CA1127572A - Polysaccharides having anticarcinogenic activity and method for producing same - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention discloses a polysaccharide having anticarcinogenic activity isolated from a liquid extract of a fruit body and/or mycelium of a strain which is capable of producing an anticarcinogenic polysaccharide and which belongs to the genus Polyporus of the class Basidiomycetes, or from a culture medium in which the said strain has been incubated.

Description

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Thi`s invention relates to polysaccharides having an anticarcinogenic activity and also to a process for producing the same.
More particularly this invention relates to polysaccharides having an anticarcinogenic activity and to a process for producing such polysaccharides from an extract of mycelium and/or fruit body of a strain which is capable of producing an anticarcinogenic polysaccharide and which belongs to the ~enus Pol~porus of the class Basidiomycetes or from a culture medium in which said strain has been incubated.
According to this inve~tion there can be used any strain of species belonging to the genus polyporus of the class basidiomycetes and which is capable of producing an anticarcinogenic polysaccharide.
However in the embodiments of this invention to ~;
be explained hereinafter there was used a strain of Poly~orus tuberaster (Fr.) which was obtained by the culti-.
vation of a fruit body (tissue) of a fungi species collected at National Park "Izumidake", 2~1iyagi Prefecture, Japan in autumn of 1973. The identification of this species was made by ~hè following books; "Coloured Illustrations of Fungi of Japan" ed. by Rokuya Imazeki and Tsugio Hongo (published by Hoiku-Sha, Osaka, Japan) 1965; "Mycological flora of Japan" ed. by Seiya Ito (published by Yoken-Do, Tokyo, Japan), vol. 2, No. 4, 1955; and Canadian Journal of ~otany 29 147-157, 1951.
Thus the characteristics of this stain are as follows. The fruit body is about 2 - 6 inches in diameter, ..
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which is approximately same as height. The cap of the fruit body is hemispherical at first but soon expanded to a structure which is slightly curved on the upper side and essentially flat on its lower side. This cap is light brown or buff in colour and is attached to the sclerotium (from which the whole fruit body grows) by st~ut stem. The cap is soft and cheese-like in texture and covered with minute buff-colour scales when matured.
On the under side of the cap is a layer of tubes, those near the stem end of fungus being shorter and shallower than else. The sclerotium of this strain is not spherical or oval but flattened in shape. The spores are colourless (but appear white in mass) and oval, smooth and the size i~ 1.2 ~
From these characteristics and by referring to the above cited books this strain was identified with Pol~porus tuberaster (Fr.). This strain was deposited at Fermentation Research Institute, Agenc~ of Industrial ~' Science and Technology, Japan under FERM-P 4641 and also at American ~ype Culture Collection under ATCC 20544.
The anticarcinogenic polysaccharide of this invention can be obtained from a liquid extract of fruit body and/or mycelium of this strain which has been incu-bated or from a filtered broth of culture in which such mycelium has been incubated.
However, it is not easy to collect such fruit body in a sufficiently large amount in natural field.
Therefore, it is advantageous to incubate the strain to obtain a large amount of the fruit body. The incubation may be conducted in a manner well known for the incubation of fungi of the class Basidiomycetes, îor example on wooddust or sawdust medium. Thus, for example, 200 g of sawdust, 100 g of ricebran and 300 ml of water are well mixed to prepare a culture medium, which is packed in a suitable vessel and sterilized. The strain which has been cultured separately in a slant culture medium is incubated on the above prepared culture medium in a con-ventional manner at 25 - 27C. for about one month to sufficiently grow the mycelium. The culture medium was then covered with soil or sand and left to stand for one to two months so that the fruit body grows thereon. The fruit ~ody so grown is collected and used as the starting material of this invention.
The mycelium to be used in this invention may be o~tained b~ a conventional cultivation method such as solid culture or liquid culture. In solid culture, for example, agar, gelatin, starch, wooddust, pulp, other con-ventional solid culture medium or a combination thereof may be used. In liquid culture there may be used a liquid culture medium contalning various nutrients which are well known in the art of cultivation of microorganisms. Thus the liquid culture medium may contain a carbon source such as glucose, maltose, lactose, sucrose, starch, molasses, etc., a nitrogen source (organic and inorganic nitrogen source material) such as pepton, yeast, yeast extract, corn steep liquor, urea, ammonium salts, etc., and one or more of organic and inorganic salts such as phosphates, magnesium salts, etc. If desired other material necessary for the growth such as vitamins may also be added. ~hese materials for solid and liquid culture media are ~ell kno~m per se in the art of cultivation of fungus, and no further ~27572 detailed explanation would be required therefor.
The liquid culture may be conducted in any conventional manner such as stational culture, shaking culture or submer~ed culture. From the economical and handling point of view, liquid culture particularly submerged culture is more advantageous than solid culture.
In conducting the liquid culture the following conditions may be used:
Initial pH 2 - 9 Incubation temp. 15 - ~5C.
Incubation period 2 - 20 days In case of submerged culture the medium is subjected to aeration at a rate of 0.1 - 2.0 I/L/min. with stirring at a rate of 30 - 500 rpm.
The mycelium grown by the solid of liquid culture is collected in a conventional manner and used as the starting material of this invention.
For example, in case of liquid culture, the mycelium -may be collected by subjecting the resulting liquid culture medium to a conventional separation procedure such as ~;
centrifugation, filtration, etc. The filtrate obtained by this separation procedure is referred to as filtered broth, which may also be used as the starting material of this invention.
According to this invention, the fruit bod~
and/or mycelium collected in the above mentioned manner is Qubjected to extraction with an aqueous solvent. In this case the fruit body and/or mycelium as such ma~ be directly subaected to the extraction. If desired, prior to such extraction, the fruit body and/or mycelium may ; ~ ' 757~, be subjected to a pretreatment such as washing with water, air drying, crushing (pulverization) or extraction with a non-polar solvent.
The aqueous solvent to be used for the extraction is water or a mixture of water and at least one water solu-ble material such as acid, base, salt or organic solvent.
In conducting the extraction the pretreated or non-pretreated fruit body or mycelium is mixed with the aqueous solvent. The temperature of the solvent is not cri-tical if maintained below 120C. Preferable temperature range, however, is from 50C. to 100C. In any case such tempera-ture may be chosen from economical view point. The extrac-tion is conducted for a period of time sufficient to effect the desired extraction. Generally, at a higher temperature the extraction time may be shorter. Within the above indi-cated preferred temperature range, the extraction is carried out preferably for a period of time from 30 minutes to 10 hours. The extraction is carried out preferably under agitation in a vessel which may be made of glass, glass-lined, e~ameled or stainless steel. The amount of the solvent may also vary over a wide range but generally 10 - 100 times the weight (on dry basis) of the fruit body and/or mycelium. The use of pulverized fruit body or mycelium is preferable for the extraction. After the extraction the mycelium or fruit body and other solid matter are removed from the liquid extract by any convenient means such as filtration or centrifugation.
The liquid extract is concentrated, for example, by vacuum evaporation or the like for further treatment. Generally the aqueous extract is concentrated to 1/3 - 1/10 of the lnitial volume.

