 1.5 1/7 grapefruit juice
(g) 66.0 66.0 Granulated sugar (g) 0.7 0.8 Citric acid (g) 0.5 0.5
Sodium citrate (g) 2.0 2.0 Flavor (g) rest rest Desalted water
Total 1000 ml 1000 ml pH 3.68 3.67 Acidity* 1.53 1.57 Brix 7.5 7.4
Agarobiose (mM)** 0.06 0.02 Agarobiose (mM)** was measured by the
method described in Example 10-(8).
[0307] TABLE-US-00013 TABLE 13 Product Product 1 2 Control Texture
mildness 4.4 4.1 3.6 smoothness 4.5 4.3 3.8 Flavor balance 4.4 4.2
3.6 General 4.5 4.3 3.7 assessment
[0308] As compared with the control, Products 1 and 2 of the
present invention were assessed to have better flavor balance,
suitable thickness as well as milder and smoother texture. Thus,
the products were drinks with novel tastes. The formation of an
oligosaccharide for an antioxidant, agarobiose, by heat treatment
in the presence of citric acid added in Products 1 and 2 of the
present invention was recognized. Thus, the novel drinks containing
an oligosaccharides for an antioxidant were provided.
[0309] According to the same manner as that described in Example
10-(8), a mounts of agarobiose formed were measured using heating
conditions at 75.degree. C. for 1 day; at 85.degree. C. for 5
minutes; at 103.degree. C. for 5 minutes; or at 122.degree. C. for
45 seconds in stead of 80-75.degree. C. for 15 minutes. As a
result, it was confirmed that the amounts of agarobiose formed were
0.03 mM, 0.02 mM, 0.04 mM and 0.05 mM, respectively, that
agarobiose was formed by heat treatment, and that the more severe
the heating conditions became, the more agarobiose was formed.
[0310] (2) Ethyl alcohol was added to Product 1 and Product 2 of
the present invention, and the control described in Example 12-(1)
at ethyl alcohol concentration of 2% v/v or 4% v/v. The total
volume was adjusted by reducing the volume of desalted water which
corresponds to that of ethyl alcohol added. Thus, alcohol drinks
were prepared.
[0311] As compared with the control, the alcohol drinks
corresponding to Products 1 and 2 were assessed to have better
flavor balance, suitable thickness as well as milder and smoother
texture. Thus, the products were drinks having novel taste.
[0312] Additionally, frozen products of the above-described alcohol
drinks exhibited good sherbet-like texture.
Example 13
[0313] (1) Commercially available agar (Agar Noble) was suspended
in 0.1 N hydrochloric acid at a concentration of 1% and the
suspension was treated at 37.degree. C. for 5 hours, 16 hours or 48
hours. The suspension thus treated was diluted 10-folds with
distilled water and analyzed with thin layer chromatography as
described in Example 9. As a result, the formation of small amounts
of agarooligosaccharides was observed by the treatment for 5 hours.
The amounts thereof were increased by the treatment for 16 hours
and were further increased by the treatment for 48 hours.
[0314] (2) Commercially available agar (Agar Noble) was suspended
in a phosphate buffered saline or distilled water at a
concentration of 1% and the suspension was heated at 121.degree. C.
for 4 hours. The suspension thus heated was diluted 10-folds with
distilled water and analyzed with thin layer chromatography as
described in Example 9. As a result, the formation of trace amounts
of agarooligosaccharides was observed in the sample heat-treated in
the phosphate buffered saline. In the sample heat-treated in
distilled water, the formation of clearly more amounts of
agarooligosaccharides was observed. The former was at about pH 7
after the heat treatment, while the latter was at about pH 5. After
cooling to room temperature, the former gelated but the latter did
not.
Example 14
[0315] (1) Agar (Agar Noble) was suspended in 0.1N HCl at a
concentration of 10% and heated at 100.degree. C. for 19 minutes.
TOYOPEARL HW40C (manufactured by Toso) column (4.4 cm.times.85 cm)
was equilibrated with water and 10 ml of the above-mentioned sample
was applied to this column. Gel filtration chromatography was
carried out using water as a mobile phase at a flow rate of 1.4
ml/min. The eluted substances were detected using a differential
refractometer and each 7 ml fraction was collected.
[0316] Peaks were recognized at elution time 406, 435, 471 and 524
minutes. The analysis of the fractions corresponding to respective
peaks with thin layer chromatography as described in Example 9
demonstrated that these were agarooctaose, agarohexaose,
agarotetraose and agarobiose in this order. The fractions were
lyophilized to obtain 30 mg agarooctaose, 100 mg agarohexaose, 150
mg agarotetraose and 140 mg agarobiose.
[0317] (2) Agarobiose and agarohexaose obtained in Example 14-(1)
were dissolved in water to prepare 100 mM aqueous solutions
thereof. To each 25 .mu.l of these solutions was added 50 .mu.l of
100 mM aqueous L-cysteine solution, followed by addition of 925
.mu.l of phosphate buffered saline. Then, the mixture was treated
at 37.degree. C. for 1 hour or 16 hours. The same reaction was
repeated except that an aqueous solution containing the same
concentration of L-lysine was used instead of the aqueous
L-cysteine solution.
[0318] 1 .mu.l of a sample from each reaction was spotted on a
silica gel sheet 60 F.sub.254, developed with
1-butanol:ethanol:water=5:5:1 and detected by orcinol-sulfate
method.
