CA1084488A - Protein-bound polysaccharides - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:
Disclosed in this invention are protein-bound polysaccharides having antitumor activities. The polysaccharides have molecular weight within a specified range and present a pattern of color reaction peculiar to the saccharides as well as a pattern of color reaction characteristic of amino acids and peptide bonds. The proton nuclear magnetic resonance spectra of the polysaccharides show peculiar absorption bands. The ratio of the polysaccharide portion to protein portion in the polysaccharides is within a specified range. The protein-bound polysaccharides can be obtained by extraction of the mycelia and/or fruit bodies of fungi belonging to Coriolus of Polyporaceae of Basidiomycetes.

Description

1084~88 :^ ' ' . . .
FIELD OF THE INVENTION:
This invention relates to the novel protein-bound polysaccharide substances having the antitumor activities.

BACKGROUND OF THE INVENTION:
Reports have been published on producing antitumor substances from various kinds of Basidiomycetes in recent years.
These substances have a polysaccharide stucture and show a significant antitumor or anti-cancer effect in intraperitoneal administrations, but they prove to be extremely low in antitumor activities when administered orally. Therefore,although these substances have been of much interest for scientific studies, they have had little practical utility.
It is also known that the extracts abtained by extracting fungi belonging to the genus Coriolus with an aqueous solvent are predominantly composed of protein-bound poly-saccharides. However, as these extracts are obtained from native Basidiomycetes and contain complicated compounds composed of various kinds of protein-bound polysaccharides, no definite elucidation has yet been made on the characters of the ac~ ve antitumor ingredients of these extracts.
SUMMARY OF THE INVENTION:
According to the present invention, it has been found that the extracts obtained from the fungi (mycelia and/
or fruit bodies) belonging to the genus Coriolus have an excellent antitumor activity in not only intraperitoneal administrations but also oral administration.
The extracts in the form of protein-bound poly-saccharides can be obtained by the steps of: extracting mycelia or fruit bodies of fungi belonging to the genus Coriolus with an aqueous solvent; removing low molecular weight substances from said extract; subjecting the obtained

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solution to fractional precipitation; collecting the thus obtained precipitate and dissolving same with water; desalting the obtained solution; passing the desalted solution through an adsorption column to adsorb the protein-bound polysaccharides and collecting the adsorbed material.
The invention thus provides protein-bound polysacch-arides having molecular weights within the range of 5,000 to 300,000 as measured by an ultracentrifugal method and presenting color reactions characteristic of saccharides in a-naphthol-sulfuric acid reaction, indolesulfuric acid reaction, anthrone-sulfuric acid reaction, phenol-sulfuric acid reaction and tryptophane-sulfuric acid reaction and also presenting color reactions characteristic of amino acids in ninhydrin reaction after hydrochloric acid hydro-lysis and peptide linkage in Lowry-Folin method, said polysaccharides having the absorption regions as measured by proton nuclear magnetic resonance spectra at 0.9 + 0.1 ppm, 1.2 + 0.1 ppm, 2.0 + 0.1 ppm, 4.5 i 0.1 ppm and 4.7 + 0.1 ppm, with broad absorption being seen at 3.0 - 4.4 ppm, and also having the polysaccharide portion/protein portion ration within the range of 55/45 to 95/5 provided that proton strength of the protein portion is at 0.5 - 2.5 ppm and that of the polysaccharide .~ portion is at 2.5 - 6.0 ppm, and further said polysaccharide portion being composed of ~-glucan which shows no absorption attributed to a-glucan at 4.9 - 6.0 ppm, and said protein portion being composed of aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, tryptophane, : 30 phenylalanine, lysine, histidine and arginine.

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According to a particularly preferred extraction procedure, the following steps are carried out: firstly, an extracted solution is preparea by extracting the fungi with an aqueous solvent, removing the extraction residue from the . :
obtained extract and concentrating the extract. The thus obtained aqueous solution is subjected to a treatment such as salting out with ammonium sulfate or ultra-filtration to get rid of the low molecular weight substances contained therein, then this refined solution has added thereto a determined amount of ammonium sulfate for salting out and the formed precipitate is collected; the collected precipitate is dissolved with :.
water and the resultant solution is desalted by dialysis; and then the obtained solution is passed through a DEAE (Diethyl aminoethyl) cellulose column, and the thus adsorbed substance on the column is then eluted out with an aqueous solution of ~
sodium chloride (saline) and the obtained elutant has further : -added thereto a determined amount of ammonium sulfate for salting out; the thus formed precipitate is collected and then again dialysed for desalting. We also succeeded in elucidating the characteristic properties of the active ingredient and attained this invention on the basis of these findings.
BRIEF EXPLANATION OF THE DRAWINGS
In the accompanying drawings:
Figure 1 is a graphic representation of the infrared absorption spectrum of the protein-bound polysaccharide :
substances according to this invention;

. -3a-Figures 2-1, 2-2, 2-3 and 2-4 show the proton nuclear magnetic resonance spectra of the above substances of this invention; and Figure 3 shows the proton nuclear magnetic resonance spectrum of PS-K treated as control.
The protein-bound polysaccharide substances ~herein-after referred to simply as present substance) according to the present invention are of a structure in which protein is bonded to the polysaccharide portion. ~he present substance is odorless, tasteless and water soluble,having light brown or brown color. Also, the present substance has no definite melting point and is gradually blackened and decomposed at a temperature of higher than around 120C.
Now the characteristic properties of the present substance are described in due order.

