Note: Descriptions are shown in the official language in which they were submitted.
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ANTITUMOR ACTIVITY STRENGTHENING METHOD OF CRUDE DRUGS,
COMPOSITION CONTAINING CRUDE DRUG STRENGTHENING ANTITUMOR
ACTIVITY, EVALUATING METHOD OF ANTITUMOR EFFECTIVENESS BY
CRUDE DRUG TREATMENT, AND EVALUATING METHOD OF ANTITUMOR
EFFECTIVENESS OF CRUDE DRUG
BACKGROUND OF THE INVENTION
This invention relates to techniques such as
strengthening method, evaluating method and the like of
antitumor activity of crude drugs such as mushrooms
containing polysaccharides having antitumor activity,
particularly to techniques for enlarging manifestation of
antitumor effects in internal administration or expecting
effectiveness of antitumor activity in internal
administration and the like.
Various kinds of mushrooms have heretofore been used
in the field of Chinese medicines, health foods or the like.
For example, it has been known that mushrooms such as
GANODERMA (REISHI) (Fomes (SARUNOKOSHIKAKE)), PORIA
(BUKURYO), maitake mushroom (MAITAKE, Grifola frondosu
Dickson), and the like have antitumor activity such as
carcinostatic activity, etc. Many researches have been
done about effective ingredients of mushrooms showing
antitumor activity, and it has generally been said that
polysaccharides contained in mushrooms show effective
antitumor activities.
As a matter of fact , in the field of Chinese
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medicines, dried mushrooms are decocted and internally used
by drinking the decoction.
Various kinds of Chinese drugs have heretofore been
used as natural medicines since they have less side effects
as compared with synthesized medicines. In Japanese
pharmacopoeia, a number of Chinese drugs have been
described as crude drugs.
On the other hand, with regard to antitumor
activities of such mushrooms, their decoctions or extracts
are ingested to experimental animals such as mouse, etc. by
providing with food, and mice of a control group to which
no Chinese drug is ingested and mice of a group to which it
is ingested are compared and observed to examine, for
example, an extinct state, a transferred state of cancer,
or vitality percentage of mice or the like whereby their
effectivenesses are inspected.
Moreover, in such experimental animals, toxicity and
effectiveness are also examined, and after confirmation of
their safeties to apply to human beings sufficiently, these
medicines are actually administered internally and
effectiveness of antitumor activities in human beings are
finally confirmed by clinical tests.
SUMMARY OF THE INVENTION
Antitumor activities such as carcinostatic property,
cancer transfer-inhibiting property, etc. of mushrooms have
heretofore been known as mentioned above, and their
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effectivenesses are widely known. They are used by
decocting a dried product of mushrooms and drinking the
decoction, or mixing an effective ingredient extracted with
an alcohol, etc. with other components such as a vitamin
preperations to make a form such as health drinks, health
foods or medical agents.
According to the administration by an internal means
such as drinking a decoction conventionally carried out,
the case where its medical effects could be obtained so
that the therapeutic effects are apparently observed is
unexpectedly little. In agaricus mushroom (AGARIKUSUTAKE,
which is also called to as °Agaricus") which has recently
attracted attention as having a large medical effect, the
same tendency can be observed. Moreover, in maitake
mushroom (MAITAKE, Grifola frondosu Dickson) which has
recently attracted attention, there is also little example
to prove its medical effects.
However, they do never show medical effects, but
there is a case where a clearly effective antitumor
activity is shown whereas it is in an extremely limited
number of examples.
Thus, the present inventor considered whether there
is any effective way to obtain the antitumor activities of
mushrooms for any person by attaining the mechanism why
differences in manifestation of antitumor effects provided
by mushrooms occur.
Even when effectiveness of mushrooms having antitumor
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activity are confirmed in experimental animals, as
mentioned above, the expected effects cannot be shown in
almost all the cases when it is actually administered
internally to human beings. For confirming the effects
thereof by actual and internal administration in clinical
experiments, it is necessary to continue internal
administration for a certain period of term by controlling
the administration system under the constant conditions and
to continue observation thereof. Thus, a significant term
would be required for obtaining a result whether or not the
effects of the mushrooms are shown by internal
administration to a patient.
Such a method is certainly effective, but, for
example, for searching out a substance having an effective
antitumor activity by screening a number of natural
products, an enormous amount of days is required so that it
is necessary to develop a technique which can evaluate an
antitumor activity within a shorter period of time.
Also, when a patient who is under a clinical test
suddenly caused complication and a treatment out of control
is applied to the patient in haste, confirmation of
effectiveness cannot be carried out with regard to the
patient. Thus, in a clinical test over a long period of
time, a large number of cases where confirmation of
effectiveness cannot be carried out occur so that it is not
necessarily easy to carry out the clinical test for a long
period of time while maintaining effectiveness of the test
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itself .
Thus, the present inventor considered that it is
necessary to develop a method which can expect
manifestation of an antitumor effect with a certain degree
when a specimen is administered to human beings without
carrying out such a clinical test.
Also, as a substance having an antitumor activity, a
large number of materials have been known as crude drugs in
Chinese medicine other than mushrooms, and it is more
preferred if the above-mentioned problems regarding
mushrooms could be solved in view of spreading all of the
crude drugs.
An object of the present invention is to effectively
develop antitumor activities of crude drugs such as
mushrooms and the like containing polysaccharides having
antitumor activities.
Another object of the present invention is to expect
antitumor activities of crude drugs such as mushrooms and
the like when they are administered to human beings without
carrying out clinical tests.
Another object of the present invention is to provide
a composition containing crude drugs such as mushrooms and
the like containing polysaccharides having antitumor
activity so as to effectively manifest their antitumor
activities.
The above-mentioned and other objects and novel
characteristic feature of the present invention are
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described in the following present specification in detail.
The present invention relates to an antitumor
activity strengthening method which is applied to a crude
drug containing polysaccharides having antitumor activity
and comprises a roasting step of roasting the above-
mentioned crude drug by far infrared rays and a
fermentation step of fermenting by adding microorganisms to
strengthen the antitumor activity of the above-mentioned
crude drug than the case of not effecting the roasting and
fermentation.
Subsequent to the above-mentioned fermentation step,
the present invention comprises a step of making an oily
agent in which the above-mentioned fermentated crude drugs
are enclosed with an oily component obtained from plants
such as roasted sesame or the like by far infrared rays.
The present invention further comprises using a
mushroom which contains B-glucan.
The present invention further comprises using at
least one mushroom selected from the group consisting of
agaricus mushroom (AGARIKUSUTAKE, Agaricus blazei), maitake
mushroom (MAITAKE, Grifola frondosu), shiitake mushroom
(SHIITAKE, Cortinellus Shiitake), matsutake mushroom
(MATSUTAKE, Tricholoma matsutake), shimejitake mushroom
(SHIMEJITAKE, Lyophyllum decastes) and enokitake mushroom
(ENOKITAKE, Flammulina velutipes).
When the above-mentioned agaricus mushroom
(AGARIKUSUTAKE) is used, the present invention comprises
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applying roasting to raw agaricus mushroom by far infrared
rays.
The antitumor activity strengthened crude drug-
containing composition of the present invention comprises a
crude drug to which the antitumor activity strengthening
method of the crude drug with any of the above-mentioned
constitutions is applied.
The present invention is an evaluating method of
effectiveness on an antitumor activity of the treatment to
be applied to the crude drug containing polysaccharides
having an antitumor activity, which comprises adding the
crude drug to which the above-mentioned treatment is
applied to a system which forms lipid peroxide by
irradiating ultraviolet rays to an unsaturated fatty acid
such as docosahexaenoic acid and evaluating an effect of
the treatment for strengthening the antitumor activity of
the above-mentioned crude drug to be large as an increased
ratio of a formed amount of the above-mentioned lipid
peroxide based on an increased ratio of the concentration
of the above-mentioned crude drug by the treatment being
large.
The present invention comprises mushrooms which
contain B-glucan being used for the above-mentioned crude
drug.
The present invention is an evaluating method of
effectiveness of the crude drug for evaluating
effectiveness of an antitumor effect shown by the crude
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drug containing polysaccharides having an antitumor
activity without carrying out clinical tests, which
comprises adding the crude drug to a system which forms
lipid peroxide by irradiating ultraviolet rays to an
unsaturated fatty acid such as docosahexaenoic acid and
evaluating and expecting an effect of
antitumor activity by internal
administration of the above-mentioned crude drug to be
large as an increased ratio of a formed amount of the
above-mentioned lipid peroxide based on a ratio of the
concentration of the above-mentioned crude drug being large.