[0319] As a result, the spots of agarobiose and agarotetraose were
disappeared in the sample from the reaction for 1 hours with
L-cysteine. In the samples from the reaction for 16 hours with
L-cysteine and L-lysine, the spots of agarobiose and agarotetraose
were disappeared.
[0320] When each sample was analyzed with normal phase HPLC as
described in Example 8-(2), the results were consistent with those
obtained with the thin layer chromatography.
[0321] (3) According to the same manner as that described in
Example 2-(1), an antiproliferation activity against HL-60 cells
was measured by placing 10 .mu.l of each sample from the reaction
prepared in Example 14-(2) into a well of a 96 well microtiter
plate.
[0322] As a result, it was observed that the activities were
disappeared in the sample whose spots of agarobiose and
agarotetraose were disappeared. Namely, antiproliferation
activities in the samples from the reaction of agarobiose and
agarotetraose with L-cysteine for 1 hour and from the reaction of
agarobiose and agarotetraose with L-cysteine or L-lysine for 16
hours were reduced to about 1/10 relative to the same
concentrations of agarobiose and agarotetraose.
Example 15
[0323] (1) A suspension of 2.5 g of K-carrageenan (manufactured by
Sigma, C-1263) in 50 ml of 0.1 N HCl was heated at 100.degree. C.
for 16 minutes. The resultant solution was cooled to room
temperature, neutralized to about neutral pH with NaOH, filtrated
through Cosmonice filter and separated with normal phase HPLC as
follows. [0324] Column: PALPAK type S (4.6.times.250 mm,
manufactured by Takara Shuzo, CA8300) [0325] Solvent A: aqueous 90%
acetonitrile solution [0326] Solvent B: aqueous 50% acetonitrile
solution [0327] Flow rate: 1 ml/min. [0328] Elution: solvent A (10
minutes)>linear gradient from solvent A to solvent B (40
minutes)>solvent B (10 minutes) [0329] Detection: absorbance at
215 nm [0330] Column temperature: 40.degree. C. [0331] Amount of
sample applied: 50 .mu.l
[0332] The separation pattern of normal phase HPLC is shown in FIG.
27. That is, FIG. 27 illustrates normal phase HPLC chromatogram of
acid decomposition product of K-carrageenan. The horizontal axis
represents the retention time (min.) and the vertical axis
represents the absorbance at 215 nm.
[0333] Each elution peak was fractionated, collected, evaporated to
dryness under reduced pressure and dissolved in 100 .mu.l of water.
Each fraction was sterilized by filtration and, according to the
same manner as that described in Example 2-(1), an
antiproliferation activity against HL-60 cells was measured. As a
result, in groups to which the fractions from peaks at 27.797,
33.905 to 34.784, and 36.226 to 36.654 min. were added, apoptosis
corpuscles were observed. The absorbance at 590 nm thereof was
lower than that of the control group to which water was added, and
cell proliferation was inhibited.
[0334] The fraction from the peak at elution time 27.797 min. was
separated 12 times under the above-mentioned HPLC conditions and
the fractions were combined and evaporated to dryness under reduced
pressure to obtain an apoptosis-inducing and carcinostatic
substance.
[0335] (2) Mass spectrometry of the apoptosis-inducing and
carcinostatic substance described in Example 15-(1) was carried out
using DX302 mass spectrometer (manufactured by Nippon Denshi).
Glycerol was used as a matrix and the measurement was performed
with negative ion mode.
[0336] FAB-MS
[0337] m/z 403 [M-H].sup.-
[0338] 495 [M+glycerol-H].sup.-
[0339] The results are shown in FIG. 28. That is, FIG. 28
illustrates mass spectrum of the apoptosis-inducing and
carcinostatic substance. The horizontal axis represents the m/z
value and the vertical axis represents the relative intensity.
[0340] A nuclear magnetic resonance spectrum of the
apoptosis-inducing and carcinostatic substance obtained in Example
15-(1) was measured with JNM-A500 nuclear magnetic resonance
apparatus (manufactured by Nippon Denshi).
[0341] In FIG. 29, .sup.1H-NMR spectrum of the apoptosis-inducing
and carcinostatic substance is shown. In FIG. 29, the horizontal
axis represents the chemical shift value and the vertical axis
represents the signal intensity.
[0342] Based on these analytical results of mass spectrometry and
.sup.1H-NMR, the apoptosis-inducing and carcinostatic substance
described in Example 15-(1) was identified as .kappa.-carabiose
[.beta.-D-galactopyranosyl-4-sulfate-(1>4)-3,6-anhydro-D-galactose].
[0343] In view of the above, it has been found that the
apoptosis-inducing and carcinostatic substance obtained in Example
15-(1) is .kappa.-carabiose.
[0344] (3) .kappa.-Carabiose obtained in Example 15-(1) was
dissolved in water at a concentration of 1.56 mM, 10 .mu.l of the
solution was placed in a well of a 96 well microtiter plate and,
according to the same manner as that described in Example 2-(1), an
apoptosis-inducing activity and an antiproliferation activity were
measured. As a result, apoptosis corpuscles were observed under an
optical microscope and, as compared with a control group to which
water was added, cell proliferation in the group to which
.kappa.-carabiose was added was suppressed by about 70%. Therefore,
.kappa.-carabiose induced apoptosis in HL-60 cells and inhibited
cell proliferation at 156 .mu.M.