_4 -PPYS: _AL AND CHEUICAI. PROPER'rIES: ¦
~1) Color reactions .
Color reaction tests were conducted on the aqueous : -solutions of the present substance to obtain the results :
shown in Table 1 below.
¦ Table 1 :

Color reaction Color Results - . . .
~-naphtol-sulfuric acid .
reaction (molish reaction) Purple . Saccharides Indole-sulfuric acid reaction Brown Saccharides (Dische reaction) Anthrone-sulfuric acid Greenich .
reaction blue Saccharldes Phenol-sulfuric acid reaction Brown Saccharides Tryptophane-sulfuric Purplish .
acid reaction brown Saccharides Lowry-Folin process Blue Peptide bonds, . tyrosine, . . tryptophane, cysteine Ninhydrin reaction after hydrochloric acid Purplish hydrolysis blue a-amino acids (6N HCl, 110C, 20 hr) . . .__ It i9 apparent from the above-shown results of the color reaction tests that the present substance contains saccharides and protein. :

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(2) Solubility ¦
The present substance is soluble in water and almost insoluble in methanol, pyridine, chloroform, benzene and hexane.
(3) Hygroscopicity In order to determine hygroscopicity of the present substance, several specimens were placed in each desiccator maintained at respective given humidity as shown in Table 2 ~elow by ta~ing a saturated solution of salts therein, and the moisture content of each specimen was determined by measuring the change of weight with time. The results are shown in Table 2.

Table 2 (Change o~ moisture uptake with time at each relative humidity) (hr) Moisture content (%) con- _ _ ditions \ 1 3 7 24 48 72 96 144 98% RH 20C 10.0 12.5 16.0 22.5 25.5 27.0 27.0 27.0 79~ R~ 25C g.0 9.5 11.7 15.0 15.5 16.0 16.6 17.0 52% RH 20C 9.0 9.5 11.7 15.0 15.3 15.5 15.7 16.0 32% RH 16.4C 8.0 8.5 9.0 10.0 12.5 12.5 12.5 12.5 . _ _ ~ .. .
Note: Saturated solution used:

at 32% RH, a saturated solution of sodium chloride-potassium nitrate, at 52~ RH, a saturated solution of sodium bichro-mate, at 79% RH, a saturated solution of ammonium chloride, at 98% RH, a saturated solution of lead nitrate.

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As understood from Table 2, each specimen of the present substance suffered little change of appearance, and there were seen no phenomena such as deliquescence which is caused by moisture abosrption.

(4) pH value The pH value-of the present substance was measured by dissolving 1 gr. of specimen in 100 ml of water. It was found that it has pH value within the range of 6.6 to 7.2.
Thiæ indicates that the present substance is substantially neutral.

(5) Optical rotation Optical rotation of the present substance was measured by using a 0.25% aqueous solution of specimen to determine the specific rotation ~a]25. It was within the range of 0 to !5 30, and it is as~umed that the present substance was predominantl composed of ~-glucan whose specific rotation is in the neigh-borhood of 0.

(6) Infrared absorption spectrum An infrared absorption spectrum of the present subs-~0 tance as measured according to the potassium bromide disk method . i8 shown in Fig. 1 of the accompanying drawings. The broad absorption band at 3600 to 3200 cm 1 as noted in Fig. 1 is considered attributable to ~OHIs which are hydrogen-bonded at various degrees. This can be assumed, for example, from the ~5~ fact that such broad absorption band diminishes or vanishes ~0844~8 .

when the hydroxy groups in the polysaccharide portion of specimen are methoxylated. Absorptions at 1600 cm 1 and lS30 cm are considered ascribable to aeformation vibration of -NH2 and -NHl respectively, and it is recognized that such phenomenon originates from the protein portion in the specimen. On the other hand, the broad absorption bands at 1200 to 1000 cm 1 are considered due to unsymmetric stretching vibration of C-O-C linkage in the pyranose rings in the polysaccharide portion. Also, a characteristic pattern of absorption resulting from ~-linkage of glucose in the polysaccharide portion is observed at 890 cm 1, but there is hardly seen characteristic absorption resulting from a-linkage at 840 cm 1.
S~RU TURAL CHARACTERISTICS:
(1) Structure of polysaccharide portion In order to determine the structural characteristics of the polysaccharide portion of the present substance, 10 mg of specimen was added with 3% hydrochloric acid-methanol to I perform methanolysis at 100C for 16 hours and then, after ¦ trimethylsilation according to a ordinary method, subjected to a gas chromatographic analysis. The results showed that glucose accounts for more than 99% of the total saccharides, and other saccharide components such as mannose, galactose, -xylose and fucose are very scanty. In order to ascertain the type of glucose (D type or L type), glucose crystals were separated from the hydrolyzates of the present substance.

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¦ It was found that the melting point of the separated glucose is within the range of 143 to 145C, and when the glucose crystals were mixed with a standard D-glucose, no depression of melting point was observed. Therefore, the glucose S composing the polysaccharide portion of the present substance was identified as D-glucose.
(2) CHARACTERISTICS OF MODE OF LINKAGE OF saccharides consti-tuting the polysaccharide portion.
The position of glycosidic linkage was determined in the following way. The linkage patterns of G + (G ~means glucose structure skeleton), +4~1 "+4~31 " ~4~6+' '3Gl'~ and +3~6~ were confirmed from analysis of the monosaccharides obtainec according to the periodate oxidation method or Smith's decom-position method, and their constitution ratios were determined ~5 by the methylation experiments according to Haworth's method.
As for identification, the saccharides obtained from hydrolysi~
of the methylated compounds were identified by gas chromato-graphy as alditol-acetate and methylglucoside, and further, the individual hydrolyzates were isolated by column liquid chromatoqraphy and then crystallized or led into crystalline derivatives thereof for confirmation. The molar ratios of the respective linkage in the present substance are shown in Table 3 bèlow by indexing the molar ratio of Gl+ linkage as 1.
The molar ratios in Table 3 were determined from the area ratios on the gas chromatograph of alditol-acetate.

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~ Table 3 I . . _ Hydrolyzates of methylated sugars Linkage Molar ratio I
¦ 2,3,4,6-tetra-0-methyl-G - G ~ 1 2,3,6-tri-0-methyl-G ~4G1~ 3 - 12 2,3-di-0-methyl-G ~4G1~ 0.5 - 3 2,6-di-0-methyl-G ~4Gl~ 0.1 - 2.5 2,4,6-tri-0-methyl-G '3G1~ 2 or less 2,4-di-0-methyl-G ~ G ~ 2 or less _ ~6 ;
As apparent from the above table, it is considered that the polysaccharide portion of the present substance is mostly composed of ~-1,4 linkages, but there also exist ~-1,3 linkages and many branches in this polysaccharide portion.
It may be construed from this that the polysaccharide portion of the present substance is of a structure where side chains are bonded to the main chains of cellulose and ~-1,3 linkages exist therein scatteredly.
(3) Characteristics of protein portion The protein portion of the present substance was hydrolyzed according to a ordinary method, and the amino acids composing the hydrolyzate were analyzed by using an amino acid analyzer. The results are shown in Table 4 below.

