U.S. patent application number 09/731291 was filed with the patent office on 2001-04-12 for active oxygen scavenger and cancer chemopreventer from grifola.
Invention is credited to Odaira, Yasuo, Tazawa, Kenji, Watanabe, Masataka.
Application Number | 20010000229 09/731291 |
Document ID | / |
Family ID | 27790309 |
Filed Date | 2001-04-12 |
United States Patent
Application |
20010000229 |
Kind Code |
A1 |
Tazawa, Kenji ; et
al. |
April 12, 2001 |
Active oxygen scavenger and cancer chemopreventer from Grifola
Abstract
The present invention relates to an active oxygen scavenging
agent and a cancer chemopreventing agent both comprising a dried
Grifola, a dry Grifola powder and/or a Grifola extract, and to a
food or animal feed comprising the active oxygen scavenging agent
or the cancer chemopreventing agent.
Inventors: |
Tazawa, Kenji; (Toyama,
JP) ; Odaira, Yasuo; (Niigata, JP) ; Watanabe,
Masataka; (Tochigi, JP) |
Correspondence
Address: |
DAVIDSON, DAVIDSON & KAPPEL, LLC
485 SEVENTH AVENUE, 14TH FLOOR
NEW YORK
NY
10018
US
|
Family ID: |
27790309 |
Appl. No.: |
09/731291 |
Filed: |
December 6, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09731291 |
Dec 6, 2000 |
|
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09421572 |
Oct 20, 1999 |
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Current U.S.
Class: |
424/195.15 |
Current CPC
Class: |
A61K 36/07 20130101 |
Class at
Publication: |
424/195.15 |
International
Class: |
A61K 035/84 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 20, 1998 |
JP |
297781/1998 |
Sep 28, 1999 |
JP |
275197/1999 |
Claims
What is claimed is:
1. An active oxygen scavenging agent comprising a dried Grifola, a
dry Grifola powder and/or a Grifola extract.
2. The active oxygen scavenging agent of claim 1, wherein the dried
Grifola is produced by drying a fresh Grifola at a temperature
ranging from 50 to 90.degree. C.
3. The active oxygen scavenging agent of claim 1, wherein the dry
Grifola powder is produced by: drying a fresh Grifola at a
temperature ranging from 50 to 90.degree. C. to give a dried
Grifola; and then grinding the dried Grifola into powder using a
milling apparatus.
4. The active oxygen scavenging agent of claim 1, wherein the
Grifola extract is produced by extracting a fresh Grifola, a dried
Grifola and/or a dry Grifola powder with water or hot water.
5. The active oxygen scavenging agent of claim 1, wherein the
Grifola extract is produced by: extracting a fresh Grifola, a dried
Grifola and/or a dry Grifola powder with water or hot water to give
an extract; adding an alcohol to the extract; leaving the resultant
solution to stand; and then removing matters floating on and/or in
the solution and/or adhered to the wall surface of the vessel in
which the solution is contained, from the solution.
6. The active oxygen scavenging agent of claim 1, wherein the
Grifola extract is produced by: extracting a fresh Grifola, a dried
Grifola and/or a dry Grifola powder with water or hot water to give
an extract; adding an alcohol to the extract; leaving the resultant
solution to stand; removing matters floating on and/or in the
solution and/or adhered to the wall surface of the vessel in which
the solution is contained, from the solution; removing the alcohol
from the solution; and then drying the resultant solution.
7. The active oxygen scavenging agent of any one of claims 1 to 6,
wherein the active oxygen to be scavenged with the scavenging agent
is a hydroxyl radical and/or a superoxide anion radical.
8. A food or animal feed with active oxygen scavenging activity,
comprising the active oxygen scavenging agent of any one of claims
1 to 7.
9. A cancer chemopreventing agent comprising a dried Grifola, a dry
Grifola powder and/or a Grifola extract.
10. The cancer chemopreventing agent of claim 9, wherein the target
cancer to be treated by the cancer chemopreventing agent is
gastrointestinal cancer.
11. The cancer chemopreventing agent of claim 9, wherein the target
cancer to be treated by the cancer chemopreventing agent is
colorectal cancer.
12. The cancer chemopreventing agent of claim 9, wherein the dried
Grifola is produced by drying a fresh Grifola at a temperature
ranging from 50 to 90.degree. C.