Example 16
[0345] (1) A suspension of 4.5 g of commercially available agar
powder (manufactured by Wako Pure Chemical Industries, Ltd.) in 150
ml of 0.1 N HCl was heated with a microwave oven. The resultant
solution was held on a boiling bath for 10 minutes. After heating,
the solution was allowed to cool to room temperature and insoluble
materials were removed by centrifugation. The supernatant was then
collected and adjusted to pH 6.8 with 1 N sodium hydroxide. To 150
ml of the supernatant was added the equal volume of ethyl acetate,
and the mixture was stirred vigorously and partitioned between the
ethyl acetate phase and the aqueous phase. The partitioned aqueous
phase was evaporated to dryness with an evaporator and the residue
was dissolved in 150 ml of water again. Insoluble materials were
removed by centrifugation to obtain a supernatant. The ethyl
acetate phase was evaporated to dryness with an evaporator,
dissolved in 100 ml of ion-exchanged water and adjusted to pH 6.5
with 1 N sodium hydroxide.
[0346] The ethyl acetate phase and the aqueous phase were
sterilized by filtration with a filter of 0.2 .mu.m pore size
(manufactured by Corning), diluted 10-, 20- and 30-folds with water
and, according to the same manner as that described in Example
2-(1), an antiproliferation activity against HL-60 cells was
measured. As a result, an antiproliferation activity was observed
in the aqueous phase, but was not in the ethyl acetate phase.
[0347] 50 ml of the aqueous phase solution thus prepared was
subjected to gel filtration with Cellulofine GCL-25 column
(41.times.905 mm). The eluent was 0.2 M NaCl containing 10%
ethanol. The elution pattern is shown in FIG. 30. That is, FIG. 30
illustrates the results of gel filtration with Cellulofine GCL-25
column. In FIG. 30, the vertical axis represents the saccharide
content in the eluate measured by phenol-sulfuric acid method
(absorbance at 490 nm: closed circle) and the horizontal axis
represents the fraction number (10 ml/fraction).
[0348] 1 .mu.l of each eluted fraction was spotted on a silica gel
sheet 60 F.sub.254 (manufactured by Merck) and developed with
1-butanol:acetic acid:water=4:1:2. Orcinol reagent [prepared by
dissolving 400 mg of orcinol monohydrate (manufactured by Wako Pure
Chemical Industries, Ltd.) in 22.8 ml of sulfuric acid and adding
thereto water to make the total volume up to 200 ml] was sprayed
and the sheet was heated on a hot plate heated at 150.degree. C. to
observe the spots.
[0349] Every 5 fractions of Fraction Nos. 40 to 120 whose spots
were confirmed by the above-mentioned TLC analysis were combined
and sterilized by filtration. Then, an antiproliferation activity
against HL-60 cells was measured. As a result, Fraction Nos. 86 to
90 had the strongest antiproliferation activity.
[0350] (2) Fraction Nos. 86 to 88 were recovered, and evaporated to
dryness with an evaporator to obtain 0.94 g of powder. The powder
obtained was dissolved in 30 ml of 90% ethanol, and then white
precipitate was removed using 5C filter (manufactured by ADVANTEC).
The resultant was subjected to gel filtration with Sephadex LH-20
column (35.times.650 mm). The eluent was 90% ethanol. The elution
pattern was shown in FIG. 31. That is, FIG. 31 illustrates the
results of gel filtration with Sephadex LH-20 column. In FIG. 31,
the vertical axis represents the saccharide content in the eluate
measured by phenol-sulfuric acid method (absorbance at 490 nm:
closed circle) and the horizontal axis represents the fraction
number (10 ml/fraction).
[0351] Each eluate fraction was analyzed with a silica gel sheet as
described above.
[0352] Components detected by TLC analysis were roughly divided
into five groups, i.e., Fraction Nos. 30 to 35, 36 to 40, 41 to 44,
45 to 48 and 49 to 53. For each group, 125 .mu.l portions from the
respective fractions of the group were combined and evaporated to
dryness with an evaporator. The residue was dissolved in 500 .mu.l
of ion-exchanged water and its antiproliferation activity against
HL-60 cells was measured.
[0353] Fraction Nos. 36 to 40 which had the strongest
antiproliferation activity against HL-60 cells were evaporated to
dryness with an evaporator, and dissolved in 5 ml of
1-butanol:acetic acid:water=4:1:2. The solution was applied to
column (20.times.710 mm) packed with silica gel 60 F.sub.254 and
washed with 1-butanol:acetic acid:water=4:1:2. As the eluent,
1-butanol:acetic acid:water=4:1:2 was used (3 ml/fraction).
[0354] Each eluted fraction was analyzed by using a silica gel
sheet as described above. As a result, components detected were
roughly divided into five groups as follows: Fraction Nos. 46 to 52
(group 1), 60 to 70 (group 2), 72 to 84 (group 3), 86 to 94 (group
4) and 96 to 120 (group 5).
[0355] Each group was evaporated to dryness with an evaporator,
dissolved in 5 ml of ion-exchanged water and filtrated through a
filter having pore size of 0.45 .mu.m (manufactured by IWAKI). An
antiproliferation activity against HL-60 cells of each solution
obtained was measured. As a result, an antiproliferation activity
was observed in the groups 3, 4 and 5.
[0356] Regarding the structure of the substance contained in the
groups 4 and 5, it was confirmed to be agarobiose by TLC analysis
and mass spectrometry.
[0357] As for the structure of the substance contained in the group
3, it was confirmed to be
.beta.-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose
by mass spectrometry and NMR analysis. FIG. 32 illustrates the mass
spectrum of
.beta.-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose.
The horizontal axis represents the m/z value and the vertical axis
represents the relative intensity (%). And, FIG. 33 illustrates the
.sup.1H-NMR spectrum of be
.beta.-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose.