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Table 4 (Amino acids constituting protein portion) :
. . .
Kinds of amino acid Weight (%) ; Aspartic acid13 - 19 Threonine 6 - 10 Serine . 6 - 11 Glutamic acid12 - 18 ::.
Proline trace - 8 ~ ~ :
Glycine 6 - 9 .
, Alanine 6 - 13 Cysteine trace .
Valine 5 - 11 Methionine 1 - 4 Isoleucine 3 - 5 Leucine . 6 - 8 Tyrosine trace - 3 Phenylalanine3 - 6 ' . Tryptophanetrace - 2 Lyslne 2 - ~ :
Histidine trace - 2 Arginine 2 - 4 Ammonia 2 - 6 .

As noted from the above table, the protein portion of the present substance contains 18 kinds of amino acids, of which . .

the acidic amino acids and neutral amino acids are predominant, and the basic amino acids are very scanty in quantity. It is also characteristic of the present substance that aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine and leucine put together account for more than 70~ of ; ~-all the kinds of amino acids found in the protein portion.
Although the presence of glucosamine was also confirmed in the amino acid analysis, the quantity of this substance was less than l~ ~y weight of the total amount of protein.
As for linkage of these amino acids to the polysaccharid ;
portion it is assumed that the amino acids are tightly bound ;
in the form of the oligopeptide or peptide to the polysaccharide portion. This assumption can be derived from the results of various kinds of tests such as mentioned below.
Firstly, when tested according to Sevag's method which is often used for removing protein mixed in the specimen, the specimens of the present substance produced no precipitate.
This method is practiced by adding l/5 volume of chloroform and lt25 volume of n-butanol to the aqueous solution of each specimen (it is desirable to adjust pH of the solution at 4 to 5) and ~ `
then shaking thoroughly the mixture, and the solution is carefully observed whether there is produced any gel-like precipitate or not. If the polysaccharide portion and protein in the specimen exist in the form of a mere mixture, protein is denatured and gelled to precipitate between the water layer and ~ _ , . . .. .... .. . . .... . . . .. . .. .

10~448B
the chloroform layer, but no such precipitation occurs in case the polysaccharide and protein are bound.
In another precipitation test conducted similarly by using trifluorotrichloroethane, there was here again witnessed no formation of precipitates of the specimens of the present substance.
Also, the specimens of the present substance showed no change of protein content in the test in which protease, a proteolytic enzyme, was acted to the specimèns. ~
Judging from the results of various kinds of tests such -as above-mentioned, it is supposed that, in the substance of this invention, the polysaccharide and protein portions are not ~ -merely mixed forms each other but are bound chemically to each other.
lS As regards the mode of linkage of the polysaccharide portion and protein portion in the present substance, there are generally known the following types of sugar and protein linkage:
N-acylglycosilamine type linkage, O-glycoside type linkage and glycosideester type linkage, but in the case of the present substance, in view of the facts that the linkages can hardly be broken with weak alkali and that the presence of glucosamine is noticed in an amino acid analysis after hydrolysis of the present substance, it is considered that N-acylglycosilamine type linkage are predominant in the present substance.

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(4) Proton nuclear magnetic resonance spectra ~N.M.R.) N.M.R. (lOOMHz) of the present substance was measured by using heavy water as solvent and adopting sodium 2,2-dimethyl-2-silanopentane-5-sulfonate tD.S.S.) as internal standard.
The results are shown in Fig. 2. In Fig. 2, if it is assumed that absorption at 0.5 to 2.5 ppm under the D.S.S. standard ;~
is attributable to side chain protons of the protein portion and that absorption at 2.5 to 6.0 ppm is attributable ~
to protons of the polysaccharide portion, and if the properties ~ `
0 of the present substance under the above-stated definitions is expressed by way of proton strength ratio between the two, it is noted that such ratio is within the range of 95/5 to 55/45.
In the above measurements of N.M.R., in order to eliminate the influence of residual light water in heavy water, there were '5 used the values after correction under the supposition of the Lorenz's curve.
In absorption of the polysaccharide portion, the prominent rise of absorption at 2.5 to 4.1 ppm is due to the -methylene protons at 2- to 6-positions in the pyranose rings in the sugar chain. For reference, there is shown in Fig. 3 the N.M.R. spectra of a product obtained from an extract of the fungi belonging to Coriolus yersicolor (F .) Q~l. (Polystictus versicolor Fr.) by subjecting the extract to filtration under pressure, heat sterilization and spray drying. (Such product is reported by the name of PSR, so it is hereinafter referred to as PS-K.) The greatest difference between PS-Kand present substance as noticed from the comparison of Fig. 2 and Fig. 3 , 10~34'188 '' .
' is that, in the case of the present substance, there is observed absolutely no absorption in the range of 4.9 to 6.0 ppm origi-nating from ~-linkages. This fact endorses that the polysaccha-ride portion of the present substance is constituted from the , 5 ~-D-glucan alone.
It is known that, in Fig. 2, absorption at 4.5 ppm is concerned with ~ 4) and ~ 6) in the methine protons at l-position while abosrption at 4.7 ppm is associated with ~-tl ~ 3) and B-(l ~ 2) in the methine protons in l-position, ~0 so that it is possible to determine the ratio of ~ 4) and ~-(1 ~ 6)/~ 3) and ~ 2), but since there is also involved the branch stxucture, the above-stated methylation method must be employed for elucidation of the fine structure.
As regards the protein portion, it is hardly possible 'S to presume the structure of such protein portion only by the ~: I measurements of the nuclear magnetic resonance spectra. However, as the present substance shows absorption at certain given ¦ regions as mentioned below, this method is considered very ¦ favorable for identification of the present substance.
: '0 I The present substance has the characteristic to show `` ¦ absorption at 0.9 + 0.1 ppm, 1.2 + 0.1 ppm, 2.0 + 0.1 ppm, 4.5 + 0.1 ppm and 4.7 + 0.1 ppm, no absorption in the region of , 4.9 to 6.0 ppm, and broad absorption in the region of 3.0 to 4.4 ppm : !
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10~4~88 ~5) Molecular weight : :
The molecular weight of the present- substance, as ~: .
measured by an ultracentrifugal method, was within the range :
of 5,000 to 300,00~, and the average molecular weight was within the range of lO,OOO to lOO,OOO. The values obtained according :
to the other measuring methods, such as fractionating by the :~
ultra-filtration, also pointed to the range of lO,OOO to lOO,OOO.
Therefore, it may be supposed that the average molecular weight of the present substance is within the range of lO,OOO to 1,0 100,000. . .
It was thus ascertained that the present substance is a novel substance obtained from the protein-bound polysaccha-rides (PS-K) originating from fungi belonging to the Coriolus .
and not containing ~-glucan as measured by N.M.R., therefore, the present substance should be distinguished from PSK. It is easy to identify the substance by specifying the absorption region by N.M.R~ measurements, and in this respect, we believe that the identification techniques relating to the substance of this invention will prove to be a precious guide for the study of complicated high molecular substances derived from natural . . .
materials.
As the present substance is obtained by separating from the mucopolysaccharide substances (PS-K),it is quite natural that it should have a high antitumor activity, and such activity can b obtained even if the substance is administered orally.