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The extract obtained as described above is then subjected to purification to be explained below, resulting in the precipitation and recovery of the intended poly-saccharide. The term "liquid extract" as used herein means a filtrate or centrifugate resulting from the removal of the mycelium and/or fruit body and other solid matters from the extract.
~ he filtered broth may also and preferably be used for the purification to be explained below, resulting in the precipitation and recovery of the intended polysaccharide;
The term "filtered broth" as used herein means a filtrate containing the active ingredient, i.e. polysaccharide and obtained by the removal of mycelium and other solid matters from the cultured broth, i.e. culture medium in which the strain has been incubated by liquid culture in the manner as explained before. The filtered broth is concentrated, for example, by vacuum evaporation or the like for further treat-ment. Generally the filtered broth is concentrated t-o 1/3 - 1/10 of the initial volume. The concentrated filtered broth is then subjected to the purification.
The liquid extract and the filtered broth may be subjècted to the purification separately, or the liquid extract and the filtered broth may be combined together so that the mixture is subjected to the purification.
The purification may be conducted in any of the following procedures.
(A) Precipitation of the desired substrance by the addition of a highly polar organic solvent (such a~ lower alcohols and ketones e.g. methanol, ethanol, propanol, butanol, acetone, etc.) or by ~2757~
salting out (by the addition of water-soluble inorganic salts such as ammonium sulfate, sodium chloride, potassium chloride, etc.).
(B) Removing acids,ions and low molecular weight substances by any of dialysis, gel filtration (by the use of dextran or poly-acrylamide gel such as those available under the trade marks Sephadex and siogel), ion-exchange resin treatment (by ~he use, for example, of various commercial anion and cation exchange resins such as those avail-able under the trade marks Amberlite, Dowex and Duolite) 7 ultrafiltration and a combina-tion thereof, to produce a substantially pure solution from which the desired active sub-stance is recovered.
(C) Treatment for removal of free proteins, such as Sevag method, trifluorotrichloro-methanemethod, protease treatment, etc.
These procedures are well known per se in the art. If de-sired two or more of them may be combined. Generally, how-ever, the liquid extract or filtered broth, after concen-tration, is subjected to a procedure selected from ion-; exchange resin treatment, dialysis, gel filtration, ultra-filtration and a combination thereof to effect decoloration, deacidification and removal of low molecular weight substan-ces. From such purified solution the desired active sub-stance may be recovered by a proper procedure such as freeze drying. If desired the above mentioned procedure(s) may be repeated to obtain desired extent of purification.
The substance thus obtained has the following characteristics:
(1) This substance is white or greyish white powder and non-dialyzable.

