Note: Descriptions are shown in the official language in which they were submitted.
:~a0~672C~
This invention relates to novel polysaccharides, and
more part1cularly, to polysaccharides having antitumor and
other significant pharmacological activities, and to a process
for the preparation of such polysaccharides.
There have been reports in recent years of antitumor
substances being obtained from various kinds o~' Basi~iomycetes.
These substances show a significant antitumor effect in
intraperitoneal administrations, but they prove to be extremely
low in antitumor activity when administered orally. Therefore,
although these substances are o~ academic interest, they have ''
little practical utility.
In the course of studies on extracts rom various
kinds of Basidiomycetes and cultures-thereof by means of
aqueous solvents, the inventors have found that the refined
products fxom the extracts obtained ~rom the fungi o~
..... ;, :.
~asidiomycetes belonging to the genus Coriolus of Polyporaceae
or cultures thereof (including the media used for cultivation)
have excellent antitumor activity not only in intraperitoneal '
administrations but also in oral administrations, and further
studies have been made on the active components of these
extracts.
According to one aspect of the invention there is
provided a process for producing a polysaccharide substance
comprising the steps of extracting mycelia and/or fruit bodies
of fungi of the genus Coriolus ~ith an aqueous solvent,
saturating the extract solution thus obtained by adding
ammonium sulfate ater removing substances with molecular
weights of less than 5,000, collecting the resulting precipitate,
dissolving the precipitate in water, desalting the solu~ion
. 30 thus obtained, passing the solution through a column packed with
an lon exchanger to adsorb and remove the nitrogenous substance , ' ''' '
?
concentrating the obtained solution, and, if desired, drying it.
According to another aspect of the invention there
is provided a polysaccharide substance having molecular weight
of from 5,000 to 300,000 as measured by ultracentrifugation and
developing color reactions characteristic of saccharides in the
~-naphthol-sulf~ric acid reaction, indo1e-sulfuric acid reaction,
phenol-sulfuric acid reac~ion and tryptophane-sulfuric acid reac-
tion,the elemental analysis of said substance showing 43.5--
45.3% carbon, 5.7 - 6.7% hydrogen a.,d remaining percent of oxygen,
the specific rotation ~]D5Of said substance being 70 to
180, said substance showing specific absorption at 840 cm in
infrared absorption spectrum and showing absorption at
3.7 + 0.1 ppm, 3.8 + 0.1 ppm, 5.0 ~ 0.1 ppm and 5.4 + 0.1 ppm
in nuclear magnetic resonance absorption spectrum, said substance
being soluble in water but insoluble in pyridine, chloroform and
hexane, the saccharide portion of said substance being composed
principally of D-glucose, the pattern of bonding of said D-
glucose in said substance being ~-bonding,
The ion exchanger in the column may be, or example,
a cellulose material such as diethylaminoethyl (DEAE) cellulose.
The accompany drawings show the infrared absorption
spectra and nuclear magnetic resonance (NMR) absorption spectra
o~ polysaccharide substances according to this invention.
In particular:
Figures 1 to 4 show the infrared absorption spectra of
the polysaccharide substances obtained from the ~ungi of the
strains Coriolus versicolor ~Fr.) QuelO (formerly called Poly-
stictus versicolor Fr.) (Fig. 1), Coriolus hirsutus_(Fr.) QuelO
(formerly called Polystlctus hirsutus Fr.)(Fig. 2), Coriolus
consors (Berk.) Imaz. (formerly called Irpex consors Berk.)
(Fig. 3) and Coriolus par~a~enus (Fr.)_Pat. (formerly called
3 -
6~Zll
Polystictus pargamenus Fr . ~ (Fig ~ 4 );
Figures 5 to 8 respectively show the NMR absorption
spectra of the above-mentioned substances; and
Figure 9 shows an NMR absorption spectrum of a known
polysaccharide substance (reported as "PS-K") obtained by
filtering and sterilizing under pressure and then spray-drying
the extract from the Basid~iomycetes belonging to the genus
Coriolus, such PS-K being treated as a control in this invention.
The polysaccharides according to this invention
(hereinafter referred to simply as the inventive substance) are
white powders, and are tasteless and odorless. The characteristic
properties of the inventive substance are described hereinbelow.
~1) PHYSICAL AND CHEMICAL PROPER'rIES:
Elemental Analysis
.
An alemental analysis of the inventive substance by
Yanagimoto Manufacturing Company's Model CHN-CORDER MT-2
Analyzer revealed the following composition: 43.5 to 45.3% -
carbon, 5.7 to 6.7% hydrogen, and the remainder oxygen. ..
Color Reactions
Color reaction tests were conducted on the
a~ueous solutions of the inventive substance the results shown
in Table 1 below were obtained.
~L0~6'7Z~
Table_l
. v_ __ . __ , . .
Color reaction Color Results
. ~ . _ _ _ _
~-naphtol-sulfuric acid .
reaction (~lolisch reaction) Purple Saccharides
Indole-sulfuric acid reac-
tion (Dische reaction) Brown Saccharides
Anthrone-sulfuric acid Greenish Saccharides
reaction blue
Phenol-sulfuric acid
reaction Brown Saccharides
Tryptophane-sulfuric acid Purplish Saccharides
reaction brown
. __ .. . ,
It is apparent from the above~shown color reaction
test results that the inventive substance does indeed contain
; ~saccharides.
pH Value
1 gr. of the inventive substance was dissolved in 100 cc
o~ water and the pH value of the solution was measured by using
'
~- 5 _
~ '
~'
72a~
HITACHI-HORI~A Manufacturing Company's M 5 pH Meter. It was
within the range of 6.5 to 7.2.