The present invention comprises either of the above-
mentioned antitumor activity strengthening methods being
applied to the above-mentioned crude drug.
The present inventor has carried out various studies
on an anti-active oxygen inhibiting substance in Chinese
drugs, and through such studies, he has confirmed that
active oxygen or lipid peroxides provide remarkable effects
on human body and these substances pertains to breakage of
tissues in human body whereby they becomes the cause of
occurring various infectious diseases.
In the tissues of human body, an enzyme called SOD
(superoxide dismutase) exist to protect the tissues of
human body from such an active oxygen and to remove the
active oxygen. An amount of the SOD decreases with ages
and removal of the active oxygen in the body cannot be
carried out. Decrease thereof is particularly remarkable
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with the age of 40 or more, and removal of the active
oxygen taken in the body cannot sufficiently be carried out
and it has been clarified in recent years that it becomes
the cause of various kinds of adult diseases.
Also, when an excessive active oxygen is formed in
the body by chronic or acute stimulation or by a chemical
substance, etc., removal of the active oxygen cannot be
sufficiently carried out with the amount of SOD existed in
the body from the beginning so that the person is suffered
from various diseases.
Thus, to various diseases caused by not sufficiently
removing the active oxygen due to lack of SOD, it has been
considered to effect treatment by administering SOD to
solve lack of SOD. However, according to such a treatment
method, effects can be admitted in the case of an injection
medicine, but not remarkable effect can be admitted in the
case of an internal medicine so that it is the present
status that the effects have not yet been proved.
With regard to the point that the effects as an
internal medicine are not manifested, the present inventor
has found that SOD is unstable to gastric juice and the
molecular weight of SOD is 30000 or more and it is caused
by not being absorbed from digestive organs as such. Also,
he has found that there is an action limit that SOD only
acts on superoxide (OZ-) among the existing four kinds of
active oxygens.
The present inventor has earnestly studied about a
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substance which can remove an excessive active oxygen in
the human body and is capable of internally administering,
and as a result, he has found a composition which comprises
plants oil obtained from a roasted plants which is added to
a substance obtained by roasting plant seeds or their germs
under suitable conditions and then fermented by adding
microorganisms can be used as an active oxygen removing
agent which is effective as an internal use (see Japanese
Patent No. 2,125,887).
In the above-mentioned plant seeds or their germs,
low molecular weight anti-active oxygen substances such as
flavonoids, polyphenols, tannin, tocopherol, vitamin B2,
etc. inherently exist. The present inventor has found that
such low molecular weight anti-active oxygen substances
chemically bind mutually or with the other component,
generate a molecular compound or form a complicated complex
or a macromolecular compound by adsorption or inclusion,
and when they are ingested in such a state, an anti-active
oxygen suppressing action expected from a low molecular
state anti-active oxygen substance cannot be obtained.
That is, even when it is ingested in such a state,
much persons nowadays cannot digest an anti-active oxygen
substance to a low molecular weight substance by gastric
juice so that the anti-active oxygen suppressing action
cannot be obtained. The present inventor stated such a
point in "Food Industry", vol. 35, No. 14, "Development and
Improvement of DDS, SOD-like Function Foods from Natural
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Plants and Seeds and Their Pharmacological and Biochemical
Consideration".
The present inventor studied to effectively utilize
the above-mentioned anti-active oxygen substance contained
in plant seeds or their germs for treatment or prevention
of diseases and how to activate the above state anti-active
oxygen substances.
As a result, he has found that by heating and
roasting plant seeds or germs under mild conditions, the
anti-active oxygen substance contained in the plant seeds
or their germs is partially liberated from the above-
mentioned complex to form an original low molecular weight
substance or is partially activated by generating an active
functional group due to chemical change. By effecting such
treatments, the anti-active oxygen action is significantly
strengthened as compared with that before roasting.
On the other hand, many of the antitumor active
functions such as carcinostatic property or cancer-transfer
inhibiting property of mushrooms are considered to be
generally based on polysaccharides such as B-glucan, etc.,
but such B-glucan is different from the above-mentioned low
molecular weight anti-active oxygen substance contained in
the plant seeds or germs, and it is out of the objects of
the above-mentioned series of studies on active oxygen by
the present inventor.
However, the present inventor considered whether the
fact that, for manifestation of the antitumor effects of
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mushrooms, when it is internally administered by drinking a
decoction, as mentioned above, it shows remarkable effects
for some person but the effects could never be obtained for
the other many peoples could be explained by the similar
thought as the fact of low molecular weight of the above-
mentioned anti-active oxygen substance.
That is, whereas the action and mechanism showing
antitumor activity of B-glucan have not yet sufficiently
been elucidated, as mentioned above, the fact that there
are differences in appearance of antitumor effects when
mushrooms are internally ingested means there are patients
to whom an effective ingredient which manifests an
antitumor activity of mushrooms easily acted and patients
to whom hardly acted.
According to the internal administration form,
mushrooms internally ingested are treated by gastric juice
sooner or later. Accordingly, he considered that whether
the effective ingredient which manifests antitumor activity
of the above-mentioned mushrooms effectively act or not
markedly related to such a gastric juice treatment. That
is, the present inventor considered that there are
individual differences in gastric juice treatment and this
individual differences would cause differences in
manifestation of the antitumor effects.
Thus, the present inventor set forth the following
hypothesis between easiness in manifestation of antitumor
action in internal use and a gastric juice treatment in
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view of the clinical fact that there are individual
differences in strength of the gastric juices.
That is, the effective ingredient showing an
antitumor activity contained in mushrooms which becomes an
object of the gastric juice treatment exists in the state
at which the effects are difficultly manifested, and as
mentioned above, it is changed to the state in which the
effects are easily manifested due to the gastric juice
treatment with difference in individuals. Thus, if a
person has a strong gastric juice by nature, an ingredient
having effects on an antitumor activity contained in
ingested mushrooms becomes the state in which the action
can be easily shown whereby the antitumor effects are
manifested.
However, in a person who has a weak gastric juice by
nature, an ingredient having effects on an antitumor
activity does not become the state in which the action can
be easily shown whereby the antitumor effects are not so
manifested.
Much persons nowadays do not have strong gastric
juice which can change the effective ingredient showing the
antitumor activity of mushrooms to an extent that an action
thereof is easily manifested, and as a result, it can be
considered that a number of examples which can confirm the
effectiveness of the antitumor activity of mushrooms with a
certain extent can be scarcely observed by internal
administration.
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That is, it can be explained that, in the case of a
person who has a strong gastric juice by nature, whereas
components which are effective for antitumor activity
contained in mushrooms are hand in hand like a chain
(whereas it is a simile expression) by, for example,
polymerization and exist in the unmovable state, i.e., in
the non-active state at which they cannot activate, the
components are cut by the gastric juice to change to the
effective components in the free activated state whereby
the antitumor activity can be shown in the body.
It can be considered that much persons nowadays
cannot cut the chain in the inactivated state in which the
effective ingredients which are effective for cancer are
connected with each other as a chain with gastric juice as
mentioned above. Thus, it can be so explained that the
ingredient effective to cancer are absorbed by the
intestines in such a non-activated state that they are hand
in hand so that they cannot show their effects in a body.
Thus, the present inventor considered that if a
treatment which can previously activate the effective
ingredient which is in the non-activated state easily can
be applied to mushrooms, the above-mentioned problems would
be solved.
The present inventor has tried various means to
activate the effective ingredients of such mushrooms in a
non-active state, but he could not readily reach an
effective means. This is because a sufficient knowledge
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cannot be obtained about the state of the effective
ingredients how they are in the non-activated state since
the actual system is too complex to evaluate.
The present inventor has carried out various
experiments as mentioned above, and as the final experiment,
he tried to apply the above-mentioned method invented by
himself as an activating means of active oxygen suppressing
substances such as flavonoids, polyphenols, etc. whereas
they are quite different composition from B-glucan.
When such a method is to be applied, the means is for
activating flavonoids and the object to be applied is quite
different from B-glucan so that the non-activated state is
considered to be quite different therefrom as a matter of
course, and thus, diversion of the method for activating B-
glucan has been considered to be difficult at the beginning.