13. The cancer chemopreventing agent of claim 9, wherein the dry
Grifola powder is produced by: drying a fresh Grifola at a
temperature ranging from 50 to 90.degree. C. to give a dried
Grifola; and then grinding the dried Grifola into powder using a
milling apparatus.
14. The cancer chemopreventing agent of any one of claims 9 to 11,
wherein the Grifola extract is produced by extracting a fresh
Grifola, a dried Grifola and/or a dry Grifola powder with water or
hot water.
15. The cancer chemopreventing agent of any one of claims 9 to 11,
wherein the Grifola extract is produced by: extracting a fresh
Grifola, a dried Grifola and/or a dry Grifola powder with water or
hot water to give an extract; adding an alcohol to the extract;
leaving the resultant solution to stand; removing matters floating
on and/or in the solution and/or adhered to the wall surface of the
vessel in which the solution is contained, from the solution;
removing the alcohol from the solution; and then drying the
resultant solution.
16. A process for producing a cancer chemopreventing agent,
comprising: mixing a water-soluble excipient with a Grifola extract
produced by extracting a fresh Grifola, a dried Grifola and/or a
dry Grifola powder with water or hot water; and then spray-drying
the resultant mixture.
17. A process for producing a cancer chemopreventing agent
comprising: mixing a water-soluble excipient with a Grifola extract
produced by extracting a fresh Grifola, a dried Grifola and/or a
dry Grifola powder with water or hot water to give an extract,
adding an alcohol to the extract, leaving the resultant solution to
stand, and then removing matters floating on and/or in the solution
and/or adhered to the wall surface of the vessel in which the
solution is contained from the solution; and then spray-drying the
resultant mixture.
18. A food or animal feed for the chemoprevention of cancer,
comprising the cancer chemopreventing agent of any one of claims 9
to 17.
Description
BACKGROUND OF THE INVENTION
1. 1. Field of the Invention
2. The present invention relates to an active oxygen scavenging
agent and a cancer chemopreventing agent both which can prevent
biological damage in living bodies caused by excess active oxygen
and can also prevent the development of cancer, and also relates to
a food or animal feed comprising the active oxygen scavenging agent
and/or the cancer chemopreventing agent.
3. 2. Description of the Prior Art
4. Oxygen is essential for most of the animals and plants on the
earth to live. However, excess oxygen exerts a harmful action
called "oxygen toxicity", which is likely to damage a living body,
and therefore upon recently has been focused on as a causative
substance of the acceleration of aging and various diseases or
disorders specific to adult people, including cancer.
5. Oxygen found in the atmosphere normally takes the form of a
stable, so-called "triplet oxygen". Upon taking up into a living
body, the oxygen is partially converted into a highly reactive
substance called "active oxygen". Active oxygen is necessary for
killing pathogenic viruses or bacteria invading into a living body,
and plays an extremely important role in biophylaxis. However, it
has become evident that excess active oxygen in a living body
adversely acts on proteins, lipids and even nucleic acids to cause
various kinds of damage in the living body, and therefore is a
cause of the acceleration of aging and the development of various
diseases, such as arteriosclerosis, diabetes, Alzheimer's disease
and Crohn's disease.
6. The active oxygen species produced in a living body include: in
a narrow sense, superoxide anion radical (O.sub.2.sup.-), hydrogen
peroxide (H.sub.2O.sub.2), hydroxyl radical (.OH) and singlet
oxygen (.sup.1O.sub.2); and in an broad sense, in addition to the
above four species, alkoxy radical (LO.), lipid peroxy radical
(LOO.), nitric oxide (NO) and peroxy nitrite (ONOO.sup.-) which is
a reaction product of nitrogen oxide and a superoxide.
7. As the scavengers for these active oxygen species, the following
substances are known:
8. (1) superoxide dismutase (SOD)--for superoxide anion
radicals;
9. (2) catalase, glutathione peroxidase, ascorbate peroxidase, and
the like-- for hydrogen peroxide;
10. (3) carotenoids, tocopherol and the like--for singlet oxide;
and
11. (4) tocopherol, flavonoids, ascorbic acid, glutathione,
carotenoids and the like--for alkoxy radicals and lipid peroxy
radicals.
12. These scavengers, however, have disadvantages. For example,
superoxide dismutase (SOD) is difficult to be applied to
experiments that use cells, animals and plants, because it is a
protein. Other enzymatic scavengers, such as catalase for hydrogen
peroxide, are also difficult to handle for the same reason as
mentioned for SOD. Carotenoids, tocopherol and the like for singlet
oxide have poor selective activity.