Specific Rotation
A 0.25~ aqueous solution of the inventive substance was
prepared and its optical rotation was measured by using
Yanagimoto Manufacturing Company's YANACO OR-50, and the
specific rotation [~]Ds was determined therefrom. It was
within the range of 70 to 180.
Molecular Weight
The molecular weight of the inventive substance, as
measured by an ultracentrifugal method, was within the range of
; 5,000 to 300,000, and the average molecular weight was within
..~ ~ , ... .
the range of 10,000 to 100,000. The values obtained according
to the other measuring methods, such as fractionating by
ultrafiltration, also pointed to -the range of 10,000 to 100,000.
Therefore, it may be supposed a~ the average molecular weight
of the inventive substance is within the range of 10,000
to 100,000. The measurement was made by using SPINCO-E
Ultracentrifuge.
Solubility
The inventive substance is easily soluble in water
but insoluble in pyridine, chloroform, benzene and hexane.
Infrare~ Absorption Spectrum
The frared absorption spectra of the inventive
substance as measured according to the potassium bromide tablet
me-thod, are shown in Figures 1 to ~ of the accompanying
drawings. No
- 6- :::
. : . . , . . .
.. .
6720
significant difference was noted between the specimens. The
spectrum showed absorptions at 3600 to 3200 cm , 2920 to
2900 cm -1, 1660 to 1610 cm~l, 1460 cm~l, 1410 cm~l, 1360 cm~l,
1230 cm~l, 1150 cm~l, 1080 cm~l, 1060 to 990 cm 1, 925 cm 1,
840 cm 1, 755 cm 1 and 705 cm 1 The broad absorption band at
3600 to 3200 cm 1 as seen in Figs. 1 to 4 is considered
attributable to vOH's which are hydrogen-bonded to various
degrees. This can be assumed, for example, from the fact
that this broad absorption band vanishes when the hydroxy
groups in the saccharide portion of the specimen are
O-methylated. Another broad absorption at 1200 to 1000 cm
may be attributed to the unsymmetric stretching vibration
o~ C-O-C linkage in the pyranose rings in the saccharide portion.
Absorption at 840 cm 1 is ascribable to ~-bonding of
saccharides. This absorption is therefore indicative of
~-bonding of saccharides in the present substance. The
measurements were made by using Nippon Bunkosha's DS-701
Spectrum Analyzer.
Proton Nuclear Magnetic Resonance (NMR) Absorption Spectrum
The NMR (100 MHz) absorption spectrum of the inventive
substance was measured by usin~ Varian's Model A Spectrum
Analyzer. Heavy water was used as the solvent while adopting
sodium 2,2-dimethyl-2-silanopentane-5-sulfonate (D.S.S.)
as an internal standard The results are shown in Figs. 5 to 8.
Absorption at 2.5 to 6.0 ppm in Fi~s~ 5 to 8 is
attributable to protons in the saccharide portion while
absorption ` ~
''
-7-
36'7;~
at 5.0 ppm is ascribable -to ~-(1~6) bonding and that at
5.4 ppm to ~ 4) and ~ r3) bonding. By way of control,
an NMR absorption spectrum is shown in Fig. 9 of a known
product obtained by filtering and sterilizing under pressure
and then spray-drying the extract from the sasidiom~cetes
belonging to the genus Coriolus of Polyporaceae. The
product is reported by the name oE PS-K, so it is hereinafter
referred to as PS-K. The most prominent difference between
PS-K and the inventive substance as noticed from the
comparison of the absorption spectra shown in Figs. 5 to 8
and that shown in Fig. 9, is that, in the case of the
inventive substance, although an absorption at 4.9 to 6O0
ppm originating from ~-bonding is Observed, no absorption
is seen in the range of 4.4 to 4.9 ppm attrubutable to ~-
bonding. This indicates that the saccharide component of the
inventive substance are all ~-bonded. As regards the
` quantitative measurement of the mode of bonding of the
saccharides in the inventlve substance, a certain
; assumption may be made from the ~-(1~6) bonding observed at
5.0 ppm and the ~ 4) and the ~-(1-~3) bonding observed at 5.4
ppm, but since the ~-(1-~4) bonding and ~-(1-~3) bonding overlaps
at 5.4 ppm, the accurate quantitative measurement of the
respective bonds is difficult. Further, since the inventive
substance has a branch structure, the minute structural
elucidation of the substance must he resorted to the methyl-
ation method described below.
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(2) STRUCTURAL CHARACTERISTICS:
In order to identify the saccharide component in the
saccharide portion o~ the present substance, 10 mg of a
specimen o~ the present substance was mixed with 3% hydorchloric
acid methanol for methanolysis at 100C for 16 hours and ~hen,
after trimethylsilation according to an ordinary method,
sub]ected to a gas chromato~raphic analysis. The results
showed that glucose is predominent and other saccharides such
as mannose, galactose, xylose and fucose are very scarce.