However, the experimental results were quite
different from the expectations by the present inventor and
showed that the activation means of the active oxygen
suppressing substance is to be effective for changing B-
glucan having the different structure from that of the
active oxygen suppressing substances such as flavonoids,
etc. to be easily activated.
That is, the present inventor has found that
mushrooms which have previously been roasted by far
infrared rays are used in the experiment, antitumor effects
are improved when it is internally administered as compared
with the case where mushrooms which are not roasted by far
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infrared rays are used. Moreover, it is found that when
the mushrooms roasted by far infrared rays are fermented by
adding microorganisms, particularly "Koji" (Aspergillus
oryzae) or yeast, the antitumor effects are more improved.
As the mushrooms, mushrooms containing
polysaccharides having an antitumor activity, for example,
mushrooms such as agaricus mushroom (AGARIKUSUTAKE) which
has recently been particularly attracted attention as for
its medical effects may be used. In addition, there may be
used mushrooms which have heretofore been said to have
potent antitumor activity such as Fomes (SARUNOKOSHIKAKE),
PORIA (BUKURYO), etc. without problem.
Incidentally, with regard to agaricus mushroom
(AGARIKUSUTAKE), it can be found that even when it is
carried out far infrared rays roasting and fermentation
treatment under various conditions after harvest and drying,
sufficient effect cannot be obtained. Thus, it is
preferably subjected to treatment under non-dried
conditions, i.e., under a raw state, for example, without
leaving long after harvest.
Moreover, agaricus mushroom (AGARIKUSUTAKE) is easily
spoiled, and if it takes a long time until the far infrared
rays roasting treatment, it shall not be allowed to stand
for a long term in a raw state. Thus, in such a case, it
is preferred to once store in a refrigerator at a low
temperature and the treatment such as the far infrared rays
roasting, etc. is subjected to within three days from the
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harvest.
Moreover, after the above-mentioned far infrared rays
roasting and fermentation treatment, it is found that the
' antitumor effects are more improved when the resulting
material is subjected to oily treatment. For example, an
oily component obtained from plants such as sesame, etc.
subjected to roasting by far infrared rays is added thereto
to cover the resulting material after fermentation
treatment with the oil component.
As described above, it was initially never expected
that the means for making the anti-active oxygen substance
contained in plant seeds or germs a low molecular weight
compound could be also effectively applied to manifestation
of antitumor activity of mushrooms containing
polysaccharides having antitumor activities which have
quite different structure, etc. from the anti-active oxygen
substance. However, it can be clarified that significant
effects can be obtained by actually applying to the
material through the experiments at this time whereby the
present invention has been accomplished.
The present inventor has also found that an antitumor
activity which is worth attention can be manifested by
carrying out the above-mentioned treatment even when the
mushrooms have not attracted specific attention in the
point of its antitumor activity. For example, it was found
that when maitake mushroom (MAITAKE, Grifola frondosu
Dickson), etc. are subjected to roasting by far infrared
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rays and fermentation treatment by microorganisms, an
antitumor effect comparable to or more than that of
agaricus mushroom (AGARIKUSUTAKE) can be obtained.
The antitumor effect of mushrooms such as maitake
mushroom (MAITAKE, Grifola frondosu Dickson) has heretofore
been known, but it has generally been not considered that
it has an effect comparable to that of agaricus mushroom
(AGARIKUSUTAKE) which has now been attracted attention. By
applying the treatments with the above-mentioned
constitution of the present invention to mushrooms, the
antitumor effects of mushrooms such as maitake mushroom
(MAITAKE, Grifola frondosu Dickson) inherently possessed
are sufficiently manifested and the antitumor activity
comparable to that of agaricus mushroom (AGARIKUSUTAKE) is
shown.
That is, potential antitumor activity possessed by
mushrooms could not sufficiently be brought out and its
effectiveness could not be clearly confirmed as of today.
However, by applying the treatment of the present invention
to mushrooms, their antitumor activities are clearly shown
and the antitumor activity can be manifested with a degree
which could never be expected.
In any of mushrooms, B-glucan is more or less
contained and the above treatment is considered to be a
method which is capable of strongly promoting its action
and manifestation. Thus, in addition to maitake mushroom
(MAITAKE, Grifola frondosu Dickson), the treatment of the
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present invention is applied to mushrooms which has usually
been used as raw materials for meals such as shiitake
mushroom (SHIITAKE, Lentinus edodes), matsutake mushroom
(MATSUTAKE, Tricholoma matsutake), shimejitake mushroom
(SHIMEJITAKE, Lyophyllum decastes), enokitake mushroom
(ENOKITAKE, Flammulina velutipes), etc. As a result, it
was found that the method can strongly develop potential
antitumor activity contained therein.
When the above-mentioned antitumor activity
strengthening method of mushrooms of the present invention
is applied, B-glucan of mushrooms which can be usually and
simply available with inexpensive can be activated without
using expensive and rare mushrooms, and even when an amount
of B-glucan is less than that of the rare mushrooms,
significantly effective antitumor activity can be developed
as compared with the case where the rare mushrooms are used
with the conventional method. This lead to reduction of
economical burden for a patient.
Also, the composition containing mushrooms to which
such antitumor activity strengthening method is applied is
significantly heightened its antitumor activity as compared
with the conventional composition in which mushrooms which
are said to have an antitumor activity are dried and
powdered, and simply mixed.
Moreover, in the course of effecting the above-
mentioned series of researches of mushrooms, mushrooms are
added by stepwisely increasing the concentration thereof to
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the system in which lipid peroxide is formed by irradiating
ultraviolet rays to an unsaturated fatty acid such as
docosahexaenoic acid, etc. and amounts of the formed lipid
peroxides are examined with the respective mushrooms at
various kinds of concentrations. As a result, it could be
found that the larger the degree of increasing the formed
amount of lipid peroxide by the mushrooms is, the stronger
the antitumor action by the internal administration shows.
That is, various kinds of mushrooms are added to the
system which forms the above-mentioned lipid peroxide with
the same concentration, and amounts of the respective lipid
peroxides formed are measured. When the amount of the
formed lipid peroxide is larger, then the antitumor effects
can be shown more potently when it is actually provided by
internal administration.
If such a method is employed, whether the antitumor
effects are manifested or not when the mushrooms are
internally used can be expected without internal
administration actually. Even when a clinical test is not
carried out to a patient for subjecting to experimental
internal administration, the method can be used as a method
for evaluating the antitumor effectivity (or effectiveness)
by internal administration.
Moreover, for example, various kinds of treatments
are applied to mushrooms, and formed amounts of lipid
peroxide are compared by adding the mushrooms to which the
respective treatments are each carried out, the antitumor
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effectivities of the respective treatments can be evaluated.
Also, the above-mentioned explanation is made with
respect to mushrooms, the method may be applied to a crude
drug containing polysaccharides having antitumor activity
other than mushrooms. Application to at least a crude drug
containing B-glucan is effective.
The method can be applied to crude drugs other than
mushrooms such as natural crude drugs including crude drugs
derived from plants, crude drugs derived from animals and
crude drugs derived from minerals. Also, many of Chinese
medicines use crude drugs naturally grown, but it may be
crude drugs harvested in a farm under suitable conditions
or may be those grown by an artificial culture such as
water culture, etc.
Moreover, as crude drugs, other than the crude drugs
described in, for example, Japanese pharmacopoeia, etc., if
it shows effective antitumor activity when the present
invention is applied, such natural products or culture
crops can be used as crude drugs mentioned in the present
specification .
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the following, embodiments of the present
invention is described in detail by referring to Examples.
With regard to an antitumor activity strengthening
method of crude drugs, a composition containing antitumor
activity strengthened crude drugs, an evaluating method of
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antitumor effectivity of crude drug treatment, and an
evaluating method of an antitumor effectivity of crude
drugs, explanation will be made by using mushrooms which
has been well known as crude drugs containing B-glucan
which is a polysaccharide having an antitumor activity.
(EMBODIMENT 1)
In the embodiment 1 of the present invention, an
antitumor activity strengthening method of crude drugs and
a composition containing an antitumor activity
strengthening crude drugs are described.