The horizontal axis represents the chemical shift value (ppm) and
the vertical axis represents the signal intensity (%).
[0358] It was found that, like agarobiose,
.beta.-D-galactopyranosyl-(1>4)-3,6-anhydro-2-O-methyl-L-galactose
had a strong antiproliferation activity against HL-60 cells.
Example 17
[0359] (1) Inhibitory activity of the saccharides derived from agar
obtained in Example 3 against lipid peroxide radical production was
measured as follows.
[0360] Staphylococcus aureus 3A (National Collection Of Type
Culture, NCTC 8319) was inoculated into 5 ml of brain heart
infusion medium (manufactured by Difco, 0037-17-8) and cultured at
37.degree. C. overnight. The bacterial cells were collected by
centrifugation, washed with phosphate buffered saline 3 times and
then suspended in phosphate buffered saline at a concentration of
1.times.10.sup.7 colony forming units/ml. A mixture of 100 .mu.l of
the cell suspension, 100 .mu.l of an aqueous sample solution, 100
.mu.l of aqueous 1 mg/ml methemoglobin (manufactured by Sigma
M9250) solution, 600 .mu.l of phosphated buffered saline and 100
.mu.l of aqueous 50 mM tert-butyl hydroperoxide (manufactured by
Katayama Kagaku, 03-4990) solution were reacted at 37.degree. C.
for 30 minutes. To the reaction mixture was added 1 ml of
2.times.NMP medium [prepared by dissolving 8 g of nutrient broth
(manufactured by Difco, 0003-01-6), 5 g of trypton (manufactured by
Difco, 0123-17-3), 5 g of NaCl, 10 g of mannitol (manufactured by
Nacalai Tesque, 213-03) and 0.035 g of phenol red (manufactured by
Nacalai Tesque, 268-07) in distilled water to make the volume up to
500 ml. The pH was adjusted to 7.5 with NaOH and then the mixture
was sterilized by filtration] to stop the reaction. The resultant
mixture was diluted every 3-folds with NMP medium (prepared by
diluting 2.times.NMP medium 2-folds with sterilized water) to
prepare 12 serial dilutions and 160 .mu.l of each dilution was
placed in each well of a 96-well microtiter plate. The plate was
incubated at 37.degree. C. overnight. Color of the medium was
observed with the naked eye and the sample contained in a well in
which the color of the medium changed from red to yellow by growth
of the bacterium was identified as that having an activity of
inhibiting lipid peroxide radical production.
[0361] The results are shown in Table 14. In Table 14, + represents
the sample in which the growth of the bacterium was observed, and -
represents the sample in which the growth of the bacterium was not
observed. The concentration shown in the uppermost line of the
table is that of the sample in the reaction mixture in which the
sample was reacted with tert-butyl hydroperoxide and the bacterial
cells at 37.degree. C. for 30 minutes. TABLE-US-00014 TABLE 14 0.1
mM 1 mM Agarobiose + + Agarotetraose - + Galactose - -
[0362] As seen from the above results, a strong activity of
inhibiting lipid peroxide radical production was found in
agarobiose and agarotetraose. Similar activity was confirmed in
carabiose and 3,6-anhdro-2-O-methyl-L-galactose.
[0363] (2) A suspension of 5 g of commercially available agar (Ina
agar type S-7, manufactured by Ina Shokuhin Kogyo) in 45 ml of 50
mM citric acid was heated at 93.degree. C. for 155 minutes and
adjusted to pH 6 with NaOH to prepare a sample (citric acid treated
sample). Likewise, a suspension of the same agar in 45 ml of 100 mM
hydrochloric acid was heated at 95.degree. C. for 13 minutes and
adjusted to pH 6 with NaOH to prepare a sample (hydrochloric acid
treated sample). Both samples were diluted with water to give 1-,
2-, 4-, 8-, 10- and 100-fold dilutions and, according to the same
manner as that described in Example 17-(1), an activity of
inhibiting lipid peroxide radical production inhibitory activity
thereof was determined. As a result, in both of the citric acid
treated sample and the hydrochloric acid treated sample, the
activity was confirmed up to 10-fold dilutions and both had
equivalent activities of inhibiting lipid peroxide radical
production.
Example 18
[0364] Inhibitory activity of agarobiose against lymphocyte
blastgenesis induced by Concanavalin A (Con A)
[0365] A spleen was taken out from a ddY mouse (Nippon SLC; male, 7
weeks old), finely minced and suspended in RPMI-1640 medium (Gibco)
containing 10% fetal bovine serum (HyClone) to obtain a single cell
suspension. The cell suspension was seeded into a plastic Petri
dish, incubated at 37.degree. C. for 2 hours in a carbon dioxide
incubator. Adhesive cells adhered to the Petri dish were removed
and non-adhesive cells were used as spleen lymphocytes. 200 .mu.l
of 2.times.10.sup.6 cells/ml spleen lymphocytes suspension was
seeded into each well of 96 well microtiter plate. Agarobiose at
varying concentration was added to the wells other than the control
well. Furthermore, to all the wells was added 5 .mu.g of Con A
(Nacalai Tesque) and the plate was incubated at 37.degree. C. for
one day in a carbon dioxide incubator. After incubation, 1 .mu.Ci
of .sup.3H-thymidine was added to each well and incubation was
continued for additional one day. Then, its uptake into cells was
measured using a liquid scintillation counter.