In order to ascertain the form (either D form or
L ~orm) of the glucose which is the principal saccharide com-
ponent in the saccharide portion o~ the present substance,
glucose crystals were separated from the hydrolyzates
of the present substance. It was found that the separated
glucose had a melting point of 143 to 145C and showed no drop
of melting point when mixed and melted with a standard D-glucose
preparation. This identified the glucose in the saccharide
portion of the present substance as D-glucose.
Then the position of the glycosidic linkage was
determined in the ~ollowing way~ The linkage patterns of
Gl-~ (Gl-~ means glucose structure Skeleton), ~Glt,-~4G~
~ 3Gl~ and -~3~ were confirmed from an analysis of the
monosaccharides obtained according to the periodate oxidation
method or Smith's decomposition method, and their ratios
were d~term~ned by the methylation experiments according
~o Haworth's method. As for identification, the saccharides
obtained by hydrolysis of the methylation products were
identiEied by gas chromatography as
~' ~,'.
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_g_ .
15 67ZCJ
alditol~acetate and me-thyl glucoside. The individual hydro-
lyzates were isolated by column liquid chromatography and
then cryst~llized or converted into crystalline derivatives for
confirmation.
The molar ratios of the respective linkages in the
present substance are shown in Table 2 below by indexing the
molar ratio of Gl~ linkage as 1. The molar ratios in
Table 2 were determined from the area ratios on the gas
chromatograph of alditol-acetate.
Table 2
'
.... ~ ,
Hydrolyzates of methylated sugars Linkage Molar Ratio
. . :.'.: '
2,3,~,6-tetra-O-methyl-G Gl~
; 2,3,6-tri-O-methyl-G -~4Gl-~ 3-5 to 8.5
2,3,-di-O-methyl-G -~4Gl-~ 0.5 to 2
2,6-di-O-methyl-G ~4~ 0.1 to 2.5
2,4,6-tri-O-methyl-G ~3Gl~ 2 or less ;
2,4,-di-O-methyl-G ~3~1~ 0.8 or less
- -
As apparent from the above table, it is considered that
the polysaccharide portion of the present substance is mostly
composed of ~-(1~4) bonds, but there also exist ~-(1-~3) bonds ;
and numerous branches in the polysaccharide portion. This may
be
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construedas indicating that the present substance is of a
structure where side chains are bonded to the main chains
of glucan of ~ 4) bonding,with the glucan portions of
3) bonding of the similar structure being scattered
therein.
The process for the preparation of the polysacc-
harides according to this invention is described below.
The polysaccharides of this invention, as described above,
are extracted from the fruit bodies or mycelia of the natural-
or artificial~cultured fungi of Basidiomycetes belonging ;~
to the genus Coriolus Of Polyporaceae of the Aph~llophorales
~ order.
For identification of the fungi used in thisinvention, we referred to "COLOURED ILLUSTR~TIONS OF FUNGI
OF JAPAN" by Roakuy Imazeki and Tsuguo Hongo (Hoikusha Press)
and "FUNGI OF JAPAN" by Seiya Ito (Yokendo Press). According
to this literature, the starting material used for the
extraction of the polysaccharide substances of this invention,
that is, the ~ungi of Basidiomycetes from which the cultuxes
for the extraction are obtained are the ones belonging to
-the genus Coriolus, such as for example Coriolus consors
- (Berk.) Imaz., Coriolus versicolor (Fr.) ~uel., Coriolus
.
hirsutus (Fr.) Quel. and Coriolus pargamenus (Fr.) Pat. These
ungi are deposited, under the Deposit Nos. shown in the
Eollowing table, in the Fermentation Research Institute
~F.R.I.) of Agency of Industrial Science and Technolog~,
a deposit organisation designated by the Japanese Government.
--11-- : '
- . . .. .
- ~ 10867Z0
l Table 3
I ._
. ,... . . .
No. Genus Strain Deposit No.
I . .
1 Coriolu~
- - Imaz.~ CM~166 F.R.I. Dep.`No. 988
2 "Coriolus vers~cQlQr ~r.l ~
CM-103 " No.2414
3 " " CM-104 " Nou2415
4 " " CM-105 " No.2416
" " CM-106 " No.2417
6 " " CM-107 " No.2418
, 7 " " CM-108 " No.2419
8 " " CM-109 " No.24201
Il 9 " " CM-llO " No.2421
" " CM-lll " No.2422
11 " " CM-112 " No.2423
12 " " CM-113 " No.2424
13 " " CM-114 " No.2425
CM-115 " No.2426
~0 !l 15 " Coriolus hirsutus_~c~ `
!l ~ :
¦i CM-151 " No.2711
16 "
~I Pat.