In the antitumor activity strengthening method of
crude drugs, mushrooms containing B-glucan are firstly
roasted by far infrared rays, and then, microorganisms such
as "Koji", etc. are added thereto and fermentation is
carried out under the predetermined humidity and
predetermined temperature. After completion of the
fermentation step, the resulting material is suitably
formed to be fine powder, and the fine powder is coated
with an oily component obtained by squeezing and pressing
sesame which had been roasted by far infrared rays to
prepare an oily agent.
Also, a material obtained by successively subjecting
mushrooms containing B-glucan to the above-mentioned far
infrared rays roasting step, the fermentation step by
microorganisms, and the step of making oily agent becomes a
composition which shows an effective antitumor activity by
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an internal administration containing mushrooms to which
the antitumor activity strengthening method is applied.
In the following, the above-mentioned present
invention is to be explained in more detail by referring to
Examples.
(EXAMPLES)
As mushrooms to be used in the present Example,
mushrooms containing B-glucan were used. Particularly when
mushrooms which contain much amounts of B-glucan and
generally have antitumor effects are used, the antitumor
effects are strongly manifested whereby it is preferred.
In the present Example, as such mushrooms, PORIA
(BUKURYO), agaricus mushroom (AGARIKUSUTAKE), POLYPORUS
(CHOREIMAITAKE, Polyporus umbellatus FRIES), maitake
mushroom (black, MAITAKE, Grifola frondosu Dickson),
matsutake mushroom (MATSUTAKE, Tricholoma matsutake) and
shiitake mushroom (SHIITAKE, Cortinellus Shiitake) were
used. With respect to agaricus mushroom (AGARIKUSUTAKE),
it was found by the present inventor's experiments that it
shows potent antitumor activity when it is used in a raw
state than the case where it is used after drying, so that
it was stored in a refrigerator at a low temperature (4°C)
after the harvest and used within 3 days from the harvest.
With regard to the other mushrooms than the agaricus
mushroom, not so significant differences in effectiveness
were observed when they are used in the raw state and in
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the dried state which has heretofore been provided as a
Chinese medicine to which the antitumor activity
strengthening method of the present invention is applied so
that in the present Example, dried materials were used with
regard to PORIA (BUKURYO), POLYPORUS (CHOREIMAITAKE,
Polyporus umbellatus FRIES), maitake mushroom (black,
MAITAKE, Grifola frondosu Dickson), matsutake mushroom
(MATSUTAKE, Tricholoma matsutake) and shiitake mushroom
(SHIITAKE, Cortinellus Shiitake).
Incidentally, in the present embodiment, two kinds of
maitake mushrooms were used. Those produced in Japan are
those mainly cultured in Niigata prefecture which is a snow
country and which are formally called as snow country
maitake mushroom (Yukiguni maitake) or black maitake
mushroom (Kuromaitake), and in the present specification,
it is mentioned as maitake mushroom (black).
On the other hand, maitake mushroom (MAITAKE, Grifola
frondosu Dickson) obtained in China is mentioned as
POLYPORUS (CHOREIMAITAKE, Polyporus umbellatus FRIES) in
the present specification and distinguished from the above-
mentioned maitake mushroom (black) depending on necessity.
In particular, in POLYPORUS (CHOREIMAITAKE, Polyporus
umbellatus FRIES), a root is mainly used.
The first step of the antitumor activity
strengthening method of the present Example is a far
infrared rays-roasting step in which the above-mentioned
various kinds of mushrooms are roasted by far infrared rays.
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CA 02282234 1999-09-16
The far infrared rays to be used in such a far infrared
rays-roasting step are preferably far infrared rays having
a wavelength of about 4 to 14 um. Mushrooms are roasted by
using an apparatus such as a pan, a kiln, etc. in which a
ceramics which irradiates far infrared rays having a
wavelength with such a range is coated at the inner surface
thereof, an apparatus such as a pan, a kiln, etc. prepared
by mixing ceramic powder which irradiates far infrared rays
with stone, gravel, sand, etc. of metal oxides.
For effecting such a roasting, other than agaricus
mushroom (AGARIKUSUTAKE), those which are once dried as
used in the usual Chinese medicines are used. With regard
to the agaricus mushroom (AGARIKUSUTAKE), it is used in a
raw state as mentioned above.
The above-mentioned mushrooms are roasted in the
above-mentioned apparatus, for example, in an earthen pot
prepared from far infrared rays irradiating substances such
as granite, ceramics, Tenshouseki stone, etc. and
irradiating far infrared rays with a wavelength of 4 to 14
um with the extent that they are not burned for 30 to 90
minutes while gradually and well stirring.
As the roasting method, it is not limited by the
above-mentioned method, and it may be any method so long as
it promotes liberation of B-glucan of mushrooms. For
example, as a far infrared rays source to be used for
roasting, in addition to the above-mentioned apparatuses,
optional ones may be used so long as it comprises a
- 25 -
CA 02282234 1999-09-16
material which irradiates far infrared rays with a
wavelength of 4 to 14 um such as platinum electromagnetic
wave fiber, etc.
Subsequent to such a far infrared rays roasting step,
a fermentation step is provided as a second step. After
applying the far infrared rays roasting treatment as
mentioned above, by using fermentation bacteria such as
"Koji" or yeast, fermentation is carried out in a room
containing moisture at 30 to 36°C for 48 to 72 hours. It
may be carried out by using a fermentation apparatus to
shorten the fermentation time.
As microorganisms to be used for fermentation,
microorganisms other than "Koji" may be used.
Also, for effecting fermentation, in addition to
"Koji", a substance having fermentating power such as
matured papaya, juice of pineapple, the rind of a fig, the
rind of a grape, a bark of a young bamboo, etc. may be used.
Moreover, digestive enzymes such as diastase, pancreatine,
etc., proteolytic enzymes derived from microorganisms such
as protease, pepsin, trypsin, etc., lytic enzymes of
polysasccharide such as hemicellulase, or a precursor
substance which forms the above-mentioned digestive enzymes
or proteolytic enzymes, etc. may be used for fermentation.
However, according to the experiments by the present
inventor, more preferred results can be obtained when
fermentation is carried out by using microorganisms, and
when fermentation is carried out "Koji" among the
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CA 02282234 1999-09-16
microorganisms in the point of strengthening antitumor
activity. After completion of such a fermentation step,
the starting materials were pulverized.
For pulverization, commercially available pulverizer
may be used, but a machine which generates high temperature
at the time of pulverization shall be preferably avoided.
It may be carried out by using a pulverization method which
does not generate high temperature such as stone mill.
The materials of starting mushrooms to which the far
infrared rays roasting step and fermentation step are
applied are further subjected to an oily agent-making step.
This procedure for making oily agent is to enclose the
above-mentioned fine powder of the starting material fine
powder after fermentation with an oily component from
sesame to which far infrared rays roasting is applied.
As the oily component to be used for making oily
agent, there may be used an oil (hereinafter referred to as
sesame paste oil) collected from roasted sesame. The
sesame paste oil is an oil obtained by subjecting raw
sesame to far infrared rays roasting at a temperature not
exceeding 100 C slowly for a long time, and grinding down
and compressing the sesame after roasting. In such a
sesame paste oil, fine solid materials formed by grinding
down the sesame are remained as such so that it takes a
paste-like appearance.
To such a sesame paste oil is added the above-
mentioned pulverized mushrooms after fermentation to
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CA 02282234 1999-09-16
enclose the finely pulverized mushrooms with an oily
component of the sesame. By enclosing the mushrooms,
permeation force thereof to the cells at the diseased
portion can be strengthened as compared with the case where
no such an enclosure with the paste oil is carried out.
Such an oily agent-making step is an important step to
provide an effective target directivity in the viewpoint of
drug delivery system (DDS) which is attracted attention in
recent years.
Incidentally, the oily agent-making step is an
important step as mentioned above, and it is inherently
preferred to provide such a step, but if there is some
difficulty that to make an oily agent cannot suitably be
carried out, such an oily agent-making step may be omitted.
The reason why the above can be said is that, as mentioned
below, it is inspected by the test that if the far infrared
rays roasting step and the fermentation step are certainly
carried out, effectiveness of strengthening antitumor
activity becomes significantly large even when the oily
agent-making step is not carried out as mentioned below.
Also, for making an oily agent, more preferred
results can be obtained when a mixed oil in which the
above-mentioned sesame paste oil and an oil collected from
raw sesame are mixed with a suitable ratio is used.