[0366] The results are shown in FIG. 34. FIG. 34 illustrates the
relation between the agarobiose concentration and the
.sup.3H-thymidine uptake in lymphocyte blastgenesis induced by Con
A. The horizontal axis represents the agarobiose concentration and
the vertical axis represents the .sup.3H-thymidine uptake (cpm).
The open bar and the shaded bar represent the .sup.3H-thymidine
uptake without stimulation and with stimulation by Con A,
respectively. As seen from FIG. 34, agarobiose exhibits the
dose-dependent inhibitory activity against mouse lymphocyte
proliferation stimulated by mitogen, and almost completely inhibits
the proliferation at 100 .mu.g/ml. Thus, the inhibitory activity of
agarobiose against lymphocyte activation has been recognized. For
3,6-anhydrogalactopyranose, agarotetraose, agarohexaose,
agarooctaose, carabiose and 3,6-anhydro-2-O-methyl-L-galactose,
similar activities have also been recognized.
Example 19
Inhibitory Activity of Agarobiose Against Mixed Lymphocyte
Reaction
[0367] Spleens were taken out from a BALB/c mouse (Nippon SLC;
male, 6 weeks old) and a C57BL/6 mouse (Nippon SLC; male, 6 weeks
old) and spleen lymphocytes were obtained by the above-described
method. Each cell suspension was adjusted to a concentration of
2.times.10.sup.6 cell/ml, 100 .mu.l portions from respective
suspensions were mixed together and seeded in a 96 well microtiter
plate. Agarobiose at varying concentration was added to the wells
other than the control well, and the plate was incubated at
37.degree. C. for 4 days in a carbon dioxide incubator. After
incubation, 1 .mu.Ci of .sup.3H-thymidine was added to each well,
and the plate was incubated for additional 1 day. Its uptake into
cells was measured using a liquid scintillation counter.
[0368] The results are shown in FIG. 35. That is, FIG. 35
illustrates the relation between the agarobiose concentration and
the .sup.3H-thymidine uptake in the mixed lymphocyte reaction. The
horizontal axis represents the agarobiose concentration and the
vertical axis represents .sup.3H-thymidine uptake (cpm). The open
bar and the shaded bar represent .sup.3H-thymidine uptake in case
where cells from either one of the lines were used independently,
and in case where mixed cells from both of the lines were used,
respectively. As is seen from FIG. 35, agarobiose has the
dose-dependent inhibitory activity against lymphocytes activation
by stimulation with an alloantigen, and almost completely inhibits
the lymphocytes activation at 10 .mu.g/ml. Thus, the inhibitory
activity against lymphocyte activation of agarobiose has been
recognized. For 3,6-anhydrogalactopyranose, agarotetraose,
agarohexaose, agarooctaose, carabiose and
3,6-anhydro-2-O-methyl-L-galactose, similar activities have also
been recognized.
Example 20
[0369] (1) RAW 264.7 cells (ATCC TIB 71) were suspended in phenol
red-free Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum (manufactured by Gibco) and 2 mM L-glutamine
(manufactured by Life Technologies Oriental, 25030-149) at a
concentration of 3.times.10.sup.5 cells/ml, and 500 .mu.l portions
thereof were seeded to respective wells of a 48-well microtiter
plate and incubated at 37.degree. C. for 12 hours in the presence
of 5% CO.sub.2. To each well were added 10 .mu.l of 25 .mu.g/ml
lipopolysaccharide (LPS, manufactured by Sigma, L-2012) and 10
.mu.l of aqueous 5000, 1500, 500, 150 or 50 .mu.M agarobiose or
neoagarobiose (manufactured by Sigma, G4410) solution, and the
plate was incubated for additional 12 hours. Then, concentration of
NO.sub.2.sup.- produced by oxidation of NO in the medium was
measured. As control groups, a group to which LPS was not added and
a group to which agarobiose or neoagarobiose was not added were
provided.
[0370] After incubating as described above, 100 .mu.l of 4% Greece
reagent (manufactured by Sigma, G4410) was added to 100 .mu.l of
the medium, and the mixture was allowed to stand for 15 minutes at
room temperature. Then, the absorbance at 490 nm was measured.
NO.sub.2.sup.- concentration in the medium was calculated with
reference to a calibration curve prepared by using NaNO.sub.2 at
given concentrations dissolved in the same medium as that described
above. All the measurements were carried out in triplicate.
[0371] As a result, agarobiose dose-dependently inhibited NO
production induced by LPS, while neoagarobiose did not. The results
are shown in FIGS. 36 and 37. That is, FIG. 36 illustrates the
NO.sub.2.sup.- concentration in the medium incubated under the
respective incubation conditions with addition of agarobiose. FIG.
37 illustrates the NO.sub.2.sup.- concentration in the medium
incubated under respective incubation conditions with addition of
neoagarobiose. In FIGS. 36 and 37, the horizontal axes the
represent incubation conditions and the vertical axes represent the
NO.sub.2.sup.- concentration (.mu.M).
[0372] When 3,6-anhydrogalactopyranose, carabiose, agarotetraose,
agarohexaose, agarooctaose and 3,6-anhydro-2-O-methyl-L-galactose
were used instead of agarobiose, the similar results were
obtained.