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We will now describe the antitumor activity of the present substance by showing the results of various kinds of animal tests.
Acute toxicity:
Acute toxicity in mice and rats The mice used in this test were of ICR-JCL s-rain, 4 to 5 weeks old, weighing 21 to 24 gr., and the rats were of Donryu strain, 4 to 5 weeks old, weighing 100 to 150 gr. The ;
substance of this invention was administered through the follow-ing four routes: intravenous, subcutaneous, intraperitoneal and oral. Observations 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 5 below. No death was caused by administration 1 of the substance even with the maximum dosage in both rats and s ¦ mice, and it was practically impossible to calculate the numerlc 1 values for LD50.

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. ':~.- . , , ¦ Table 5. Acute toxicity in mice and rats ~ . .
I Kind of Route of LD50 (mg/kg) animal administration Female Male . ~ l . _ "
Intravenous > 1,300 > 1,300 ¦ Mice Subcutaneous> 5,000 > 5,000 ¦ Intraperitoneal > 5,000 > 5,000 l Oral ~ > 20,000 > 20,000 .. l : .
Intravenous > 600 > 600 ¦ ~ Rats Subcutaneous> 5,000 > 5,000 , l Intraperitoneal > 5,000 > 5,000 Oral > 20,000 > 20,000 :~ I _ . _ ,'"''"
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5 j Antitumor activity tests (1) In vitro antitumor activity (1)-1 In vitro inhibitory activity against growth of ascites hepatoma AH-13 cells 50% growth inhibitory concentration IC50 was examined.
0 The substance of this invention, diluted stepwise, was added to a suspension of ascites hepatoma AH-13 celIs, and after 48-hour culture, the living c~lls were counted by a staining methc d to determine growth inhibition. The result re~ealed that IC50 ' of the present substance was 100 ~gJml.
(1)-2 In vitro activity against growth of Ehrlich ascites ¦ cells of mlce ':''' ~1 :
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~OB4488 . , The influence of the substance of this invention on uptake of 3H-uridine and 3H-thymidine by Ehrlich ascites cells of mice (hereinafter shortened as EC cells) was examined. EC
cells (5 x 105/ml) were incubated in Eagle MEM culture media containing 500, 1,000 and 2,000 ~g/ml of the substance of this invention and 0.5 Ci/ml of 3~-uridine or 3H-thymidine at 37C for 120 minutes. As control, sterile physiological saline was added in place of the substance of this invention. As a result, the substance of this invention could reduce the uptake of 3~-uridine into RNA of EC cells to about 70~ of the control at 500 ~g/ml of the substance of this invention after 120 minutes~
to about 65% of the control at 1,000 ~g/ml, and to about 60% of the control at 2,000 ~g/ml. It could also reduce the uptake of 3H-thymidine into DNA of EC cells to about 65% at 500 ~g/ml, about 60% at 1,000 ~g/ml and about 50% at 2,000 ~g/ml, after 120 minutes, respectively.
(2~ In vitro and in vivo antitumor activity test ~Suppression activity of the substance of this invention against growth of Ehrlich ascites cells) ~
The suppression activity of the substance of this ;
invention against growth of Ehrlich ascites cells of mice (hereinafter shortened as EC cells) was investigated. The EC
cells (5 x 106 cells/ml) were incubated in a ~anks' solution containing 5,000 ~g/ml of the substance of this invention at 37C
~5 for hours, and aft-r incubation, EC cells were inoculated ' -19- I

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into the peritoneal cavities of JCR-JCL female mice (10 mice in : each group) at the rate of 106 cells per mouse and the deaths were examined during the period of 20 days.
As a result, no death was seen during the period of 20 days after inoculation in the mice to which EC cells treated with the substance of this invention has been inoculated. There were some mice who had no accumulation of abdominal dropsy.
On the other hand, all of the mice of the control group ; ~ inoculated with the e~tn~ EC cells died of tumor during the ` 10~ 20-day period after inoculation.
(3) In vivo antitumor activity tests -The results of the in vivo antitumor activity tests ; on mice and rats are as shown in Table 6 ~elow. The substance ~ of thl ~entlon revealed hlyh ant tumor aotlvity ln each case.