By mixing the oil collected from raw sesame, a size
of an oil droplet ca be made small, whereby a penetrating
power to the target cells can be markedly heightened as
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CA 02282234 1999-09-16
compared with the case that the mushrooms are enclosed with
the sesame paste oil having a high viscosity and a large
oil droplet size.
The oil collected from the raw sesame means an oil
obtained by grinding down raw sesame as such and
compressing the resulting material, then removing solid
material, which corresponds to a commercially available
usual sesame oil.
A ratio of mixing the sesame paste oil and the above-
mentioned sesame oil obtained from raw sesame may vary
depending on the amount of the starting fine powder to be
added so that it cannot regulate sweepingly but suitably 1
to 3 parts by weight of the sesame oil based on 1 part by
weight of the sesame paste oil. Also, a mixing ratio of
the mixing oil and the starting fine powder may be, for
example, about 4 to 5 parts by weight of the starting fine
powder based on about 1 part by weight of the mixing oil.
The mushrooms in which their antitumor activities had
been strengthened by the above-mentioned manner were added
to a system which forms lipid peroxide by irradiating
ultraviolet rays to docosahexaenoic acid and an amount of
the formed lipid peroxide was examined. In addition, their
antitumor effects by internal administration were also
measured by clinical tests.
Also, to compare the antitumor activity strengthening
method of the above-mentioned explanation, formed amount of
the above-mentioned lipid peroxide, and effectiveness in
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CA 02282234 1999-09-16
the clinical tests were examined with respect to the case
where the far infrared rays roasting alone was applied to
the above-mentioned mushrooms, the case where the
polysaccharide lytic enzyme fermentation alone was applied
to the same, the case where the "Koji" fermentation alone
was applied to the same, the case where both of the far
infrared rays roasting and the "Koji" fermentation were
applied to the same, and the untreated case where no
treatment was applied, respectively.
For effecting the above-mentioned polysaccharide
lytic enzyme fermentation, various polysaccharide lytic
enzymes can be used, but in the present Example,
hemicellulase which is a representative one among them was
used.
Incidentally, in the case where the far infrared rays
roasting alone is applied to mushrooms, the case where the
polysaccharide lytic enzyme fermentation alone was applied
to the same, the case where the "Koji" fermentation alone
was applied to the same, and the case where both of the far
infrared rays roasting and the "Koji" fermentation were
applied to the same, at the time at which the respective
treatments were carried out, the material was pulverized to
fine powder with the procedure as mentioned above, and used
in the following experiments.
The degree of pulverization of the respective samples
is so adjusted that an average pulverized particle size
becomes the same degree as others to exclude the effect on
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CA 02282234 1999-09-16
the pulverized particle size. In the case of untreated,
the agaricus mushroom (AGARIKUSUTAKE) was used with a raw
state and finely chopped, and the other mushrooms than the
above, they were used by making fine powders after drying.
Measurement of the formed amount of the above-
mentioned lipid peroxide was carried out as mentioned below.
A predetermined amount of docosahexaenoic acid was placed
in a plural number of test tubes, and the above-mentioned
mushrooms weighed previously with different amounts were
added to the respective docosahexaenoic acid and they were
mixed. In addition, ultraviolet rays were irradiated to
the contents in the respective test tubes and amounts of
the formed lipid peroxides were measured.
Docosahexaenoic acid was used by previously preparing
a diluted solution in which the raw liquid was diluted with
ethanol 10-folds and the diluted solution was further
diluted to become 200-folds as the final diluted
concentration.
The samples of the mushrooms to which the above-
mentioned treatments (including untreated) were applied was
added to predetermined ethanol so as to become the
concentration of the mushroom of 60 mg/ml, and the ethanol
was allowed to stand at normal temperature for about 2 to 6
weeks whereby extraction with ethanol was carried out. For
using the sample in the test, the extract was used by
diluting the final concentration of 0.6 mg/ml.
By mixing 0.05 ml of the diluted docosahexaenoic acid
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CA 02282234 1999-09-16
solution prepared by the manner as mentioned above, 0.1 ml
of a mushroom sample and 0.85 ml of ethanol in a test tube,
the total amount was made 1 ml. This test tube was
irradiated by ultraviolet rays for 3 hours to form a lipid
peroxide. The formed lipid peroxide was measured by the
TBA reaction (thiobarbituric acid reaction).
By heating the formed lipid peroxide under acidic
conditions, liberated malondialdehyde (MDA) was reacted
with thiobarbituric acid (TBA) and the formed absorption
maximum substance was determined to obtain the formed
amount of the lipid peroxide indirectly.
For effecting the TBA reaction, 0.2 ml of a 7~ sodium
dodecylsulfate, 2 ml of O.1N hydrochloric acid, 0.3 ml of
phosphotungstic acid and 1 ml of the reagent prepared by
mixing 0.67 of TBA and acetic acid with a ratio of 1:1
were added to the respective test tubes to which
ultraviolet rays had been irradiated, and the TBA reaction
was carried out by maintaining at 95°C for 12 minutes after
the addition.
Thereafter, the mixture was cooled to room
temperature, the TBA reaction layer was extracted with n-
butyl alcohol, and the fluorospectrophotometric method was
applied to the n-butyl alcohol layer to measure the above-
mentioned MDA amount. For effecting the measurement, light
with a wavelength of 515 nm was used as an exciting light
and fluorometry was carried out at a wavelength of 535 nm.
The fluorospectrophotometer used was F2000 manufactured by
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CA 02282234 1999-09-16
Hitachi Ltd. using a 400 V photomultiplier, and the
measurement was carried out by making the band pass for an
excited light irradiation of 20 nm and the band pass at the
fluorescence measurement of 20 nm.
With regard to the effects of docosahexaenoic acid on
the mushrooms to which the above-mentioned respective
treatments were applied by ultraviolet rays irradiation in
the lipid peroxide forming system, they were examined in
relation with the MDA value (which is the same as in MDA
amount, shown by absorbance) measured by the above-
mentioned manner. The results are shown in Table 1.
On the other hand, among the above-mentioned
mushrooms, with regard to PORIA (BUKURYO), agaricus
mushroom (AGARIKUSUTAKE), maitake mushroom (MAITAKE,
respective of black maitake mushroom (Kuromaitake) (snow
country maitake mushroom (Yukiguni maitake)) and POLYPORUS
(CHOREIMAITAKE, Polyporus umbellatus FRIES) were used),
clinical tests were carried out about breast cancer,
stomach cancer and lung cancer in the respective cases of
untreated, the polysaccharide lytic enzyme fermentation
treatment, "Koji" fermentation treatment, far infrared rays
roasting treatment + "Koji" fermentation treatment, far
infrared rays roasting treatment + "Koji" fermentation
treatment + oily agent-making treatment. The results are
shown in Tables 2, 3 and 4.
In Table 2, treatment effects of the above-mentioned
mushrooms to which various kinds of methods are applied on
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CA 02282234 1999-09-16
breast cancer patients are shown. Tables 3 and 4 show
treatment effects on stomach cancer patients and lung
cancer patients of the various kinds of mushrooms to which
the same treatments were carried out as in Table 2.
Incidentally, in Tables~2 to 4, effective ratios calculated
from the following formula are also shown.