[0373] (2) A suspension of 5 g of commercially available agar (Ina
agar type S-7, manufactured by Ina Shokuhin Kogyo) in 45 ml of 0.1
N HCl was treated at 95.degree. C. for 13 minutes. After cooling to
room temperature, the suspension was neutralized with NaOH and
filtered through 0.22 .mu.m MILLEX-GP filter (manufactured by
Milipore, SLGPR25LS). For this sample (agar decomposition product
with hydrochloric acid) and agar decomposition oligosaccharide
solution as described in Example 11-(1) (agar decomposition product
with citric acid), according to the same manner as that described
in Example 20-(1), an activity of inhibiting NO production was
measured. Namely, 10 .mu.l of 25 .mu.g/ml LPS and 10 .mu.l of a
20-fold dilution of the sample mentioned above were added to wells
of a 48 well microtiter plate containing RAW264.7 cells which had
been incubated in the wells. The measurement was carried out with
the culture medium. As control groups, a group to which LPS was not
added, a group to which a sample was not added and a group to which
2.5 mM citric acid was added were provided. All the measurement
were carried out in duplicate.
[0374] As a result, both of the agar decomposition product with
hydrochloric acid and the agar decomposition product with citric
acid inhibited the NO production induced by LPS. The results are
shown in FIG. 38. That is, FIG. 38 illustrates the NO.sub.2.sup.-
concentration in the medium cultured with addition of the agar
decomposition product with hydrochloric acid or agar decomposition
product with citric acid. In FIG. 38, the horizontal axis
represents the incubation conditions and the vertical axis
represents the NO.sub.2 concentration (.mu.M).
[0375] (3) According to the same manner as that described in
Example 20-(2), an inhibitory activity against NO production was
evaluated by using an aqueous 100 mM galactose (manufactured by
Nacalai Tesque, code 165-11) or 100 mM 3,6-anhydro-D-galactose
(manufactured by Funakoshi, code G0002) solution.
[0376] As a result, 3,6-anhydro-D-galactose inhibited NO
production, while galactose did not. The results are shown in FIG.
39. That is, FIG. 39 illustrates the NO.sub.2.sup.- concentration
in the medium cultured with addition of 3,6-anhydro-D-galactose or
galactose. In FIG. 39, the horizontal axis represents the
incubation conditions and the vertical axis represents the
NO.sub.2.sup.- concentration (.mu.M).
[0377] (4) RAW 264.7 cells were suspended in the Dulbecco's
modified Eagle's medium described in Example 20-(1) at a
concentration of 3.times.10.sup.5 cells/ml, and 500 .mu.l portions
thereof were placed in respective wells of a 48 well microtiter
plate. The plate was incubated for 37.degree. C. for 10 hours in
the presence of 5% carbon dioxide. To the wells was added 10 .mu.l
of aqueous 5,000 .mu.M agarobiose solution and incubated for
additional 1, 2, 4 or 6 hours. Then, the culture supernatant was
removed from the well and to each well were added 500 .mu.l of
fresh Dulbecco's modified Eagle's medium and then 10 .mu.l of
aqueous 2.5 .mu.g/ml LPS and aqueous 800 U/ml interferon-.gamma.
(IFN-.gamma., sold by Cosmobio, GZM-MG-IFN) solution. The plate was
incubated for 1 hour. Then, the culture supernatant was removed
from the well and to each well was added 500 .mu.l of fresh
Dulbecco's modified Eagle's medium and the plate was incubated for
additional 16 hours. The concentration of NO.sub.2.sup.- produced
by oxidation of NO in the medium was measured according to the same
manner as that described in Example 20-(1). As control groups, a
group to which neither LPS nor IFN-.gamma. was added and a group to
which agarobiose was not added were provided. All the measurements
were carried out in duplicate.
[0378] As a result, the longer the pre-incubation time was, the
higher the inhibition of NO production by agarobiose was. Namely,
by addition of agarobiose to a cell culture medium beforehand, NO
production induced by LPS and IFN-.gamma. could be inhibited and
prevented. The results are shown in FIG. 40. FIG. 40 illustrates
the NO.sub.2.sup.- concentration in the medium cultured under
respective incubation conditions. In FIG. 40, the horizontal axis
represents the incubation conditions and the vertical axis
represents the NO.sub.2.sup.- concentration. For
3,6-anhydrogalactopyranose, agarotetraose, agarohexaose,
agarooctaose, carabiose and 3,6-anhydro-2-O-methyl-L-galactose, the
similar activities have also been recognized.
Example 21
[0379] (1) Agar powder (manufactured by Wako Pure Chemical
Industries, Ltd.) was added to 50 mM citric acid solution at a
final concentration of 3%. The resultant was heat-treated at
95.degree. C. for 160 minutes to prepare an oligosaccharide
solution for a carcinostatic test.
[0380] Male nude mice (SPF/VAFBalb/cAnNCrj-nu, 4 weeks old) were
purchased from Nippon Charles River and pre-bred for 1 week. Human
colon cancer cell line HCT116 (ATCC CCL-247) were transplanted
subcutaneously to the mice at 1.5.times.10.sup.6 cells/mouse.
[0381] After 2 weeks from the transplantation of the colon cancer
cell line, the above oligosaccharide solution for the carcinostatic
test which was adjusted to pH 6.5 just before use was freely given
to the mice as drinking water for 5 days per week. The average of
daily intake per one mouse was 3.5 ml. Furthermore, MF manufactured
by Oriental Yeast was freely given to the mice as feed.
[0382] After 4 weeks from the beginning of administration of
oligosaccharides, the solid cancer was removed from each mouse that
received oligosaccharides and the weight of each solid cancer was
compared with that of a control to which normal water was given.
This test was carried out using 10 mice per one group.
[0383] As a result, a significant activity of inhibiting cancer
cell growth was observed in the group to which the sample for the
carcinostatic test was administrated orally, and a strong
carcinostatic activity was observed in the group to which the
oligosaccharides derived from agar was administrated orally.