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l . :' Preparation of the present substance :
Now the process for preparation of the substance of the present invention is described. As starting material, it ~-l is possible to use not only the specified strains of Coriolus versicolor (Fr.) Qu~ Polystictus versicolor Fr.) -- Fermenta-~ , tion Research Institute of Agency of Industrial Science and -Technology Deposit FERM-P No.2412-2426 (Strain Nos. CM-101-115 Strains) -- but also other strains of Coriolus versicolor (Fr.) Quel. and strains belonging to other Species such as Coriolus con_ors (Berk.) Imaz. tIrpex consors Berk.], Coriolus pubescens (Fr.) Qu~l. [Polyporus pubescens Schum. ex Fr.; ,Tyromyces pubescens (Fr.) Imaz.~, Coriolus biformis (Klotz.) Pat. [polystictus biformis (Kl.) Fr.], Coriolus hirsutus (Fr.) ~ . .~
Quel. [Polystictus hirsutus Fr.], Coriolus conchifer (Schw.) Pat.
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1.5 [Pol~stictus conchifer (Schw.) Sacc.; Poronidulus conchifer (Schw.) Murr.], Coriolus pargamenus (Fr.) Pat. [Polystictus , -- ~ .
~arqamenus Fr ]. However, it is most advantageous for industrial production to use the above-mentioned CM-101 strain as it can provide the highest yield of production of the present ~0 substance.
The substance of this invention is contained in the extracts of the fruit bodies and mycelia of Coriolus.
The fungi are precultured and the mycelial mat grown on the surface of culture medium are ~5 homoge ized with physiological salLne to prepare the seeds for ¦ ~;
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production culture. The seeds are subjected to stationary cultur~
or submerged culture to develop the mycelia, which are extracted by using an aqueous solvent such as hot water or dilute alkalih~
solution. The obtained extract, after removing the extraction residue therefrom, is concentrated and then subjected to salting out with ammonium sulfate or ultra-filtration to eliminate the low molecular weight substances. The thus refined extract is ;~ concentrated up to 5-10~ by weight. This concentrated solution is further added with ammonium sulfate in an amount corresponding to 25% of its saturation value for the purpose of conducting fractional precipitation, and the formed precipitate is removed.
The resultant solution is again added with ammonium sulfate in an amount corresponding to 40% of its saturation value and the : produced precipitate is collected. The collected precipitate ig dissolved with water and then desalted by dialysis. The resultant solution is treated through a DEAE cellulose column, and the adsorbed substance on the column is washed with water and then eluted with 1 N saline. The obtained elutant is again added with ammonium sulfate in an amount corresponding to 40% of its saturation value to form a precipitate, and the formed precipite is again dissolved with water and then desalted by dialysis. The resultant refeined product is dried to obtain - the object substance of this invention.
As reviewed above, tne protein-bound polysaccharide substance according to this invention shows a very excellent '.' ',` . . '' ,, i1 ! . . -8448~ ' ' ~... ' effect when used ~s an orally ~dmiAistered antitumor agent.
The present substance also gives immunopotentiation through the host and is effective for preventing or minimizing decline of immunity or physical strength of the patients who have undergone various kinds of treatments such as chemotherapy, radiotherapy, surgical operation or blood transfusion no matter whether they are affected by cancer or not. Furthermore, the present substance is also effective for protecting the patients against infectious diseases such as hepatitis or pnèumonia which may be caused by infection of virus or bacteria as a result of loss or decline of immunity or physical strength.
Also, the substance of this invention, when orally administered to the patient, not only bring about improvements l of the general physical conditions but also work marvelously in 1 expediting the intestinal activity and promoting appetite of the patient. The present substance also proves extremely helpful ¦ in promoting the bowel's action of a patient who is bed ridden for a long time.
I The present invention is now described in further detail ¦ by way of some preferred embodiments thereof.
A ~ EXAMPLES 1 - 4:
Each strain~ of CM-101 (Fermentation Research Institute of Agency of Industrial Science and Technology I Deposit FERM-P. No. 2412), CM-102 (FERM-P. No.2413) and CM-103 l ~FERM-P. No. 2414) belonging to Coriolus versicolor (Fr.) Quel.
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(Polystictus versicolor Fr.), was inoculated in a 200 ml erlenmeye flask containing 30 ml of a culture medium having the following l compositions: 5% glucose, 0.2% peptone, 0.3% yiest extract, ¦ 0.1% KH2PO4 and 0.1% MgSO4 7H2O, and cultured stationally at 25 to 27C for 10 days, and the mycelial mat grown on the surface of culture medium were homogenized with physiological saline to thereby prepare the seeds. The seeds were then inoculated in each of the 1.0 litre culture flasks containing 200 ml of the same culture medium and incubated at 25 to 27C for 25 days to thereby 0 ~ obtain the mycelia. The mycelial yield~ ~e 4 to 4.3 gr. per flask for CM-101, 2.0 to 2.5 gr. for CM-102, and 2.7 to -3.2 gr. for CM-103.
Then, 3 litres of distilled water was added to each of 100 gr. of the prepared mycelia, and each of the mycelia was extracted under agitation at 98C for 3 hours. ~fter completion ¦ of extraction, each of the solution was separated into extract l and extraction residue, and the extraction residue was further ¦ subjected to the same extraction treatment as mentioned above by using each aqueous solvent shown in Table 7 below. Each of extracts was collected and then subjected to concentration.
The concentrated solution of the extract was saturated with ammonium sulfate to form a precipitate. The obtained precipi-tate was again dissolved in water and subjected to desalting by '5 11 Il - 25 -- . .
!

. :. . ,: . .. .

10844~8 dialysis using a cellulose membrance. Thus obtained solution '- was concentrated up to 5~ by weight,then added with ammonium sulfate in an amount corresponding to 25% of its saturation -value. After removing the precipitate formed by the treatment mentioned above, the solution was again added with ~A ammniUn~sulfate in an amount corresponding to 40% of its saturation value, and the obtained precipitate was dissolved with water and further subjected to same desalting as mentioned above, then passed through a DEAE-cellulose column, and the adsorbed substance on the column was washed with water, then eluted with 1 mole saline. The elutant was again added with ammonium sulfate in an amount corresponding to 40~ of its ; saturation value, and the resultantly formed precipitate was collected and dissolved again with water. The obtained , solution was desalted by dialysis, and concentrated, then spray-dried to thereby obtain the object substance. The properties of the obtained substance and the results of animal tests thereof are shown in Table 7. The "alkali extraction method" using alkali as solvent, shown in Table 7 was same as the above-described process except that a l/lON
caustic soda solution was used instead of water in reextraction of the residue and pH was adjusted to the reatral value with l/lON hydrochloric acid after completion of extraction.
In Table 7, the infrared absorption spectrum measured .~!
according to the potassium bromide disk method was as diagram-matically represented in Fig. 1, that is, there were seen absorption .