Effective ratio = (Effective number + Slightly effective
number) x 100/(Effective number + Slightly effective number
+ non-effective number)
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CA 02282234 1999-09-16
Table 1 Effects of various mushrooms on MDA production
Kind of Item to be tested and MDA value (Average)Order
specimen
treatment
Control (Mushroom not 115
added)
No treatment pOZ'ld 269 4
Agaricus mushroom 283 3
Polyporus umbellatus 439 1
Fries
Maitake mushroom (Black)398 2
Matsutake mushroom 218 5
Siitake mushroom 189 6
Far infrared ppria 288 4
rays roastingAgaricus mushroom 341 3
Polyporus umbellatus 536 1
Fries
Maitake mushroom (Black)490 2
Matsutake mushroom 271 5
Siitake mushroom 233 6
Polysacchariderla 284 5
lytic enzyme Agaricus mushroom 375 3
fermentation Polyporus umbellatus 551 1
Fries
Maitake mushroom (Black)525 2
Matsutake mushroom 304 4
Siitake mushroom 265 6
"Koji" POrlB 296 5
fermentation Agaricus mushroom 399 3
Polyporus umbellatus 594 1
Fries
Maitake mushroom (Black)528 2
Matsutake mushroom 302 4
Siitake mushroom 280 6
Far infrared p~ria 334 6
rays roastingAgaricus mushroom 489 3
+
"Koji" Polyporus umbellatus 799 1
Fries
fermentation Maitake mushroom (Black)768 2
Matsutake mushroom 412 4
Siitake mushroom 381 5
Far infrared poria 362 6
rays roastingAgaricus mushroom 509 3
+
"Koji" Polyporus umbellatus 827 1
Fries
fermentation Maitake mushroom (Black)792 2
+
oily agent Matsutake mushroom 430 4
making Siitake mushroom 397 5
Respective mushrooms were added 6 mg/ml
- 35 -
CA 02282234 1999-09-16
Table 2 Treatment effects of various mushrooms on breast
cancer patients
Kind of treatment Breast Effective
cancer
(200
persons)
Specimen EffectiveSlightlyNot Ratio
(%)
effectiveeffective
No treatment Poria 0 3 37 7.5
Agaricus mushroom1 2 37 7.5
Maitake mushroom 1 3 36 10.0
Polysaccharide Porla 1 4 35 12.5
lytic enzyme Agaricus mushroom2 6 32 20.0
fermentation Maitake mushroom 4 5 31 22.5
"Koji" TlJria 1 5 34 15.0
fermentation Agaricus mushroom3 7 30 25.0
Maitake mushroom 4 6 30 25.0
Far infrared ppria 2 4 34 15.0
rays
roasting + "Koji"Agaricus mushroom4 6 30 25.0
fermentation Maitake mushroom 12 7 31 47.5
Far infrared rla 3 5 32 20.0
rays
roasting + "Koji"Agaricus mushroom6 8 26 35.0
fermentation Maitake mushroom 14 9 17 57.5
+
oily agent-making
The number in the parenthesis shows the number of total
person tested.
(Effective + Slightly effective) sample number
Effective ratio = x 100
Total sample number
Hereinafter the same as in Tables 3 and 4.
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CA 02282234 1999-09-16
Table 3 Treatment effects of various mushrooms on stomach
cancer patients
Kind of treatment Stomach Effective
cancer
(150
persons)
Specimen Slightly Not ratio
(%)
Effectiveeffectiveeffective
No treatment POrla 0 2 28 6.7
Agaricus mushroom0 2 28 6.7
Maitake mushroom 1 1 28 6.7
PolysaccharidePoria 2 1 27 10.0
lytic enzyme Agaricus mushroom2 3 25 16.7
fermentation Maitake mushroom 3 3 24 20.0
"Koji" Poria 1 2 27 10.0
fermentation Agaricus mushroom2 4 24 20.0
Maitake mushroom 3 5 22 26.7
Far infrared ppria 1 3 26 13.3
rays
roasting + Agaricus mushroom3 5 22 26.7
"Koji"
fermentation Maitake mushroom 5 7 18 40.0
Far infrared pOria 1 4 25 16.7
rays
roasting + Agaricus mushroom4 7 19 36.7
"Koji"
fermentation Maitake mushroom 6 8 16 46.7
+
oily agent-making
The number in the parenthesis shows the number of total
person tested.
- 37 -
CA 02282234 1999-09-16
Table 4 Treatment effects of various mushrooms on lung
cancer patients
Kind of treatment Lung Effective
cancer
(100
persons)
Specimen SlightlyNot ratio
(%)
Effectiveeffectiveeffective
No treatment Poria 0 1 19 5.0
Agaricus mushroom 0 2 18 10.0
Maitake mushroom 1 1 18 10.0
PolysaccharidepOrld 1 1 18 10.0
lytic enzyme Agaricus mushroom 1 2 17 15.0
fermentation Maitake mushroom 3 4 13 35.0
"Koji" Poria 1 2 17 15.0
fermentation Agaricus mushroom 1 3 16 20.0
Maitake mushroom 3 5 12 40.0
Far infrared poria 2 1 17 15.0
rays
roasting + Agaricus mushroom 3 3 14 20.0
'Koji"
fermentation Maitake mushroom 4 5 11 45.0
Far infrared poria 2 2 16 20.0
rays
roasting + Agaricus mushroom 3 4 13 35.0
"Koji"
fermentation Maitake mushroom 5 5 10 50.0
+
oily agent-making
The number in the parenthesis shows the number of total
person tested.
- 38 -
CA 02282234 1999-09-16
From the above-mentioned Table 1, it can be
understood that the MDA value which is an index of the
formed amount of lipid peroxide becomes greater, in the
same kind of mushrooms, when the far infrared rays roasting
is applied to than the untreated case, when the
polysaccharide lytic enzyme fermentation is applied to than
the case where the far infrared rays roasting is applied,
when the "Koji" fermentation is applied to than the
polysaccharide lytic enzyme fermentation, when the both of
the far infrared rays roasting and "Koji" fermentation are
applied to than the case where "Koji" fermentation is
applied, and when the three of the far infrared rays
roasting, "Koji" fermentation and oily agent-making
procedure are applied to than the case where the both of
the far infrared rays roasting and "Koji" fermentation are
applied, respectively.
Such a tendency is common irrespective of the kinds
of the mushrooms to be applied to the tests except for the
case of PORIA (BUKURYO) between the far infrared rays
roasting and the polysaccharide lytic enzyme fermentation,
and the case of matsutake mushroom (MATSUTAKE, Tricholoma
matsutake) between the polysaccharide lytic enzyme
fermentation and the "Koji" fermentation.
Substantially the same (correctly the value of one of
the treatment methods is larger about 1~ than that of the
other treatment method) MDA value can be obtained when the
far infrared rays roasting is applied to and when the
- 39 -
CA 02282234 1999-09-16
polysaccharide lytic enzyme fermentation is applied to with
regard to PORIA (BUKURYO), and when the polysaccharide
lytic enzyme fermentation is applied to and when the "Koji"
fermentation is applied to with regard to matsutake
mushroom (MATSUTAKE, Tricholoma matsutake).
On the other hand, from Tables 2 to 4 showing the
clinical test results of administering PORIA (BUKURYO),
agaricus mushroom (AGARIKUSUTAKE), maitake mushroom
(MAITAKE, black maitake mushroom (Kuromaitake) (snow
country maitake mushroom (Yukiguni maitake)), POLYPORUS
(CHOREIMAITAKE, Polyporus umbellatus FRIES)) to which the
above-mentioned respective treatments were applied, to
breast cancer patients, stomach cancer patients and lung
cancer patients, it can be understood that effective ratios
in the respective cases of breast cancer patients, stomach
cancer patients and lung cancer patients becomes higher, in
the same kind of mushrooms, when the polysaccharide lytic
enzyme fermentation is applied to than the untreated case,
when "Koji" fermentation is applied to than the case where
the polysaccharide lytic enzyme fermentation is applied,
when the both of the far infrared rays roasting and "Koji"
fermentation are applied to than the case where "Koji"
fermentation is applied, and when the three of the far
infrared rays roasting, "Koji" fermentation and oily agent-
making procedure are applied to than the case where the
both of the far infrared rays roasting and "Koji"
fermentation are applied, respectively.
- 40 -
CA 02282234 1999-09-16
For example, from Table 2, in PORIA (BUKURYO), it can
be understood that the effective ratio is increased when
the far infrared rays roasting + "Koji" fermentation + oily
agent-making procedure are applied to as compared with the
untreated case from 7.5~ to 20.0, i.e., about three times.
In agaricus mushroom (AGARIKUSUTAKE), the effective ratio
is increased from 7.5~ to 35~, i.e., about 4.9 times. In
maitake mushroom (MAITAKE), the effective ratio is
marvelously increased in manifesting property of an
antitumor effect from 10.0 to 57.5, i.e., 5.75 times.
Such a tendency is the same as in Tables 3 and 4.
From the above results, it is shown that the
treatment method which is successively combined the far
infrared rays roasting step, the "Koji" fermentation step,
and the oily agent-making step can be effectively used as
an increasing method of manifesting property of the
antitumor activity of mushrooms, i.e., as the antitumor
activity strengthening method.