[0384] The results are shown in FIG. 41. That is, FIG. 41
illustrates the carcinostatic activity of the oligosaccharides of
the present invention. The vertical axis represents the weight of
solid cancer (g) and the horizontal axis represents the control
group and the group administrated with oligosaccharide.
[0385] In one mouse of the group to which the neutralized sample
for the carcinostatic test was administrated orally, the cancer was
completely disappeared.
[0386] (2) A carcinostatic test was carried out against Ehrlich's
ascites carcinoma using the agar decomposition oligosaccharide
solution as described in Example 11-(1).
[0387] Ehrlich's carcinoma cells were injected to female ddY line
mice (5 weeks old, weighing about 25 g) intraperitoneally
(1.2.times.10.sup.6 cells/mouse) and average days of survival and
prolongation rates were calculated based on the number of survived
animals.
[0388] Mice were divided into 3 group each consisting of 8 mice.
One was a control, and other two groups received 3.3-fold dilution
and 16.7-fold dilution of the agar decomposition oligosaccharide
solution described in Example 11-(1), respectively. Namely, each
aqueous dilution of the agar decomposition oligosaccharide solution
prepared in Example 11-(1) was prepared and was freely given to the
mice from 3 days before cancer cell administration. For the group
to which the 3.3-fold dilution of the agar decomposition
oligosaccharide solution was given, the daily intake of the
dilution was 5 ml/day/mouse. For the group to which the 16.7-fold
dilution of the agar decomposition oligosaccharide solution was
given, the daily intake of the dilution was 6 ml/day/mouse. And,
for the control group, the daily intake of water was 7 ml/day.
[0389] As a result, while the average days of survival of the
control group was 11.8 days for the control group, the average days
of survival for the groups received the 3.3-fold dilution and the
16.7-fold dilution were 19.8 days and 14.4 days, and the
prolongation rates were 168% and 122%, respectively. Thus, a
significant prolongation effect was recognized.
Example 22
[0390] To Wistar line rat (male, 5 weeks old, weighing about 150 g;
Nippon SLC) were injected 100 .mu.g of ovalbumin (OA; Sigma) and 1
ml of alum (trade name: Imject Alum; Piace) intraperitoneally to
sensitized the rat. After 14 days, peripheral blood was collected
from the abdominal aorta of the rat and the serum was used as
anti-OA antibody.
[0391] The back part of Wistar line rat (male, 7 weeks old,
weighing about 200 g; Nippon SLC) was shaved and 100 .mu.l of the
anti-OA antibody was injected subcutaneously to that part to give
passive sensitization. Forty-eight hours after sensitization, 2 ml
of agar decomposition oligosaccharide solution described in Example
11-(1) or its 10-fold dilution was administrated intraperitoneally
to 4 rats of each group. To rats of a control group, 2 ml of water
was administrated intraperitoneally.
[0392] Thirty minutes after administration, PCA was raised by
injection of 1 ml of saline containing 0.1% OA and 0.5% Evan's blue
(Nacalai Tesque) to the tail vein. Thirty minutes after the
induction with antigen, rats were killed by decapitating and
bleeding, and the skin of the back site where the pigment was
leaked was removed and collected.
[0393] The collected skins were soaked in 1 ml of 1 N KCl (Nacalai
Tesque) and allowed to stand overnight. Then, the pigment was
extracted by adding 9 ml of acetone solution (Nacalai Tesque)
containing 0.6 N H.sub.3PO.sub.4 (Merck) and the absorbance at 620
nm was measured using an ELISA reader. The amount of the pigment
leaked from the skin was calculated from a calibration curve of
Evan's blue.
[0394] The results are shown in FIG. 42. That is, FIG. 42
illustrates inhibition of PCA by the oligosaccharides of the
present invention. In FIG. 42, the vertical axis represents the
amount of leaked pigment (.mu.g/site), and the horizontal axis
represents the agar decomposition oligosaccharide solution
used.
[0395] As shown in FIG. 42, one half or more pigment leakage by PCA
was inhibited by administration of the agar decomposition
oligosaccharide solution and, as compared with the control,
significant difference (p<0.05) was exhibited.
[0396] For 3,6-anhydrogalactopyranose, agarobiose, agarotetraose,
agarohexaose, agarooctaose, carabiose and
3,6-anhydro-2-O-methyl-L-galactose, the similar activities have
also been recognized.
Example 23
[0397] Mouse melanoma cell B16BL6 suspended in RPMI-1640 containing
10% FBS was placed in a 6 well plate at a concentration of
5.times.10.sup.4 cells/2 ml medium/well and incubated at 37.degree.
C. On the 2nd day, 100 .mu.l of agarobiose solution (2 mg/ml to 0.2
mg/ml) was added thereto, and on the 7th day, the medium was
changed and, at the same time, 100 .mu.l of agarobiose solution (2
mg/ml to 0.2 mg/ml) was added thereto. On the 8th day, the cells
were collected, DNA, RNA and protein were decomposed, and then the
absorbance at 400 nm was measured to examine an activity of
inhibiting melanin production.
[0398] Namely, after removing the medium by suction, 0.3 ml of
0.25% trypsin dissolved in 20 mM EDTA solution was added to each
well and the plate was incubated at 37.degree. C. for 10 minutes.