'`; -26-:' . . ., ,~

~, ~

--1 .
~ ~o84488 ¦ of vOH at 2600 - 3200 cm 1, deformation vibrations of -NH2 and -N~
¦ at 1600 cm~l and at 1530 cm~l, respectively, broad absorption at ¦1 1200 - 1000 cm~l, antisymmetric stretching vibration of pyranose ;~
¦ rings in the polysaccharid'e portion of C-O-C linkage, and peculia~
absorption at 890 cm 1 due to ~-linkage of glucose, but absorp-; tion (~-linkage) at 840 cm 1 was scarcely seen.
As there was noticed no much difference among the ; respective specimens concerning the infrared absorption spectrum, the results of Example 1 alone were shown repre-IO sentatively.
The measurements of NMR were conducted by adopting DSS as internal standard and using heavy-water às solvent.
Shown in the Table 7 are the values after correction under the supposition Lorenz's curve for eliminating the influence of residual light water in heavy water.
The molecular weights of the present substance were measured by using the ultracentrifugal method. They were within the range of 5000 to 300,000 in all the specimens.
For measurements, there were employed the sedimentation ~o equilibrium and synthetic boundary pattern using an inter-ference optical system. The experiment conditions were as follows: specimen concentration, 0.3~; solvent, l/lOM XCl;
temperature, 25C; length of solution column, 1.7 mm; speed, ; 22,000 r.p.m.; measuring time, 5 hours.
S ~The ~mino acid analysis was performed according to a ,., .' '.
,, . .

. .

- ~84488 ordinary method by adding 4 ml of 6N hydrochloric acid to 10 mg of each specimen, freezing it with dry ice acetone, sealing same in a tube under reduced pressure, hydrolyzing at 110C for 24 hours, drying and then dissolving in 30 to 40 ml of a citric acid buffer solution with pH of 2.20.
For determining the specific rotation, first the optical rotation was measured with D line (589 m~) of sodium by using 0.25~ aqueous solution of each specimen and 5 cm cells and the specific rotation [a]25 was calcula~d from the measured optical rotation ~.
The measurement of the monosaccharides composition of the present substance was made in the following way.
3 mg of each specimen was put into a 5 mm glass ampule, to which was added 10 ml of 3% hydrogen chloride methanol to perform methanolysis at 100C for 16 hours, the resultant product was neutralized with silver carbonate and filtered at room temperature. The filtrate was evaporated to dryness and then dissolved in 0.5 ml of dry pyridine.
The obtained solution was added with 0.2 ml of hexamethyldisila-zane and 0.3 ml of trimethylchlorosilane, and the mixture was allowed to stand at room temperature for 30 minutes, to perform trimethylsilation. Upon completion thereof, the mixture was dissolved in chloroform, and after removing the excess reagent by washing with water, the obtained solution was evaporated to dryness. The thus treated substance (trimethylsilate) was dissolved in carbon tetrachloride and .

.

10844~38 measured by gas chromatograph.
The mode of linkage of saccharides was determined accord ¦ ing to Haworth's method. That is, 2 gr. of each specimen was 1 dissolved in 10 ml of lN NaOH solution and, while maintaining 1 the mixture at 40 to 50C in a nitrogen stream, under violent ¦ agitation, 20 ml of dimethylsulfuric acid and 4~ ml of 30% 1-sodium hydroxide solution were added dropwise over a period of serveral hours, and after allowing the mixture to stand ¦ overnight, it was subjected to the same treatment with the ¦ same amount of methylating reagent. The reaction solution, after neutralized, was dialyzed in running water and the dialyzate was concentrated under reduced pressure and subjected to the methylation three times, and after additional neutraliza-¦ tion and dialysis, the mixture was evaporated to dryness under 1 reduced pressure. The residual substance was dissolved in 20 ml ¦ of chloroform-methanol 110 : 1) mixed solution, to which was added petroleum ether-ether (1 : 1) mixed solution to precipitate ¦ the methylated substance. Then, about 20 mg of this methylated ¦ substance was hydrolyzed with lN sulfuric acid at 100C for ¦ 16 hours, and the hydrolyzate was led into alditol-acetate ;
¦ according to an ordinary method and the molar ratio was ¦ determined from the peak area on the gas chromatograph. In 1 l order to distinguish between 2,3,6-Tri-O-Me-G and 2,3,4-Tri-I O-Me-G, 20 mg of the methylated substance was subjected to meth-1 anolysis at 100C for 16 hours in a sealed tube by using 3 '' - 29 -' I ' , .
.

, li lU844~8 l~
:~:
: :
¦ hydrogen chloride methanol. The presence of 2,3,4-Tri-O-Me-G
¦ was not confirmed in the gas chromatographic analysis of the methanolysis product. Each of the above decomposed products was identified on the gas chromatograph by using -standard. Also, each of the abo~e-mentioned decomposed products was isolated by using column liquid chromatography and either crystallized or led into a crystalline derivative.
The properties, structural characteristics and anti-tumor activites of the thus o~tained protein-bound poly-~0 sacch ide subs ances aee shown comprehensive1y n Table 7.

`
:

; I ' . ' .
~. I, .
.;:` I .
' 1. .
i 'I - 30 -. `i .

_ . ... .. .

: 1084~88 ::
~ I . .:~
,. I . . - :~
., l ; . ::~
I ~ Table 7 .. ... .
Example No. 1 2 3 4 i: . ~ .
Deposit No. FERM-P No.2412 No.2412 No.2413No.2414 ¦ ~
Strain No. CM 101 CM-101 CM-102 CM-103 -Extraction method Hot water Alkali Hot water Alkali : Yield (g) per 100 ~ 0.7 0.9 0.3 0.4 of dry fungi Molecular weight (x104) 0.8 - 25 0.5 - 281.0 - 19 0.8 - 29 ~,~ Average mol. wt. (x104) 6.8 9.0 4.7 8.9 .`' . ~., Co~lor reaction (saccharide) ~i-naphtol-sulfuric Purple Same as Same as Same as acid reaction left left left Indol-sulf. acid Brown Same as Same as Same as reaction left left left Anthrone-sulf. Greenish ~ ll ..
acid reaction blue reaction Brown ll ll ll Tryptophane-sulfuric Purplish ll ll n ~ . -.
acid reaction brown ~

. ! ~
Color reaction (protein) ~.
Lowry-Folin method Blue ll " n Ninhydrin reaction Purplish I after hydrochloric blue I acid hydrolysis (6N-HCl, 20 hr.) Spe if. rotation l 0 ~ +~ ¦ +1 . .
`` .1 / . -.