13. With respect to hydroxyl radicals (.OH), no specific scavenger
is found, because hydroxyl radicals can react with various
biocomponents in a diffusion-dependent mode. Mannitol, dimethyl
sulfoxide (DMSO), ethanol, thiourea and the like are known to be
scavengers for hydroxyl radicals. However, these substances are
insufficient for scavenging hydroxyl radicals when used singly, and
therefore the use of these substances in combination must be
considered. Thus, up to now, no satisfactory scavenger has been
developed.
14. On the other hand, a number of new methods for the diagnosis
and treatment of cancer have been developed. Nevertheless, the
number of deaths caused by cancer tends to increase on a worldwide
basis. Therefore, in parallel to the extensive studies on cancer
eradication, value is increasingly being placed upon studies on the
prevention of cancer. The methodologies for the prevention of
cancer are as follows:
15. (i) Identification and removal of carcinogenic substances;
and
16. (ii) Screening and active intake of cancer preventing
substances.
17. Since there are innumerable chemical substances in the
surroundings, it will take enormous labor and cost to identify and
screen carcinogenic substances. Even if identified, it is probably
impossible to eliminate the carcinogenic substances from the
environment. Therefore, although it is still necessary to avoid the
substances which are clearly shown to be involved in cancer
development, it has been considered that the active intake of
cancer preventing substances is very effective for cancer
prevention.
18. Actually, the researchers of the National Cancer Institute of
U.S.A. established a category "cancer chemoprevention", which is
defined as an approach to inhibit the development and growth of
cancer through administration of a certain substance. This approach
has recently been studied intensively in most countries of the
world including the U.S.A. However, as yet, no effective approach
for the prevention of cancer has been found, and such an approach
is demanded.
SUMMARY OF THE INVENTION
19. Under these circumstances, the present inventors have
extensively screened the daily foods that have the scavenging
activity against active oxygen species, particularly hydroxyl
radicals. As a result, the inventors have found that Maitake and
related mushrooms and derivatives thereof have an effective
hydroxyl radical scavenging activity, as well as a superoxide anion
radical scavenging activity and a superoxide dismutase (SOD)-like
activity. This finding leads the accomplishment of the
invention.
20. In addition to the active oxygen scavenging activity, the
inventors have also examined daily foods on the cancer
chemopreventing activity.
21. The research on the cancer chemopreventing activity takes more
labor and cost compared to a tumor-growth inhibition test that is
usually performed for the development of a anticancer agent or the
like. This is because such a research requires a far longer period
of time for the administration of test substances into test animals
and for the observation of the pathologic process, and had to be
performed under tightly controlled conditions for the test animals.
Nevertheless, through such a research, the inventors have found
that Maitake and related mushrooms and derivatives thereof have a
cancer chemopreventing activity. This finding leads the
accomplishment of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
22. FIG. 1 illustrates hydroxyl radical scavenging activity.
23. FIG. 2 illustrates superoxide anion radical scavenging
activity.
24. FIG. 3 illustrates superoxide dismutase (SOD)-like
activity.
25. FIG. 4 illustrates the comparison of the number of tumors in
cancer chemopreventing test.
26. FIG. 5 illustrates the comparison of the tumor size (by area)
in cancer chemopreventing test.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
27. "Maitake" (Grifola frondosa) is a mushroom which has
traditionally been ingested as a food in Japan, and has been proven
to be safe to eat, and known to contain immunopotentiating
substances. However, there has been no report concerning active
oxygen scavenging activity and cancer chemopreventing activity of a
dry powder and a water or hot water extract of Maitake on the basis
of scientific experiments.