~5 1I CM-161 " No.2712
I! . .. _ . _
- As mentioned above, the starting material used for the
extraction of the polysaccharide~substances of this invention
may be either naturally or artificially cultured fruit bodies or
mycelia, and in case of artificially culturing the material by
:
1 ~
I - 12 -
- - :
~ .- -
8~i'7Z~
using a liquid medium, it is possible to use an extraction
material all the culture products including not only the produced
mycelia but also the eluates in the liquid medium used for the -
culture. From the industrial viewpoint, it is recommendable
to use all the cultures including the cultured mycelia by use
of a liquid medium. The thus obtained material is then
subjected to the next step of extraction, but if desired, it may
be preserved after a suitable drying treatment such as air drying
or freeze-dryin~, for use later on. It is preferable to pulverize
the material before subjecting it to the extraction step as such
treatment elevates the extracting effect. The extraction of
the material is performed by using an aqueous solvent. The
aqueous solvent used here in water or an aqueous solution of a
small quantity, for example about 10% or less, of an organic
solvent, acid or base which is soluble in water. Preferred
examples of the organic solvent used for the purpose are methanol,
ethanol and isopropyl alcohol. The acid used here may be
hydrochloric acid, sulfuric acid, acetic acid or the like, and
the base may be ammonia, caustic soda, caustic patash, sodium
carbonate or the like. Among these a~ueous solvents, the
most preferred are water and a dilute alkali solution, particularly
an aqueous solution of caustic soda or caustic potash with a
concentration within the ran~e of 0.005 to 2N. The extraction
is carried out by using an aqueous solvent, such as mentioned
above, in an amount 5 to 200 times the quantity (on a dry basis)
o~ the culture used as the extraction material, at a
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1~l36721~3
temperature of usually 50 to 100C for a period of 20 minutes
to 20 hours. An extraction temperature of lower tha~ 50C
results in a poor extraction efficiency, so that it is
generally preferred to perform the extraction at a temperature
within the range of 80 to 98C. The number of runs of
extraction is usually less than 10, perferably 3 to 8. It is
preferable for the reason of preventing decomposltion of the active
substances to confine the total period of extraction to less
than 20 hours regardless of the number of runs of extraction.
10 On the other hand, from the viewpoint of extraction efficiency, -
it is preferable to perform the extraction for more than one
hour. The a~ueous solvent used for the extraction is selected `
from the group shown above, but it is not essential to use
only one kind of solvent; in some cases, even better results are
provided by using two or more kinds of solvents in combination,
for example a combination of water and a dilute alkali solution.
The most preferred mode of extraction in this
invention is a multi-sta~e extraction by use of a dilute alkali
solution or a combination of water and a dilute alkali solution,
such extraction being carried out by ~radually increasing the
concen~ration of the dilute alkali solution. This mode of
extraction results in a striking improvement of extraction
efficiency. Although the reason for such high extraction effect
i9 not definitely known, it is supposed that, in such a mode
of extraction, not only mere elution of the soluble substances
from the material takes place
-14-
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~67Z0
but also a mild decomposition of the material to expedite
extraction o~ the soluble active substances. It is thus considered
that, according to such a multi-stage extraction process, de-
composition occurs to promote the extraction of the active
substance each time the alkali concentr~tion is increased.
When the extraction is carried out by using an alkali solution
which is highly concentrated from the beginning,the yield of
the object substance drops sharply in the succeeding runs of
extraction. This is considered to be due to the following
reason: the fractions ~desired substances) which are to be
obtained by ~he extraction undergo excessive decomposition
due to the harsh conditions, resulting in the generation of
degenerated or low molecular substances rather than the
! preerred high-molecular polysaccharides.¦ ;
The liquid extract obtained by the above-mentioned
extraction process is neutralized according to an ordinary
method by using an alkali in the case of acid extraction and by
using an acid in the case of alkali extraction, and the thus
obtained liquid extract is then subjected to a refining
~0 treatment. The refining treatment practiced here is intended
to get rid of the low-molecular-weight substances (with molecular
weight of less that 5,000) contained in the extract, and such
treatment may be accomplished by sàlting out, dialysis,
ultrafiltration, reverse osmosis, gel filtration or
sedimentation by the use of an organic solvent, such techniques
belng employed either singly or in
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combination. From the standpoint of engineering, it is
preferred to employ ultrafiltration, which is a membrane
separation method under pressure, or reverse osmosis either
singly or in combination, but in some cases, such reEining
treatment may be practiced after previously performing a
salting-out treatment on the extract. The dialysis for the
refining treatment is usually carried out by using a cellophane
membrane, collodion diaphragm or cellulose membrane. The
salting-out agent used for the salting-out treatment may be
ammonium sulfate, common salt, potassium chloride, barium
carbonate or the like, but ammonium sulfate is most preferred.
After practicing the salting-out treatment, it is followed
by dialysis, ultrafiltration, gel filtration, reverse
osmosis or such treatment. Gel filtration is performed by using
a column packed with dextran or polyacrylamide gel. In this
case, there is usually used a filler sold by the tradename
of Cephadex Biogel. Ultrafiltration and reverse osmosis
are both fractionating methods practiced by using a membrane
under pressure, usually 0.5 to 5 kg!cm2 in the former and
20 to 35 kg/cm in the latter. When employing sedimentation,
an organic solvent such as methanol, ethanol, isopropanol or
acetone is generally used. If need be, an ion
-16-
' ' :.' .
~16~ZI~
exchan~e treatment may be practiced concurrently with the
operation mentioned above.
The above-mentioned refining -treatment in this
invention is essential ~or the reason that the low-molecular-
weight ~raction, or the substance with a molecular weight of
less that 5,000, released from the extract by the treatmen~
shows almost no inhibitory activity against Sarcoma-180 solid
type tumors in intraperitoneal administration on mice, and also
the fraction has a slight bitterness and odor. Thus , the
presence of such low-molecular fraction is undesirable in
respect of pharmaceutical potency of the desired polysaccharide
substance of this invention.