- 41 -
CA 02282234 1999-09-16
Table 5 Amount of free B-glucan in the respective kinds of
mushrooms of untreated and to which the far
infrared rays roasting and the "Koji" fermentation
treatment are applied
Kind of treatment Specimen Amount of
d-glucan
Untreated m S
s
mushroom black) 90 g/100 g
Maitake (MAITAKEU
24.4 g/100
g
Far infrared rays Agaricus mushroom(AGARIKUSUTAKE)
14.2 g/100
roasting step + Maitake mushroom(MAITAKE, g
"Koji" black)
fermentation step
30.4 g/100
g
On the other hand, amounts of free B-glucan as an effective
ingredient showing an antitumor activity were measured in
the case where the treatment to enlarge manifestation of
the above-mentioned antitumor effect and the case of
untreated. The mushrooms used were agaricus mushroom
(AGARIKUSUTAKE) and maitake mushroom (MAITAKE, black
maitake mushroom (Kuromaitake)) which showed large
effective ratios in Tables 2 to 4. Incidentally, the
analyses were carried out at the Food Analysis Center at
Suita, Osaka, Japan. The results are shown in Table 5.
From the results of Table 5, it can be understood
that amounts of free B-glucan were increased when the far
infrared rays roasting + "Koji" fermentation treatment were
applied to in either of agaricus mushroom (AGARIKUSUTAKE)
and maitake mushroom (MAITAKE). With regard to maitake
mushroom (MAITAKE), other than the black maitake mushroom
(Kuromaitake), the same effects can be obtained also in
- 42 -
CA 02282234 1999-09-16
POLYPORUS (CHOREIMAITAKE, Polyporus umbellatus FRIES)). On
the other hand, in the clinical test results in Tables 2 to
4, it is shown that the antitumor effects are higher in the
case of applying the far infrared rays roasting treatment +
"Koji" fermentation treatment than the case of untreated.
Thus, by examining the results of Tables 2 to 4 and
those of Table 5 in combination, it can be considered that
among B-glucans effective for antitumor activity of
mushrooms, particularly when an amount of free B-glucan is
a large, the antitumor effects are remarkably manifested.
The present inventor has progressed the above-
mentioned series of studies by setting forth the hypothesis
as mentioned above that, in the non-treated mushrooms, B-
glucans having antitumor action are in an unmovable state
by binding to each other to form a chain (a simile
expression) which is so-called non-activated state, and
when mushrooms are internally administered, the chain
cannot be cut by a gastric juice of a usual person whereby
the antitumor action would not be shown than expected.
However, as mentioned above, it can be found that increase
in an amount of free B-glucan markedly affects to
manifestation of the antitumor action and the results which
could be expected by the present inventors hypothesis can
be obtained.
Also, the composition produced by the far infrared
rays roasting step, the fermentation step and the oily
agent-making step which constitute the antitumor activity
- 43 -
CA 02282234 1999-09-16
strengthening method as mentioned above is, as shown in
Table 5, etc., a composition in which an amount of free B-
glucan is increased and shows the antitumor activity
effectively by internal administration. As compared with
the composition containing mushrooms (untreated mushrooms)
to which the above-mentioned antitumor activity
strengthening method is not applied, its antitumor activity
is markedly strengthened.
Also, from Table 1, it can be understood that the
order of the height of the MDA value in the mushrooms are
changed between various kinds of treatments including
untreated ones. In the order shown in Table 1, with regard
to three kinds of PORIA (BUKURYO), Matsutake mushroom
(MATSUTAKE) and Shiitake mushroom (SHIITAKE), they show a
possibility of changing the order, i.e., effectiveness of
the antitumor activity in the three treatment methods of
the untreated, "Koji" fermentation and far infrared rays
roasting + "Koji" fermentation.
For example, it can be understood that in the case of
the untreated one, the antitumor activity becomes high in
the order of Shiitake mushroom (SHIITAKE), Matsutake
mushroom (MATSUTAKE) and PORIA (BUKURYO), whereas the far
infrared rays roasting and the "Koji" fermentation are
applied, the antitumor activity becomes high in the order
of PORIA (BUKURYO), Matsutake mushroom (MATSUTAKE) and
Shiitake mushroom (SHIITAKE). It is understood that, with
applying the above-mentioned antitumor activity
- 44 -
CA 02282234 1999-09-16
strengthening method according to the present invention, it
is possible to raise an evaluated order for untreated
mushrooms which had been evaluated to be lower in the
antitumor activity.
In the field of Chinese medicine using a natural
crude drug, there is a scarce species which cannot ensure
an amount of yield stably even when it is a mushroom having
high antitumor activity. However, when the above-mentioned
method of the present invention is used, in place of such a
scarce species, it is possible to use mushrooms which can
ensure a sufficient amount of yield or can cultivate.
According to this, stabilization of supply and low price of
the product can be established whereby many persons can
effectively enjoy the antitumor effects of mushrooms.
Incidentally, in the above-mentioned explanation, as
a best embodiment, only the treatment in which three kinds
of the far infrared rays roasting treatment, the
fermentation treatment and the oily agent-making treatment
are applied is explained. However, only the far infrared
rays roasting treatment is applied, or only the
polysaccharide lytic enzyme fermentation treatment such as
hemicellulase, etc. is applied, or only the fermentation
treatment by microorganisms such as "Koji", etc., or only
the far infrared rays roasting and the fermentation
treatment are applied, a manifestation of the antitumor
effects are strengthened as compared with the non-treated
case.
- 45 -
CA 02282234 1999-09-16
Thus, when there are any reasons that the oily agent-
making cannot suitably carried out due to the conditions of
equipments, the treatments of the far infrared rays
roasting and the fermentation by microorganism can be
carried out in combination, the antitumor activity can be
markedly effectively shown than that of the non-treated
case.
Moreover, in the treatment in which to the above-
mentioned mushrooms are applied three kinds of treatments
of the far infrared rays roasting, the fermentation
treatment and the oily agent-making treatment, or to the
mushrooms are applied two kinds of treatments of the far
infrared rays roasting and the fermentation treatment, in
addition to the fermentation treatment using microorganisms
such as °Koji" or yeast bacteria, there may be used a
fermentation treatment using a polysaccharide lytic enzyme.
In the oily agent-making step in the above-mentioned
antitumor activity strengthening method, a sesame paste oil
obtained from a roasted sesame is used, but other than
sesame, plants oil obtained by roasting soy bean, corn, a
safflower, an evening primrose, rice bran, rapeseed, etc.
by far infrared rays, and then squeezing and pressing may
be used.
Also, in the composition containing mushrooms to
which the above-mentioned antitumor activity strengthening
method is applied, in addition to the materials obtained by
treating mushrooms by the above-mentioned method, any
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component which does not participate any bad effects on
showing the antitumor activity, such as a vitamin agent,
etc., may be further added.
As a composition having such a constitution, a
preparation having an active oxygen suppressing effect
which has been developed by the present inventor may be
formulated. The antitumor activity strengthening treated-
crude drug containing compound having such a constitution
may be actually provided in an agent form which can be
internally administered easily by encapsulating them in a
gelatin capsule, etc.
Moreover, by using a suitable excipient, a binder,
etc., it may be a tablet by tabletting, or may be in a
preparation form such as a granule or a pill without any
problem. Furthermore, whereas the present invention is a
method of strengthening antitumor activity by an internal
administration, it can be considered that an injection
preparation prepared by using mushrooms to which the
antitumor activity strengthening method of the present
invention is applied can be expected to have an effect of
enlarging manifestation of the antitumor effect as compared
with the case where the antitumor activity strengthening
method of the present invention is not applied to.
Incidentally, in the above-mentioned explanation, an
explanation was made about the case where mushrooms such as
agaricus mushroom (AGARIKUSUTAKE), but it is not necessary
to restrict the mushrooms only to the above-mentioned
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mushrooms, and any mushrooms than the above may be used so
long as it contains B-glucan. For example, it may be
shimejitake mushroom (SHIMEJITAKE, Lyophyllum decastes),
enokitake mushroom (ENOKITAKE, Flammulina velutipes), etc.
Moreover, a crude drug containing a polysaccharide
having an antitumor activity other than B-glucan may be
used. Furthermore, the present invention can be used as a
method for strengthening manifestation of the antitumor
effect containing a polysaccharide having an antitumor
activity such as B-glucan in a natural or cultivated
products derived from plants widely including mushrooms.
(Embodiment 2)
In the present embodiment, an evaluating method of
effectiveness on antitumor activity for a crude drug, and
an antitumor effectivity evaluating method of treated by
crude drug are explained.