28. Accordingly, the present invention provides the followings:
29. (1) An active oxygen scavenging agent comprising a dried
Grifola, a dry Grifola powder and/or a Grifola extract;
30. (2) The active oxygen scavenging agent of item (1), wherein the
dried Grifola is produced by drying a fresh Grifola at a
temperature ranging from 50 to 90.degree. C.;
31. (3) The active oxygen scavenging agent of item (1), wherein the
dry Grifola powder is produced by: drying a fresh Grifola at a
temperature ranging from 50 to 90.degree. C. to give a dried
Grifola; and then grinding the dried Grifola into powder using a
milling apparatus;
32. (4) The active oxygen scavenging agent of item (1), wherein the
Grifola extract is produced by extracting a fresh Grifola, a dried
Grifola and/or a dry Grifola powder with water or hot water;
33. (5) The active oxygen scavenging agent of item (1), wherein the
Grifola extract is produced by: extracting a fresh Grifola, a dried
Grifola and/or a dry Grifola powder with water or hot water to give
an extract; adding an alcohol to the extract; leaving the resultant
solution to stand; and then removing matters floating on and/or in
the solution and/or adhered to the wall surface of the vessel in
which the solution is contained, from the solution;
34. (6) The active oxygen scavenging agent of item (1), wherein the
Grifola extract is produced by: extracting a fresh Grifola, a dried
Grifola and/or a dry Grifola powder with water or hot water to give
an extract; adding an alcohol to the extract; leaving the resultant
solution to stand; removing matters floating on and/or in the
solution and/or adhered to the wall surface of the vessel in which
the solution is contained, from the solution; removing the alcohol
from the solution; and then drying the resultant solution;
35. (7) The active oxygen scavenging agent of any one of items (1)
to (6), wherein the active oxygen to be scavenged with the
scavenging agent is a hydroxyl radical and/or a superoxide anion
radical;
36. (8) A food or animal feed with active oxygen scavenging
activity, comprising the active oxygen scavenging agent of any one
of items (1) to (7);
37. (9) A cancer chemopreventing agent comprising a dried Grifola,
a dry Grifola powder and/or a Grifola extract;
38. (10) The cancer chemopreventing agent of item (9), wherein the
target cancer to be treated by the cancer chemopreventing agent is
gastrointestinal cancer;
39. (11) The cancer chemopreventing agent of item (9), wherein the
target cancer to be treated by the cancer chemopreventing agent is
colorectal cancer;
40. (12) The cancer chemopreventing agent of item (9), wherein the
dried Grifola is produced by drying a fresh Grifola at a
temperature ranging from 50 to 90.degree. C.;
41. (13) The cancer chemopreventing agent of item (9), wherein the
dry Grifola powder is produced by: drying a fresh Grifola at a
temperature ranging from 50 to 90.degree. C. to give a dried
Grifola; and then grinding the dried Grifola into powder using a
milling apparatus;
42. (14) The cancer chemopreventing agent of any one of items (9)
to (11), wherein the Grifola extract is produced by extracting a
fresh Grifola, a dried Grifola and/or a dry Grifola powder with
water or hot water;
43. (15) The cancer chemopreventing agent of any one of items (9)
to (11), wherein the Grifola extract is produced by: extracting a
fresh Grifola, a dried Grifola and/or a dry Grifola powder with
water or hot water to give an extract; adding an alcohol to the
extract; leaving the resultant solution to stand; removing matters
floating on and/or in the solution and/or adhered to the wall
surface of the vessel in which the solution is contained, from the
solution; removing the alcohol from the solution; and then drying
the resultant solution;
44. (16) A process for producing a cancer chemopreventing agent,
comprising: mixing a water-soluble excipient with a Grifola extract
produced by extracting a fresh Grifola, a dried Grifola and/or a
dry Grifola powder with water or hot water; and then spray-drying
the resultant mixture;
45. (17) A process for producing a cancer chemopreventing agent
comprising: mixing a water-soluble excipient with a Grifola extract
produced by extracting a fresh Grifola, a dried Grifola and/or a
dry Grifola powder with water or hot water to give an extract,
adding an alcohol to the extract, leaving the resultant solution to
stand, and then removing matters floating on and/or in the solution
and/or adhered to the wall surface of the vessel in which the
solution is contained from the solution; and then spray-drying the
resultant mixture; and
46. (18) A food or animal feed for the chemoprevention of cancer,
comprising the cancer chemopreventing agent of any one of items (9)
to (17).
47. The term "an active oxygen scavenging agent (or scavenger)" or
"a food or animal feed with an active oxygen scavenging activity"
as used herein refers to any agent, medicine, in-between product of
a medicine and a food (e.g., neutraceutical), nutritionally
supplemental food, health food, general food and animal feed that
can scavenge active oxygen species excessive for a living body of a
human or other animal.
48. The term "a cancer chemopreventing agent (or cancer
chemopreventer)" or "a food or animal feed for the chemoprevention
of cancer" as used herein refers to any agent, medicine, in-between
product of a medicine and a food (e.g., neutraceutical),
nutritionally supplemental food, health food, general food and
animal feed that can prevent or inhibit the development of cancer
in a human and an animal.