The liquid obtained by the above-mentioned refining
treatment is adjusted in concentration to 1 to 20%, pre~erably 3
to 10%, and then saturated with ammonium sulfate, and the result-
antly produced precipitate is collected. This precipitate
is then again dissolved in water and subjected to dialysis,
ultrafiltration or reverse osmosis, and a~ter adjusting its
concentration to 3 to 10%, this solution is passed through
a DEAE cellulose column to adsorb and remove the nitrogenous
component, and after repeating the above-mentioned operation if
so required, the obtained eluate is concentrated and dried.
~here is consequently obtained the desired polysaccharide
substance which is white, tasteless and odorless.
We will now describe the antitumor acitvity of the
polysaccharide substance according to this invention by ;~
sh~wlng the results of various animal tests.
,.'
-17-
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~LV86'7Z(l
ACUTE TOXICITY
~cute toxicity in mice and rats:
The mice used in this test were oE the ICR-JCL strain,
4 to 5 weeks old, weighing 21 to 24 gr, and the rats were of
the Donryu strain, 4 to 5 weeks old, weighing 100 to 150 gr.
The substance of this invention was administered by the
following four routes: intravenous, subcutaneous, intraperitoneal
and oral. Obervations were made on general symptoms, death
and body weight through the period of 7 days after administration
of the substance, and after completion of this observation
period, the animals were killed and autopsied. The results
are shown in Table 4 below. No death was caused by adminis-
tration of the substance even with the maximum dosage in both
rats and mice, and it is practically impossible to calculate
the numerical values for LD50.
Table 4
Kind of Route of LD50 (mg/kg)
animal administration
_ - ~ Female Male
Intravenous> 1,300 > 1,300
20 Mice Subcutaneous> 5,000 > 5,000
Intraperitoneal > 5,000 > 5,000
Oral >20,000 > 20,000
.
Intravenenous > 600 > 600
Rats Subcutaneous> 5,000 > 5,000
Intraperitoneal > 5,000 > 5,000
Oral >20,000 > 20,000
_ :
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ANTITUMOR ACTIVITY
The antitumor effect of the substance of this
invention was measured according to an ordinary method outlined
below.
Sarcoma-180 tumor cells were inoculated into the
peritoneal cavities of mice, and after the 7-day period in
which sufficient growth of the tumor cells took place, 106
cells were inoculated under the axially skins o~ the other
ten mice to form solid tumors. The powdered substance of this
invention was dissolved in a physiological saline solution
and administered to the mice, starting from the 24th hour
after inoculation. In the case of intraperitoneal administration,
the substance was administered at a dose of 10 mg~kg, or 0.2
ml/20 gr (mouse body weight), once every other day for a totai
o~ ten administrations, and in the case of oral administration,
the substance was administered at a dose of 1,000 mg/kg,
or 0.2 ml/20 gr (mouse body weight), once a day for a total ;
of 20 administrations. 25 days after inoculation, the tumors
were enucleated, and the tumor growth inhibition rate was
calculated from the average weight of the tumors in the
administered groups and the average weight of the tumors in
-the control group. The results are shown in Table 5 below.
The test results obtained from the above-mentioned measurements
lndicate excellent antitumor effects of the present substance
not onl~ in intraperitoneal administration but also in oral
administration. The specimens of the present substance used
~ in the test were~prepared from the extracts of the cultures
; oE the fungus strains shown 1n Table 5 below.
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As will be appreciated from the experimental results
shown above, the substance can be administered by way of oral
and intraperitoneal administrations~
One of the main characteristics of the substance of
the invention is that it has no allergenicity because it does
not contain proteins, resulting a safe intravenation, etc.
The fact that the results of experiments on mammals can be
applied to human cases is clearly shown in the literature
(Akio HOSHI and Kazuo KURETANI, Farumashia, 9:464-468, 1973~.
Accordingly, in the case of oral administration for the
treatment of gastro-intestinal-tract cancer, such as esophagus -
cancer, stomach cancer, colonial cancer and Rectal cancer, cancer
at region of head and neck, breast cancer, lung cancer,
malignant lymphoma and other tumors of adult patients, the
normal dosage, although varying depending on the number of
administratons, is preferably within the range of 1 to 3 gr a
day, and for injection, the normal dosage is preferably not
greater than 500mg.
When the substance is prepared into solid forms for
oral administration, such as tablets, grains, powder, capsules,
etc., the composition may contain a binder, inclusion agent,
shaping agent, lubricant, disintegrator, wetting a~ent or
other like adducts. The substance may be also formed into
oral liquid preparations such as internal liquid medicines,
shake mixtures, suspensions, emulsions, sirups, etc., or it
may be in the form of a dry product which is redissolved
. ... .
before use. Such liquid preparations may contain the
normally used types of additives or preservatives.
The preparations for injection may also contain
an additive or additives such as a stabilizer, buffer agent,
-21-
.; ,
~8~;7Z~ ~
preservative, isotonizin~ agent, e-tc., and they maybe provided
in the form of ampoules containin~ unit doses of the substance
or containers containing multiple doses of the substance. The
injection compositions may be prepared in the form of an aqueous
solution, suspensions, solution, or emulsion in an oil or
aqueous vehicle, and the active substance may be provided
in the form of powder which, when used, is redissolved in a
suitable vehicle, such as steri~e wa-ter not containing any
pyrogenic substance.