- 31 - I ~
'.'''` ',) '' :'' , ~ . I i i, i ,, :
. ~ :

L~ , ..
- 10~34488 . Table 7 (cont'd) ~ L -~
. : :
Infrared absorption spectra ~ :
3600 - 3200 cm 1 PresentPresent PresentPresent 1600 cm 1 " " " "
1530 cm 1 .. . . " ~ .. .. .
. 1200 - lOOOcm~l ~ n ~
890 cm 1 .. .. ,. ~ :
840 cm Absent Absent AbsentAbsent ., NMR spectra (drawing) Fig.2-1 Fig.2-2Fig.2-3 Fig.2-4 Absorption region (ppm) 0.9 + 0.1 Present Present Present Present . ' 1.2 + 0.1 .. .. .. ..

: 1 2.0 ~ 0.1 .. .. .. ., ¦ 4.5 + 0.1 n n ~ n ¦ 4.7 + 0.1 .. n n .
5.0 + 0.1 Absent Absent Absent Absent 5.4 + 0.4 .. .. n ~
3.0 i 4,4 Present . Present Present Present Absorption intensity ratio POlysaccharlde 86.1 79.8 86.2 85.5 Prot in ¦ 13.9 ¦ 20.2 ¦ 13-3 ¦ 14-5 ' - 32 -; I .
.' I ' .
I!

~-Jl : ~
~ 10844~8 ~ ~
' . . . : ~' , ¦Table 7 (cont'd) I ,., _ ._ . .. ....
Example No. 1 2 3 4 .. .. _ ., ..... _ .
., Glycoside analysis ~mc de of linkagc !l ~-. ~lG4, - 5.3 3.5 8.0 7.0 G3~ 0.7 0.1 . 2.0 0.7 , ~lG4+ 0.8 0.9 2.0 1.3 .i. t . : .-~3 2.0 0.3 1.0 0.2 t lG3~ 0.3 trace 0.2 0.4 .
:, . ,.

, lG ) 1 1 1 1 Composing mono- Glucose Glucose Glucose Glucose ¦ saccharide .
Proportion (O Over 99 Over g9 Over 99 Over 99 ., j Amino acid analysis ~ I
I Aspartic acid 18.4 14.8 13.5 14.6 ~, Threonine 7.1 8.1 7.3 9.2 . 1l Serine 6.4 6.4 7.0 7.2 '1 Glutamic acid 12.3 14.0 14.0 13.1 .
; Proline 1.7 3.8 1.5 ~ 1.8 ' il Glycine 7.6 7.4 7.8 7.1 : :~
. ¦ Alanine 7.8 9.5 8.5 8.9 :
,1 . , .

j ; ..
~
. `!
":~ Ij, .~..... .j , ,, ll ::
1~ 1084488 '." , . '~
,;,.'~ l ~ . : ' ;~; Table 7 (cont'd) .~ . ... _ .. Example i~o. l 2 3 4 .
,~. I ,_ . ....... . .. _ . Cysteine ~ + . + +
; Valine 7.5 7.4 7.6 7.6 '!" Methionine 1.4 1.5 l. ? 1. 7 ~ Isoleucine 4.6 4.3 4.7 4.7 . ~ Leucine 8.0 7.1 8.0 7.9 Tyrosine 2.2 1.8 1.8 2.0 Phenylalanine 5.0 4.3 5.2 5.0 , Tryptophane + + 0.9 +
. ~ Lysine 2.8 2.4 2.8 2.4 Histidine 1.5 1.4 1.7 1.5 . Arginine 3.4 2.9 2.8 2.8 ~; (Ammonia) 2.3 2.9 3.2 2.5 .,,j I ., . ~
I Total of aspartic acid~ 75.1 74.7 73.7 75.6 serine, glutamic acid, ~ glycine, alanine, .; ¦ valine and leucine ;

Acute toxicity (mice) (LD50 (mg/kg ) Intravenous (male) Over 1300 Same as Same as Same as . 'I . left left left Il (female) Over 7 300 " ll ~ .
~ I Subcutaneous (male) Over 5000 - ll ll ~ 1l (female) Over 5000 " . " ..
.
q' ~i .,.~ ,j I .
.`~,: ', ' !
, !' - 34 -.
.,,,",, ., I

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, ~ ' ,. :

-~ ~ :: :
~08448B

Table 7 (cont'd) I
Example No. 1 2 3 4 Intraperitoneal (male) Over 5000 Same as Same as Same as . . .
left left left . (female~ Over 5000 ......................... .
Oral (male) Over 20000 . " ll ll (female) Over 20000 " ll ..

Acute toxicity (rats) [ Dso (mg/kg)] .
Intravenous ~male) Over 600 Same as Same as Same as . left left left (female) Over 600 'l .l n Subcutaneous (male) Over 5000 . ." .l (female) Over S000 ll . .
Intraperitoneal . .
(male) Over 5000 ll .
(female) Over 5000 Oral(male) Over 2000 ll ll .
(female) Over 20000 " ~, ..
. 1 I ' ' Antitumor activity In vitro inhibition against growth of ascites hepatoma AH-13 cells . . :
IC50 ~g/ml 105 97 100 98 , .~
Il , Il , . . I
i, I

.

il lU844t~8 Table 7 (cont'd) . . . _ . ___ _ ~-I E~am~le No. 1 2 3 4 ' _ In vitro inhibitory effect against growth of Ehrlich ascites l cells (EC cells) of mice ! After 120-minute uptake of 3~-uridine into RNA of EC cells (the control is given as 100) 500 ~g/ml 71 70 72 73 1000 ~g/ml 64 65 65 63 I 2000 ~g/ml 59 60 60 59 After 120-minute uptake of 3H-thymidine into DNA of EC cells (the control is given as 100~
500 ~g/ml 63 65 64 65 1000 ~g/ml 60 - 61 59 60 ,2000 ~g/ml 50 51 52 51 '~ , . Suppres~ion against growth of Ehrlich ascites cells 300 ~g/ml All Same as Same as Same as survived left left left .