49. In the present invention, the "Grifola" refers to "Maitake"
(Grifola frondosa) or any of other related mushrooms including, for
example, "Shiromaitake" (Grifola albicans), "Choreimaitake"
(Grifola umbellata) and "Tonbimaitake" (Grifola gigantea), of which
either or both of a fruit body and a micelium may be used.
Recently, the artificial cultivation of a fruit body of Maitake has
been succeeded. Therefore, it is preferable to use a fruit body of
Maitake from the viewpoint of stable resource supply.
50. A dried Grifola used in the present invention may be prepared
by drying a fresh Grifola by any drying method selected among sun
drying, drying in a warming or heating chamber, hot air drying,
freeze drying and an appropriate combination thereof. For example,
the drying process is preferably performed at a gradually elevated
temperature range from 50.degree. C. to 90.degree. C., especially
from 60.degree. C. to 80.degree. C.
51. A dried Grifola is then ground into powder with a milling
apparatus to prepare a dry Grifola powder. When a dried Grifola is
used as it is, it may be ground into coarse or fine powder and may
be of any particle size depending on the intended use. For example,
in the application for a human body, it may be ground into powder
or fine powder having a particle size of 100 mesh or smaller;
whereas for the use in an animal feed, it may be ground into coarse
powder having a particle size of 100 mesh or larger, which may be
mixed into an animal feed product.
52. For the preparation of a Grifola extract, the extraction
treatment may be performed at room temperature or under heating for
a period of time from 5 minutes to 3 hours. For the short-time
extraction, the extraction treatment may be performed at
100.degree. C. or higher under pressure, for example, at about
120.degree. C. for from about 5 minutes to about 1 hours under
pressure using an autoclave. Water used for the extraction may be
any type, such as distilled water, purified water, ion exchanged
water, tap water or natural water. The amount of water used for the
extraction is, for example, from 4 volumes to appropriate volumes,
preferably from 10 volumes to 20 volumes, based on 1 weight of a
dried Grifola or a dry Grifola powder. When a fresh Grifola is
used, water is used in an amount from 2 volumes to appropriate
volumes, preferably from about 5 volumes to about 10 volumes, based
on 1 weight of the fresh body. After the extraction, the resultant
solution is filtered (e.g., with a filter paper or cloth) or
centrifuged, thereby yielding the intended water extract.
53. The water extract may be subjected to an additional treatment,
such as purification by the addition of an alcohol, if necessary.
For example, an alcohol may be added to the water extract in a
final concentration of 20-70% by volume, preferably 30-60% by
volume. Examples of the alcohol include methanol and ethanol. After
the addition of the alcohol, the resultant solution is left to
stand for 1 hour or longer, whereby matters floating on or in the
solution or adhered to the wall surface of the vessel in which the
solution is contained, appear. The matters are then removed from
the solution by filtration, aspiration (e.g., pipetting), straining
out with a meshed material, or the like.
54. The purified extract may be used as it is or the alcohol may be
removed therefrom before use. The purified extract may also be
condensed into a fluid extract, or dried by any conventional drying
method (e.g., condensation drying, spray drying, vacuum drying, or
freeze drying) to give a dry extract powder.
55. It has been known that the extract contains a polysaccharide
(e.g., .beta.-glucan) or a complex of a polysaccharide and a
protein. Therefore, to purify and isolate such a component, the
extract may be additionally subjected to a conventional
purification procedure, such as precipitation, chromatography and
gel filtration.
56. The active oxygen scavenging agent or cancer chemopreventing
agent of the present invention may be used as it is. Alternatively,
it may be mixed with a suitable excipient to be formulated into a
powder, tablet, granule or solution, or mixed with a food or animal
feed preparation.
57. When applied to a medicine, food or animal feed, the Grifola
extract powder is preferably used in the form of a preparation in
which it is dispersed in a suitable excipient, due to its high
hygroscopicity. In this case, the extract powder may be simply
mixed with a suitable excipient. However, it is preferable to
formulate in the following manner.
58. A Grifola extract or a purified fraction thereof is mixed with
a water- or hot water-soluble excipient (e.g., dextrin,
cyclodextrin or lactose) to yield a mixture, if necessary, water or
hot water is added to the mixture, and the liquid mixture is then
spray dried.