To the experiment system in which lipid peroxide is
formed by irradiating ultraviolet rays explained in the
above-mentioned embodiment 1 is added a crude drug for
evaluating antitumor activity, e.g., mushrooms, and a
formed amount of lipid peroxide is examined. When the
mushrooms are added with increasing their amounts, a
mushroom which has a tendency of markedly increasing the
formed amount of lipid peroxide shows a potent antitumor
activity when it is administered.
That is, when the increased ratio of the formed
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amount of lipid peroxide is large based on the increased
ratio of the concentration of the mushroom added, the
mushroom can be evaluated as it shows potent antitumor
effect when it is internally administered. Thus, a plural
kinds of mushrooms are introduced in the above-mentioned
experimental system, and when the increased ratio of the
formed amount of lipid peroxide based on the ratio of the
concentration of the mushroom added is compared to each
other, the order of effectiveness on showing antitumor
effect by internal administration of the plural kinds of
mushrooms can be determined.
In the following, the above-mentioned both of the
evaluating methods in the embodiment 2 of the invention are
explained based on the above-mentioned examples. When the
MDA values with respect to the respective treatment methods
in Table 1 mentioned above are compared to each other, it
can be understood either of the cases in which any other
treatment such as far infrared rays roasting, etc. is
carried out showed a larger MDA value as compared with the
case of non-treatment.
When either of the far infrared rays roasting, or the
"Koji" fermentation is applied, the MDA value is larger
when the mushrooms to which the "Koji" fermentation is
applied. When the "Koji" fermentation and the far infrared
rays roasting are used in combination, the MDA value is
larger than the case where either one of the far infrared
rays roasting, or the "Koji" fermentation is applied singly.
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Moreover, the MDA value when three of the far infrared rays
roasting, the "Koji" fermentation and the oily agent-making
treatments are applied is larger than that of the case
where two of the far infrared rays roasting and the "Koji"
fermentation are applied.
On the other hand, from Tables 2, 3 and 4 mentioned
above in which clinical tests were carried out with regard
to breast cancer, stomach cancer and lung cancer in the
cases of untreated, the polysaccharide lytic enzyme
fermentation treatment, the "Koji" fermentation treatment,
the far infrared rays roasting treatment + the "Koji"
fermentation treatment, and the far infrared rays roasting
treatment + the "Koji" fermentation treatment + the oily
agent-making treatment, respectively, with regard to PORIA
(BUKURYO), agaricus mushroom (AGARIKUSUTAKE), maitake
mushroom (MAITAKE, black maitake mushroom (Kuromaitake)
(snow country maitake mushroom (Yukiguni maitake)) and
POLYPORUS (CHOREIMAITAKE, Polyporus umbellatus FRIES) were
each used) among the above-mentioned mushrooms, it can be
understood that, in the same mushroom, the clinical effect
becomes high as the treatment showing high MDA value shown
in Table 1 is applied.
From such results, height of the clinical effect,
i.e., effectiveness of the antitumor effect can be judged
by using the MDA value as an index, and it can be said that
the treatment which makes the formed amount of the MDA
value high is effective for showing the antitumor effect.
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That is, as the MDA value is large, which shows an
amount of lipid peroxide formed by irradiating ultraviolet
rays to docosahexaenoic acid, the treatment can be
evaluated as it has high effectivity in antitumor activity
of mushrooms .
Also, when the results of Table 1 and those of Tables
2 to 4 are compared, in the same treatment method to
mushrooms, the order of height of the MDA value is in
agreement with the order of height of effective ratio in
clinical effects. That is, to the system which forms lipid
peroxide by irradiating ultraviolet rays to the above-
mentioned docosahexaenoic acid is added a different
mushroom to which the same treatment is applied with the
same concentration, and the formed amount of the lipid
peroxide is shown with a MDA value (amount) in the same
manner as in the above-mentioned embodiment, it can be said
that the mushroom which makes the formed amount of MDA high
is effective for showing the antitumor effect.
Accordingly, the antitumor activity of the respective
kinds of mushrooms can be expected as the mushrooms having
a large MDA value which shows an amount of lipid peroxide
formed by irradiating ultraviolet rays to docosahexaenoic
acid have high antitumor activity at the time of internal
administration. That is, without effecting clinical tests,
effectiveness of the antitumor activity of the respective
kinds of mushrooms by internal administration can be
expected.
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Incidentally, in the above explanation, an
explanation was made in the case where it is applied to
mushrooms such as agaricus mushroom (AGARIKUSUTAKE), etc.,
but as the mushrooms to be applied, it is not limited only
to the above-mentioned mushrooms. The present invention
can be applied to mushrooms other than the above so long as
it contains B-glucan.
Moreover, the present invention can be applied to
mushrooms containing polysaccharides having antitumor
activity other than B-glucan. Furthermore, the present
invention may be applied to widely those which are crude
drugs such as a natural or cultivated products derived from
plants other than mushrooms and contain polysaccharides
having antitumor activity such as B-glucan.
Also, in the above-mentioned explanation,
docosahexaenoic acid was used, but a fatty acid other than
docosahexaenoic acid can be used, which is, for example, an
unsaturated fatty acid in which an amount of lipid peroxide
formed by irradiation of ultraviolet rays by addition of
the above-mentioned mushrooms. Furthermore, such
unsaturated fatty acid derived from living body in which an
amount of lipid peroxide formed by irradiation of
ultraviolet rays by addition of the above-mentioned
mushrooms may be used in the range in which the above-
mentioned evaluation method can be applied, even when it is
not derived from a living body, i.e., a synthetic fatty
acid.
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Also, by adding mushrooms containing a carcinostatic
polysaccharide to the system which forms lipid peroxide by
irradiating ultraviolet rays to docosahexaenoic acid as
explained above with various kinds of concentrations, and
by catching the amounts of the formed lipid peroxides with
the respective concentrations, an average and suitable
administration dose in comply with the progressed stage of
the malignant tumor such as cancer can be easily expected
without carrying out clinical test about the respective
mushrooms to be used.
That is, the relationship between the concentration
of the mushroom and the MDA value in the respective kinds
of mushrooms is grasped with the above-mentioned manner,
and the MDA value and the effectiveness thereof at the
respective progress stage of cancer are previously
confirmed by the clinical test in a certain kind of
mushrooms. By effecting the above, if the concentration of
said mushroom is calculated back so as to obtain an MDA
value which had been confirmed to be effective in the
respective progress stage of tumor such as cancer, etc., an
average internal administration dose can be easily expected
from the concentration of the mushroom.
Also, in the present invention, the antitumor
activity of a crude drug can be compared through a common
index such as the MDA value so that the antitumor activity
of the various kinds of crude drugs which had been compared
by carrying out clinical tests can be realized to compare
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with each other without effecting clinical tests, i.e.,
without exerting danger by some chance to a patient.
That is, the meanings that the present inventor has
found the MDA value as a parameter which is capable of
using as a common measure for the respective kinds of crude
drugs, and mutual evaluation of the antitumor activity is
realized by correlating the MDA value and the antitumor
activity are significantly large in the point that mutual
evaluation of a number of crude drugs can be carried out
simply and easily.
According to the antitumor activity strengthening
method of crude drugs of the present invention, effectivity
in the antitumor activity of the crude drugs such as
mushrooms at internal administration can be heightened as
compared with the case in which such a method is not
applied.
According to the antitumor activity strengthening
method of crude drugs of the present invention, even in
crude drugs such as mushrooms, etc. which are not admitted
as effective when the method of the present invention is
not applied, they can be applied to as an antitumor agent
by heightening effectivity at internal administration.
The antitumor activity strengthened crude drug
composition containing crude drugs such as mushrooms to
which the antitumor activity strengthening method of the
present invention is applied has high antitumor effectivity
at internal administration as compared with the composition
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containing crude drugs to which such a method is not
applied.
According to the evaluating method of antitumor
effectivity of treatment by the crude drugs according to
the present invention, effectivity in view of the antitumor
activity by the various kinds of treatment methods applied
to the crude drugs can be simply evaluated within a short
term.
According to the evaluating method of antitumor
effectivity of the crude drugs according to the present
invention, effectivity of the crude drugs at internal
administration can be expected without effecting clinical
tests. Thus, as compared with the conventional method in
which the results can be obtained by carrying out a long
term of animal experiments or clinical tests, a search of
an antitumor effective substance by screening a tremendous
number of natural products can be carried out simply as
compared with the conventional manner.
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