As reviewed above, the polysaccharide substance
according to this invention provides very good effects when
used as an antitumor agent for oral administration. The present
substance also gives immunopotentiation through the host and
is e~fective for preventing side e~ects in chemotherapy or for
increasing sensitivity in radiotherapy. Further, the present
substance is useful for preventing suppression of immunity
or physical strength of the patients and for protecting the
patient against viral or bacterial infection to which the
patien* is susceptible due to decreased immunity. Amony other
prominent effects provided by the present substance throuyh its
oral administration are improvement of liver function, remedy
o~ intestinal disorders and promotion of urination.
The invention is now described in further detail
by way of Examples thereof, but the scope of this invention
is not limited to these Examples.
"'
-22-
' ,': .
~ 536~2a~
!¦ EXAMPLE l:
¦i CM-103 strain (F.R.I. Deposit No.2414) of Coriolus versi-
color (Fr.) Qu~l. belonging to ~he genus Coriolus was inoculated in a
Il 200 cc conical flask containing 30 cc of medium composed of 5% of
I glucose, 0.2% of peptone, 0.3% of yeast extract, 0.1% of KH2PO4
! and 0.1% of MgSO4.7H2O and subjected to 10-day stationary culture
Ij at 25 to 27C, and the mycelial mat grown on the medium surface
was homogenized with a physiological saline solution to thereby
1 prepare the seeds.
~, 20 litres of slurry of the seeds was inoculated in
l,600 litres of a medium composed of 10% glucose, 1.5% yeast
extract, 0.1% KH2PO4 and 0.1% ~gSO4 7H2O in a 2M3 vertical
fermenter and cultured at aeration rate of 0.5 ltmin per litre of
¦I medium, agitation rate of 150 r.p.m., temperature of 26C for 7
1 days. The thus obtained broth was batched off in portions of
about 500 litres, and each portion was dried by a double drum
type dryer to obtain approximately 30 kg of dry product.
100 gr of this dry product was put into a 5-litre-capacity flask
having an agitator together with 3 litres of water and maintained
at l~nternal temperature of 95 1 2~C for 3 hours while continulng
agitation, and then after cooling,lthé solution was separated
; into thq mycelial residue and extract solution. Then the;mycelial
: - , . . - - , - ~ -
residue was washed with approximately 1 litre of water, and the
wa~h was mixed with the extract solution. The mixed solution
was approximately 3.5 litres.
. .
1~ ~l0867Z~ '
The mycelial residue was added with 2 litres of 0. lN
¦I caustic soda solution and subjected to 2-hour extraction at 95 +
2C in the same way as mentioned above, followed by cooling,
I neutralization with 2N hydrochloric acid and separation into
l extract solution and mycelial residue by means of suction filt-
ration. After separation, the residue was washed with about 0.5
Il litres of water and mixed with the extract solution to obtain
j 2.2 litres of extract solution. The same treatment was performed
' with 2 litres of 0. 2N, O.3N and 0. 4N caustic soda solutions by
'll using 0.5 litres of washing water, obtaining approximately 7.2
litres of extract solution.
j The thus obtained extract solution was treated by using
Amicon's 2000 Bench Type Ultrafilter (with DM-5 membrane~
Il under operating pressure of 1.5 kg/cm2 and at temperature of 10C
ll while keeping agitation and cooling to thereby eliminate the low-
,I molecular-weight substances and neutral salts, consequently
¦ obtaining 2Q0 cc of liquid.
The thus obtained liquid was added with 160 gr of
~ ammonium sulfate and the prcduced precipitate was collected.
`20 l~his precipitate was then di-solYed~in water~and desalted by using
-~ the above-mentioned Amicon's ultraf~ilter~to obtain 190 cc of liqui ~.
This liquid~was passèd through a 10 cm-d~ameter`'and 100 cm-long
column packed with'DEAE cellulosè'(OH form) to adsorb''and remove
~he nltro~enous substance contained in the liquid, and the water
2S was passed through the column to o~ta~n 20 litres of eluate.
~ . ".
: '" ~
~ - 24 - ' ~'
: . ~ :
. ~.'
': .
.. .-, . . . ~ . .: . ,
72~
¦i The thus obtained liquid was concentxated to 180 cc by a rotary ,
evaporator and then again passed through the DEAE cellulose
column, and the resultantly obtained eluate was further concent-
,l rated by the rotary evaporator and freeze-dried to finally obtain
,, 3.0 gr of the ob~ect polysaccharide substance in a powdery form.
Il The properties of this substance were measured by the
¦ followlng methods, and also animal tests such as described below
I, we~e conducted on this substance.