59. The mixing ratio between the extract or purified fraction
thereof and the excipient may vary depending on the intended use.
However, the mixing ratio of 1:1 (on the weight basis) is
preferable, because a formulation with this mixing ratio can
considerably improve the undesirable hygroscopicity of the dried
extract and is therefore suitable for a wide range of
applications.
60. When cyclodextrin is used as the excipient, it may be of any
type, including .alpha.-form, .beta.-form and .gamma.-form, but is
preferably of .alpha.-form or .gamma.-form because of its higher
water solubility. In the case where water or hot water is added to
the mixture of the extract and the excipient to yield a liquid
preparation, it is preferable that water or hot water is added so
that the Brix value of the resultant liquid preparation becomes 50%
or lower, preferably from 30 to 40%. The liquid preparation is then
spray dried with a spray drying apparatus, thereby producing the
intended formulation.
EXAMPLE 1
61. Dry Maitake powder (A)
62. Fresh Maitake (Grifola frondosa) fruit bodies were harvested.
The stems and caps were placed on the trays in a tray drying
chamber, and then allowed to dry for approximately one day by
feeding hot air of about 60-80.degree. C. to the trays. In this
drying process, the temperature of the hot air was gradually
elevated starting from 60.degree. C. to finally 80.degree. C. The
dried stems and caps were ground to powder with a mill, thereby
giving a dry Maitake powder.
EXAMPLE 2
63. Maitake Extract (B)
64. Fresh Maitake fruit bodies (2.5 kg) were sliced into pieces of
2-3 cm thick, and then soaked in a 10-L water bath (95-100.degree.
C.) to be extracted. The resultant extract solution was filtered,
thereby giving a dark brown extract with a Brix value of 1%. The
extract was condensed at 75-80.degree. C. under reduced pressure
using a vacuum evaporator to give a condensed solution with a Brix
value of 5%. The condensed solution was spray dried with a spray
drying apparatus, thereby giving a grayish brown, dry Maitake hot
water extract powder (60 g).
EXAMPLE 3
65. Purified Maitake extract (C)
66. A dry powder (100 g) of fruit bodies of Maitake was treated in
ion exchange water (1000 mL) at 120.degree. C. for 30 minutes under
pressure at 2 atoms. The resultant solution was filtered to give a
dark brown extract solution (600 mL). The extract solution was
condensed under reduced pressure to a volume of 200 mL, and then
added with 95% ethanol (210 mL). The resultant mixed solution was
left to stand for about 18 hours, whereby brown materials floating
on and in the solution and adhered on the wall surface of the
vessel in which the solution was contained, appeared. The materials
were strained out from the solution with a wire cloth to give a
brown solution. The solution was treated under reduced pressure to
remove the alcohol therefrom. The resultant solution was further
condensed under reduced pressure (24-42 kPa) at 70-80.degree. C.
until the Brix value of the solution reached 30%, thereby giving a
dark brown condensed solution.
67. The condensed solution was spray dried with a spray drying
apparatus, whereby a brown fine powder (19 g) with sweet smell
characteristic of Maitake was obtained. The analysis of this
product revealed that it consisted mainly of carbohydrate including
polysaccharaides (e.g., .beta.-glucan) and proteins and was soluble
in water, and its aqueous solution was neutral or weakly
acidic.
EXAMPLE 4
68. Purified Maitake extract
69. A dry Maitake powder (2 kg) was treated in water (30 L) under
pressure at 120.degree. C. for 30 minutes. The resultant solution
was filtered to give a dark brown extract solution (20 L). The
extract solution was condensed under reduced pressure to a volume
of 6 L, and then added with 95% alcohol (6.5 L). The resultant
mixed solution was left to stand at a temperature of 10.degree. C.
or lower for from several hours to a half day, whereby brown
materials floating on and in the solution and adhered on the wall
surface of the vessel in which the solution was contained,
appeared. The materials were strained out from the solution with a
wire cloth to give a brown solution. The solution was treated under
reduced pressure to remove the alcohol therefrom. The resultant
solution was condensed under reduced pressure until the Brix value
of the solution reached 30%, thereby giving a dark brown condensed
solution.
70. The condensed solution was spray dried with a spray drying
apparatus, whereby a brown powder (395 g) with sweet smell
characteristic of Maitake was obtained.