In vivo antitumour activity (growth inhibition) Mouse sarcoma-180 ¦~ 10 mg/kg 98 99 100 99 .
iP ~250 mg/kg 100 100 98 98 1500 mg/kg 100 100 100 100 .IpO~ 500 mg/kg 85 84 85 86 !l1000 mg/kg 90 87 89 90 Mouse Ehrlich iP 50 mg/kg 100 100 100 100 Rat AH-13 (survival/number of rats used, after 60 days) iP 250 mg/kg 7/10 8/10 8/10 8/10 - 36 _ .. : . . . - ,:

Claims (11)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for producing protein-bound polysaccharides comprising the steps of: extracting mycelia or fruit bodies of fungi belonging to the genus Coriolus with an aqueous solvent; removing low molecular weight substances from said extract; subjecting the obtained solution to fractional precipitation; collecting the thus obtained precipitate and dissolving same with water; desalting the obtained solution;
passing the desalted solution through an adsorption column to adsorb the protein-bound polysaccharides and collecting the adsorbed material.

2. A process according to claim 1 comprising the steps of : extracting mycelia and/or fruit bodies of fungi belonging to Coriolus with an aqueous solvent;
concentrating the thus obtained extract after removing the extraction residue therefrom; subjecting the extract to salting out with ammonium sulfate or ultra-filtration to eliminate the low molecular weight substances contained therein; adding ammonium sulfate in an amount corresponding to 25% of its saturation value to the obtained solution to conduct the fractional precipitation; removing the formed precipitate from the solution; adding again ammonium sulfate in an amount corresponding to 40% of its saturation value to the obtained solution to conduct the fractional precipitation; collecting the thus obtained precipitate and dissolving same with water;
desalting the obtained solution by dialysis; passing the desalted solution through a DEAE cellulose column and washing the thus adsorbed substances on the column with water; eluting said adsorbed substances with a saline; adding ammonium sulfate in an amount corresponding to 40% of its saturation value to the thus obtained elutant to form a precipitate; dissolving the formed precipitate with water; concentrating the obtained solution after desalting same by dialysis and the spray-drying the concentrated solution.

3. The process according to claim 2 wherein said fungi are species selected from the group consisting of Coriolus versicolor (Fr.) Quel. (Polystictus versicolor Fr.), Coriolus consors (Berk.) Imaz. [Irpex consors Berk.], Coriolus pubescens (Fr.) Quel. [Polyporus pubescens Schum.
ex Fr.; Tyromyces pubescens (Fr.) Imaz.], Coriolus biformis (Klotz.) Pat [Polystictus biformis (Kl.) Fr.], Coriolus hirsutus (Fr.) Quel. [Polystictus hirstus Fr.], Coriolus conchifer (Schw.) Pat. [Polystictus conchifer (Schw.) Sacc.;
Poronidulus conchifer (Schw.) Murr.], Coriolus pargamenus (Fr.) Pat. [Polystictus pargamenus Fr.].

4. The process according to claim 2 wherein said aqueous solvent is hot water and/or dilute alkali solution.

5. The process according to claim 4 wherein said dilute alkali solution is 1/10N caustic soda solution.

6. The process according to claim 2 wherein said extraction residue is reextracted with hot water or 1/10N
caustic soda solution and in case said caustic soda solution is used, pH of the obtained extract is adjusted to the neutral value after completion of the extraction.

7. The process according to claim 1, 2 or 3 wherein said fungus is the species Coriolus versicolor (Fr.) Quel.
(Polystictus versicolor Fr.) [FERM-P No. 2412].

8. Protein-bound polysaccharides having molecular weights within the range of 5,000 to 300,000 as measured by an ultracentrifugal method and presenting color reactions characte-ristic of saccharides in .alpha.-naphthol-sulfuric acid reaction, indolesulfuric acid reaction, anthrone-sulfuric acid reaction, phenol-sulfuric acid reaction and tryptophane-sulfuric acid reaction and also presenting color reactions characteristic of amino acids in ninhydrin reaction after hydrochloric acid hydrolysis and peptide linkage in Lowry-Folin method, said polysaccharides having the absorption regions as measured by proton nuclear magnetic resonance spectra at 0.9 ? 0.1 ppm, 1.2 ? 0.1 ppm, 2.0 ? 0.1 ppm, 4.5 ? 0.1 ppm, and 4.7 ? 0.1 ppm, with broad absorption being seen at 3.0 - 4.4 ppm, and also having the polysaccharide portion/protein portion ratio within the range of 55/45 to 95/5 provided that proton strength of the protein portion is at 0.5 - 2.5 ppm and that of the polysaccharide portin is at 2.5 - 6.0 ppm, and further said polysaccharide portion being composed of .beta.-glucan which shows no absorption attributed to .alpha.-glucan at 4.9 - 6.0 ppm, and said protein portion being composed of aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, tryptophane, phenyl-alanine, lysine, histidine and arginine, when produced by the process of claim 1 or an obvious chemical equivalent.

9. The protein-bound polysaccharides set forth in claim 8, wherein more than 70% of the amino acids constituting the protein portion is composed of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine and leucine, when produced by the process of Claim 1 or 2 or an obvious chemical equivalent.

10. The protein-bound polysaccharides set forth in claim 8, wherein said polysaccharide portion has a structure in which ?4Gl? is within the range of 3 to 12, ?3Gl? is 2 or less., is within the range of 0.5 to 3, is within the range of 0.1 to 2.5, and is 0.6 or less, provided that the non-reducing end-group (G1?) of monosaccharides in methylation hydrolysis according to Haworth's method is 1, when produced by the process of claim 1 or 2 or an obvious chemical equivalent.

11. Protein-bound polysaccharides set forth in claim 8, wherein the average molecular weight is within the range of 10,000 to 100,000, when produced by the process of claim 1 or 2 or an obvious chemical equivalent.