,
~l (1) MEAS~REMENTS O~ PROPERTIES
il Infrared absor~tion spectrum
!l ;
An infrared absorption spectrum of the obtained powdery
substance as measured according to the potassium bromide tablet
,I method iS shown in Fig. 1. The tablet used in this method was
~I prepared by mixing 1 gr of KBr and about 1/3 to 1/2 spatulaful
amount of specimen according to a common method. Absorption was
noted at 3600to 3200 cm 1, 2920 to2900 cm 1, 1660to 1610 cm 1,
1460 cm 1, 1410 cm 1, 1360 cm~l, 1230 cm~l, 1150 cm 1, 1080 cm 1,
1060to 990 cm 1, 925 cm 1, 840 cm 1, 755 cm 1 and 705 cm 1,
2~ ~bsorption at 840 cm is attributable to ~bonding of saccharides
~ This attests to the~fact that the present~substance is~ composed ~
. ~ .................... : ., -, -- - -
o~ ~-bonded~glucoside.-~
- S~ecif ic rotation ~ ` `
OpticaI rotation was measured with D-rays (589 m~)
~5 o~ sodium by using a 0.25% aqueous solution of specimen and a 5 c~
,.:
- 25 _ ~ :
: .
. ~.,'.'
.. . . .. .. .. . ... . . . . .
:
. i - . ., . . . - ,, . :
10~il6720
cell, and the specific rotation ~Ds was calculated from
the rotation ~.
NMR absorption spectrum
I
1 The NMR absorption spectrum was measured by adopting
5 1I DSS as internal standard and using heavy water as solvent. The
numerical figures shown in the table are the values after cor-
¦ rection under the supposition of Lorenz's curve for eliminatingthe influence of residual light water in the heavy water.
Molecular weight
~~ .
ll The molecular weight of the present substance was
measured by using the ultracentrifugal method. The results showed
I that the molecular weight of all the specimens tested is within
¦¦ the range of 5,000 to 300,000. For measurements, there were
Il employed the sedimentation equllibrium and synthetic boundary
5 I pattern using an i~terference optical system. The experimental
conditions were as follows: specimen concentration, 0.3~; solventl,
M/lOM KCl; temperature, 25C; liquid column, 1.7mm; speed, 22,000
r.p.m.; measuring time, 5 hours.
~ ~ " ~ ~ ,
20 - (2) ~ACCHARIDES COMPONENT OF GLUCO~IDE -~
~ ~ ~The~monosaccharlde component oE thà pr~eaent substance
1 : was analyzed in the following way. -:3 mg of~specimen wa5 put into
a glass ampoule, to which was added 10 ml of 3~ hydrogen
chloride methanol for methanolysis at 100C for 16 hours, and
then hydrochloric acid was neutralized with silver carbonate and
` l
~86~2
j filtered at room temperature. The filtrate was concentrated and
evaporated to dryness and then dissolved in 0.5 ml of dry pyridine. ''
The obtainea solution was mixed with 0.2 ml of hexamethyldisilazane
' and 0.3 ml of trimethylchlorosilane, and the mixture was allowed
,I to stand at room temperature for 30 minutes, for trimethylsila-
tion. Upon completion thereof, the mixture was dissolved in
chloroform, and after removing the excess reagent by washi~g with
water, the obtained solu~ion was evaporated to drynessO The
,~ residue was dissolved in carbon tetrachloride and analyzed by gas
l, chromatography. The results of the analysis showed that glucose
accounts for more than 99% of the saccharides componen~ of the
present substance and other saccharides such as mannose, galactose,
,I xylose, fucose and the like are scanty.
,l In order to know whether the glucose which is the prin-
Ij ciple constituent of the saccharide portion of the present
~¦ substance is D form or L form glucose was separated from the
hydrolyzate of the present substance by a column and its melting
point was measured. It was 143 to 145C. This glucose showed no
drop of melting point when mixed and melted,with a standard pre-
paration of D-g1ucose. From this resu~lt,~ the~glucose in the,
... . , ~ .
I - present subs;tance was ~identi~ied -as D~glucose~
~3~ P~TTERN OF SACCHARIDE BONDING ' '
The pattern of saccharide bonding in the present sub-
stance Was determined aacording to Howorth~s method. 2 gr of
.' .
. . .. ;'
~ 27 - '~
~ ' ~
.
10867Z0
'I
~1 specimen was dissolved in 10 ml of lN NaOH solution and, while¦I maintaining the mixture at 40 to 50C in a nitrogen stream under 1l
¦I violent agitatlon, 20 ml of dimethylsulfuric acid a.nd 40 ml of
! 30~ sodium hydroxide solution were added dropwise over a period
, of several hours, and after allowing the mixture to stand over-
l~ night, it was subjected to the similar treatment with the equal
Il amount of methylating reagent. The reaction solution, after
being neutralized, was dialyzed in running water and the dialyzate
I. was concentrated under reduced pressure and subjected to the above-
ll mentioned methylation treat~.ent three times, and after additional
i~ neutralization and dialysis, the mixture was evaporated to dryness
under reduced pressure. The rèsidue was dissovled in 20 ml of a
chloroform-methanol (10:1). mixed solvent, and to this was added
~l a petroleum ether-ether (1:1~. mixed solvent to precipitate the
lS l, methylated substance. Then, about 20 mg of this methylated sub-
¦¦ stance was hydrolyzed with lN sulfuric acid at 100C for 16 hours,l :
¦ and the hydrolyzate was converted into alditol-acetate according
to an ordinary method and the molar ratio was determined from the :.
pcak axea on the gas chromatograph. For distinction between :.