EXAMPLE 5
71. Maitake Extract-containing Powder
72. Since the Maitake extract powder prepared as described in
Example 4 was hygroscopic, it was mixed with dextrin ("PINE-DEX
#100; Matsutani Kagaku Kogyo Co., Ltd.) at a mixing ratio of 1:1
(on the weight basis). The mixture was dissolved in hot water so
that the resultant solution has a Brix value of 30-40%. The
solution was spray dried with a spray drying apparatus, thereby
giving a powdery product containing a Maitake extract.
EXAMPLE 6
73. Determination of hydroxyl radical scavanging activity
74. Aqueous solutions of the Maitake-derived products (A), (B) and
(C) obtained in Examples 1-3, respectively, were prepared at
concentrations of 0.2 mg/mL, 2.0 mg/mL and 20.0 mg/mL, and then
determined for hydroxyl radical scavenging activity by the Fenton
reaction method as follows.
75. Each of the aqueous solutions (50 .mu.L, each) was added with a
1 mM FeSO.sub.4-DTAPAC (diethylenetriamine pentaacetic acid)
solution (75 .mu.L), and then a 10-fold dilution (20 .mu.L) of DMPO
(5,5-dimethyl-1 -pyrroline-N-oxide) and 0.1 mM H.sub.2O.sub.2 (75
.mu.L) were further added thereto. After stirring for 2 seconds,
the resultant solution was taken in a flat cell, and determined for
the amount of generated hydroxyl radicals in terms of DMPO-OH using
an electron spin resonance (ESR) spectrometer (JES-FR30) [Japan
Electron Optics Laboratory Co., Ltd. (JEOL)]. The sweeping was
started 60 seconds after the addition of H.sub.2O.sub.2.
76. The conditions for the spectral analysis by ESR spectrometer
were as follows:
1 magnetic field sweep width: 335.6 mT; magnetic field modulation:
0.1 mT; amplification rate: 125; sweep time: 2 min.: response time:
0.1 sec.; and temperature of measurement: room temperature.
77. The results are shown in FIG. 1. As a result, it was found that
the products (A) and (C) exerted 70% or higher hydroxyl radical
scavenging activity at 20 mg/mL and the product (B) exerted even at
2 mg/mL, and the highest activity was observed for the product (C)
at 20 mg/mL.
EXAMPLE 7
78. Superoxide anion radical scavenging activity
79. Superoxide dismutase (SOD)-like activity
80. Using the same aqueous solutions of the Maitake-derived
products (A), (B) and (C) as used in Example 6, the superoxide
dismutase (SOD)-like activity of each solution was determined by
the spin trap method using ERS spectrometer (JES-FR30; JEOL) as
follows.
81. A hypoxanthine (HPX) solution (2 mM), a DETAPAC solution (5.5
mM), a series of SOD solutions (0.1-50 U/mL) and a xanthine oxidase
(XOD) solution (0.4 U/mL) were separately prepared using 0.2 mM
phosphate buffer solution as a solvent. A trapping agent DMPO (15
.mu.L, 9.2 M) was added to each of the HPX solution (50 .mu.L), the
DETAPAC solution (35 .mu.L) and each of the SOD solutions (50
.mu.L), and the resultant mixed solution was further mixed with the
XOD solution (50 .mu.L). The resultant solution was measured for
the spectrum of generated O.sub.2.sup.--adducts in a quartz
cell.
82. The signal intensity of the O.sub.2.sup.--adducts was
calculated as a relative value to the signal intensity of the
internal standard Mn, and a calibration curve was prepared using
the determined values at different concentrations of SOD. In the
same manner, each of the aqueous solutions (50 .mu.L, each) of the
Maitake-derived products (A), (B) and (C) was also measured for the
spectrum of generated O.sub.2.sup.--adducts in a quartz cell. The
inhibition rate (%) relative to a control (distilled water) was
determined, and the scavenging rate (%) was calculated as a SOD
concentration (corresponding to the SOD activity of the test
solution) from the calibration curve.
83. The results are shown in FIGS. 2 and 3. As a result, it was
found that all of the products (A), (B) and (C) showed high
superoxide anion radical scavenging activity and SOD-like activity
at 20 mg/mL, and both the activities were highest in the product
(C).
84. The results of Examples 6 and 7 clearly demonstrate that all of
the dry Maitake powder, Maitake extract and purified Maitake
extract according to the present invention have hydroxyl radical
scavenging activity, superoxide anion radical scavenging activity
and SOD-like activity.