Then, 2 ml of the fresh medium was added to the well and the cells
were suspended. The suspension was collected into a test tube. The
medium was then removed by centrifugation and the cells were
suspended in 2 ml of PBS and centrifuged again. After removing the
supernatant, 30 .mu.l of 50 mM sodium acetate buffer (pH 5.0)
containing 5 mM manganese chloride and 1 .mu.l of U/ml DNase I
(manufactured by Takara Shuzo) were added to the cells and
thoroughly mixed. The mixture was incubated at 37.degree. C. for 2
hours to decompose DNA. Then, 1 .mu.l of 10 mg/ml ribonuclease A
(manufactured by Sigma) was added to the mixture and the resultant
mixture was incubated at 50.degree. C. for 1 hour to decompose RNA.
Finally, 100 mM Tris-hydrochloric acid buffer (pH 7.8) containing
100 .mu.g/ml proteinase K (manufactured by Sigma), 0.1% Triton x
and 10 mM EDTA was added thereto to make the total volume up to 200
.mu.l for 2.times.10.sup.6 cells, and the mixture was incubated at
37.degree. C. for 16 hours and then the absorbance at 400 nm was
measured.
[0399] The result was shown in Table 15. As shown in Table 15,
activity of the inhibiting melanin production was recognized at
agarobiose concentrations of 50 and 100 .mu.g/ml and the
beautifying/whitening effect of agarobiose was recognized. For
agarotetraose, agarohexaose, agarooctaose, carabiose and
3,6-anhydro-2-O-methyl-L-galactose, the similar activities have
also been recognized. TABLE-US-00015 TABLE 15 Agarobiose Absorbance
at 400 nm .mu.g/ml mean .+-. SD 100 0.383 .+-. 0.007 50 0.392 .+-.
0.172 10 0.521 .+-. 0.256 control 0.487 .+-. 0.038 Note: The
measurement was carried out in triplicate; 100 .mu.l of the medium
was added to the control.
[0400] As described above, according to the present invention,
there is provided the functional substances which are useful as
active ingredients for compositions for inducing apoptosis,
carcinostatic compositions, antioxidants such as inhibitors of
active oxygen production, inhibitors of lipid peroxide radical
production and inhibitors of NO production, and immunoregulators,
and which are the members selected from the group consisting of the
compounds selected from the group consisting of
3,6-anhydrogalactopyranose, a aldehyde and a hydrate thereof, and
2-O-methylated derivatives of the 3,6-anhydrogalactopyranose, the
aldehyde and the hydrate, and the soluble saccharides containing
said compounds, for example, agarobiose, agarotetraose,
agarohexaose, agarooctaose, carabiose,
3,6-anhydro-2-O-methyl-L-galactose, etc. produced by acid
decomposition under acidic condition below pH 7 and/or enzymatic
digestion of substances containing the above-mentioned
compounds.
[0401] These substances are useful as active ingredients of
pharmaceutical compositions such as compositions for inducing
apoptosis, carcinostatic compositions, antioxidants for medical use
such as inhibitors of active oxygen production, inhibitors of NO
production, etc., immunoregulators, and anti-allergic agents. And,
the foods or drinks comprising, produced by adding thereto and/or
diluting saccharides selected from these saccharides are useful for
functional foods or drinks having an activity such as an activity
of inducing apoptosis, a carcinostatic activity, an antioxidant
activity such as an activity of inhibiting active oxygen
production, an activity of inhibiting NO production, an
immunoregulatory activity and an anti-allergic activity. Thus,
there is provided foods or drinks which induce apoptosis in cells
in lesions in patients suffered from cancers or viral diseases and,
therefor, are effective in preventing or ameliorating the disease
states of these diseases. In a case of a cancer of a digestive
organ such as colon cancer and stomach cancer, among others, since
apoptosis can be induced in tumor cells upon oral intake of the
above-mentioned compounds of the present invention in foods or
drinks, the foods or drinks of the present invention have excellent
effects on the prevention or amelioration of the disease state of a
cancer of a digestive organ. Furthermore, the above-mentioned foods
or drinks are useful foods or drinks for opposing oxidative stress
on the basis of their antioxidant activities such as the activity
of inhibiting the active oxygen production.
[0402] In addition, the functional substances of the present
invention are also useful as saccharides for an antioxidant for
inhibition of active oxygen production, and the foods or drinks
comprising, produced by adding thereto and/or produced by diluting
the saccharides for an antioxidant of the present invention are
useful as those for ameliorating the disease states of diseases
caused by oxidizing substances in a living body such as active
oxygen. Furthermore, the foods or drinks of the present invention
are effective for amelioration or prevention of constipation by the
activity of their active ingredients, i.e., a member selected from
the group consisting of the compound selected from the group
consisting of 3,6-anhydrogalactopyranose, an aldehyde, and
2-O-methylated derivatives thereof and/or the saccharide containing
said compound.
[0403] The saccharides for an antioxidant provided by the present
invention are useful as novel functional saccharides which provide
antioxidant activities such as an activity of inhibiting active
oxygen production to foods or drinks.
[0404] The functional substances of the present invention have a
freshness keeping activity and are very useful for keeping taste or
freshness of foods or perishables.
[0405] Furthermore, the cosmetic compositions comprising the
saccharides of the present invention are useful as those for
beautifying/whitening or moisturizing.
[0406] According to the present invention, there is also provided
acidic foods or drinks comprising, produced by adding thereto
and/or produced by diluting the functional substances. In the
production of such foods or drinks, factors which influence to the
contents of the functional substances have been substantially
eliminated and, therefore, very useful foods or drinks having high
contains of the functional substances are obtained. Moreover, the
acidulant prepared in the presence of an organic acid is also
useful as a novel acidulant having good taste and functions.
* * * * *