2P . 2,3,6-Txi-O-Me-G and 2,3,4-Tri-O~Me-G, 20~mg of:the methylated
, ,, - . .. . ... .. . - - -
substance was subjected to.methanolysis at 100C or:16 hours in
a seale.d tube by ùsing 3% hydrochloric mèthanol-. ~2,3,4-Tri-
: ~ O-Me-G Was not detected in the gas~chromatographlcjànalysis of
the methanolys~s product. For confirmation, each of the de-
compose.d products was identified on the gas chromatograph by using
. - 28 - .` :
. : ' '
67~
1, a standard preparation, while each hydrolyzate was isolated by
using column li~uid chromatography and either crystallized or
converte~ into a crystalline derivative.
I The properties, structural characteristics and anti-
,I tumor activities of the thus obtained polysaccharide substances
' of this invention are shown comprehensively in Table 6.
.,
.
Il
EXA~PLE 2:
.
,' The seeds of the strains Coriolus consors (Berk.) Imaz.
10 li CM-166 (F.R.I. Deposit No.988), Coriolus pargamenus (~r.) Pat. CM-~
161 (F.R.I. Deposit No.2712) and Coriolus hirsutus (Fr.) Qu~l. CM-
, 151 (F.R.I. Deposit No.2711) belonging to the genus Coriolus of
the Basidiomycetes were prepared by the same procedure as EXP~IPLE
il
15 ',
:! !
Peptone 3 gr
Yeast extract 5 gr
Potassium primary phosphate 0.1 gr
Potassium secondary phosphate 0.1 gr
20 Magnesium sulfate (heptahydrous) 0.05 gr
GIueose~ 20 gr
~ Malt extr~ct ~ ; ~ 10 gr
- ~ ~ Water ~ -1 litre
-- ...... : .. . - ~
pH 6.0
~5 Eaeh 200ee of the a~ove-mentioned medium was pipetted
into 400 culture bottles (l-litre volume), and
- 29 -
. .
. : , , :.
- 1086'720
il 1 cc of the seeds was inoeulated in the eaeh medium
followed by 25~day incubation at 25 to 30c and then drying to
obtain 980 gr of dry mycelia from Coriolus consors (serk.) Imaz.
l' CM-166, 1,200 gr :Erom Coriolus pargamenus (Fr.) Pat. CM-161 and
'~, 1,500 gr from Coriolus hirsutus (Fr.) Quel. resnectively.
Then, 100 gr of the thus obtained dry mycelia from each
strain and 2 litres of 0.4N caustic soda solution were contained . .
in an extraetion vessel equipped with a heating/cooling jacket :
Il and an agitator and subjected to 2-hour extraction'under agita- '
l~ tion by adjusting the jacket temperature such that the internal
temperature of the vessel would be maintained at 90 to 95C.
The obtained extraet was eooled to room temperature, then added
portionwise with 2N hydrochlorie aeid under agitation, and after
Il pH adjustment to 7, separated into extract solution and mycelial
.I residue by a centrifugal separator.
Then the myeelial residue was added with 2 litres of
0.4N eaustie soda solution and subjeeted to similar 2-hour
e~traction at 90 to 95C, followed by cooling, neutralization
. and centrifu~al separation to obtain extract solution and.mycelial .
~ .
. 20 ~ -.residue. The latter~was further sub~eeted..to.a;similar~.extrae-.:
~:` . tion operat'ibn for one hour with 0.4N caustic soda solution, then .'
.. . cooled, neutralized and centrifugally:separated into extract
. '''solùtion and mycelial residuè,.the latter being once again sub- :.
jected to l-hour extraction with 0.4N caustic soda solution,
cool~n~ eutralization and centrifugal separation.
_ 30 _ :~
.~ . ~ .
. , '
.. ... .... . . .. .. .. . ., . . . . . ... . l :
~ 6'72(J
¦ The total four runs of e~traction operations gave
Ij
approximately 8.4 litres of extract solution. This extract
solution was concentrated to 3-litre volume, then put into a
~I cellulose tube for dialysis (Visking tube by Union Carhide) and
I subjected to 6-day running water dialysis with tap water, and
the solution in the tube was concentrated to obtain 190 cc of
~ liquid.
- ~ The thus obtained concentrated liquid was then added
with 160 gr of ammonium sulfate and the produced precipitate was
'. collected, dissolved in water and further subjected to 6-day
; dialysis by using the Visking tube, and then the solution in the
tube was concentrated to 410 cc.
'I
This concentrated liquid was treated by the same manner ;
as EXAMPLE 1, that is, passed through a DEAE cellulose column to
lS ,l adsorb and remove the nitrogenous component in the liquid and
the obtained solution was concentrated by a rotary evaporator and
dried, consequently obtaining the object polysaccharide substances
in dry powdery form in amounts of 6.0 gr from Coriolus consors (Be: -k.
~ Imaz., 6.5 gr from Coriolus pargamenus (Fr.) Pat. and 5.5 gr from
20- Coriolus hirsutus (Fr.);`Quel., re~spectlvely.; The propertIes~of th
. obtained substances, resùlts of animal tests, infrared absorption
. ~ ",
. : ~pectra, NMR absorption spectra, molecular weight, specific rotatil ~n,
~c~ccharides component and pattern o`saccharide bonding are shown .
in Table 6 together with those o the ~roduct of EXAMPLE 1.
~, . ~ - . .
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i N ~I N O .
1~ Z ~ DO l
ii 4_1 .
ll ~ ~ '
l i ~ Cl~ o U~ = _ = = ~D O I .
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a~ O Q~ I
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