EXAMPLE 8
85. Cancer chemoprevention test
86. (1) Test method
87. In this test, male Donryu rats (4-week old, 12-13 rats per
group) were used as the test animals. In the test groups, a feed
blend containing 5% of the Maitake extract-containing powder
prepared as in Example 4 through Example 5 (corresponding to 2.5%
of the Maitake extract) in a base feed was fed to 12 rats
(hereinafter, referred to as "A-5" group); and a feed blend
containing 20% of the dry Maitake powder prepared as in Example 1
in the base feed was fed to 12 rats (hereinafter, referred to as
"A-20" group). In the control group, only the base feed was fed to
13 rats (hereinafter, referred to as "A-C" group). These three
groups were examined for cancer chemopreventing effect in the
following manner.
88. Azoxymethane (AOM) was dissolved in physiological saline, and
the resultant solution was administered subcutaneously into the
back of the rats at a dose of 7.5 mg/kg per rat at intervals of a
week for 10 times, and thirty weeks after the final administration,
all of the rats were sacrificed. Until the rats were sacrificed,
the rats were individually observed the physical conditions and
measured for the body weight once a week. After the rats were
sacrificed, the large intestine was dissected, and then the number,
the location and the size (longer diameter.times.shorter diameter)
of the tumors developed in the colon were determined. In addition,
tissues from one or more regions in the colon where the tumors were
developed were dissected, fixed in a 10% formalin solution,
embedded in paraffin, and then stained by any one of
hematoxylin-and-eosin (H.E.) and periodic acid-Schiff stain (PAS)
methods for histologic examination.
89. The base feed used in this experiment was a commercially
available feed specified for mice, rats and hamsters, "MF"
(Oriental Yeast Co., Ltd.) having the following composition.
2 General composition (in 100 g): Water 7.8 g Crude proteins 23.8
Crude lipids 5.1 Crude ash 6.1 Crude fibers 3.2 Soluble
non-nitrogenous matters Total 100.0 (calorie: 357 kcal/100 g)
90. (2) Test results
91. (i) Comparison of the number of tumors
3 TABLE 1 Number of tumors/rat A-C A-5 A-20 Average 3.4 1.9 0.3
Standard deviation 2.4 1.2 0.5 Standard error 0.67 0.34 0.13
92. As shown in Table 1 and FIG. 4, in the control group [A-C], the
average number of tumors per rat was 3.4; whereas in the test group
[A-5] [i.e., a group fed a feed blend containing 5% of the Maitake
extract powder (corresponding to 2.5% Maitake extract)], the
average number of tumors per rat was 1.9 (55.9% relative to the
number in the control group), and in the test group [A-20] (i.e., a
group fed a feed blend containing 20% of dry Maitake powder), the
average number of tumors per rat was 0.3 (8.8% relative to the
number in the control group). These results clearly demonstrate
that the administration of the Maitake extract and the dry Maitake
powder of the present invention can effectively chemoprevent and
inhibit the tumor development.
93. The t-test revealed that there was a significant difference
between the result for [A-20] and the result for [A-C] with a level
of significance of less than 1 %.
94. (ii) Comparison of the tumor size (by area)
4TABLE 2 Tumor size (mm.sup.2)/rat A-C A-5 A-20 Average 78.1 61.9
31.7 Standard deviation 58.5910425 60.6022177 78.2575391 Standard
error 16.3 17.5 22.6
95. As shown in Table 2 and FIG. 5, in the control group [A-C], the
average tumor size per rat was 78.1 mm.sup.2; whereas in the test
groups, the average tumor size per rat was 61.9 mm.sup.2 in [A-5]
[i.e., a group fed a feed blend containing 5% of the Maitake
extract powder (corresponding to 2.5% Maitake extract)], and 31.7
mm.sup.2 in [A-20] (i.e., a group fed a feed blend containing 20%
of dry Maitake powder).
96. These results clearly demonstrate that the Maitake-derived
products of the present invention can effectively reduce the tumor
size.
97. (iii) Change in body weight
98. For the verificational purpose, the change in the average body
weight per rat was determined. However, no significant difference
was observed between the test groups and the control group.
99. In the above Examples, tests were performed on the colorectal
cancer development. However, the Maitake extract-containing powder
and dry Maitake powder of the present invention will also have
effective chemopreventing activity against other intestinal cancer
including gastrointestinal cancer, as well as other kinds of
cancer.
* * * * *