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(2) Molisch reaction and Anthrone reaction are positive.

(3) Iodide reaction, Elson-Morgan reaction and Biuret reaction are negative.

(4) Ninhydrin reaction is weak positive or negative.

(5) This substance is soluble in water and in an aqueous solution of a substance soluble in water e.g. organic solvent, acid, base salt and the like, but insoluble in an organic solvent such as petroleum ether, ethyl ether, benzene, - acetone, ethanol, methanol, etc.

(6) ~his substance is without a sharp melting point and carbonized upon strong heating. ;~

(7) The hydrolyzate of this substance obtained -by dissolving this substance in 0.1 N aq. sulfuric : acid and heating the solution (containing 0.5 by weight of this substance) at 100C. for 5 hours contains at least one or more of galactose, glu-cose, mannose, fucose and xylose, the kind(s) of the sugar varying depending upon the kind or extent -of the purification.
From the above characteristics this substance is believed to be a high molecular wei~ht polysaccharide com- -posing of the sugar mentioned above and having a molecular weight of 8000 or higher.
~ he polysaccharide of this invention exhibit no antimicrobial activity against bacteria, fungi and yeasts such as StaPhylococcus aureus, scherichia coli, acillus subtilis, Asper~lllus niger, Candida albicans, etc.

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Therefore this substance is auite different from anti-bacterial anticancer agents such as those obtained from species belonging to the genus Actinomycetes.
The polysaccharide of this invention exhibits neither cytotoxicity nor side effects commonly seen in connection with the use of conventional agents such as decrease in number of leucocyte, anemia of liver and other organs, atorophy of ~pleen, loss of body weight and loss of appetite. The acute toxicity (~D50) of this polysaccharide in mica is more than 2500 mg/kg when intraperitoneally injected.
The polysaccharide of this invention has an anti-carcinogenic activity. The anticarcinogenic activity has been confirmed and determined by the following test.
Thus a mouse of IRC-JCL strain weighting 20 ~ 2 g. was inoculated intraperitoneally with solid type Sarcoma-180 çancer or Ehrlich cancer cells. After one week from the inoculation a sufficient increase of ascitic fluid was observed in the mouse. ~he cancer cells therein were subcutaneously transplanted at axillary area of the other mice at a rate of 4,000,000 cells per each mouse. These mice were divided into several groups, each composing of eight individuals. ~he first group (control group) was administered only with saline, while each of the other groups was administered with the polysaccharide of this invention.
The first administration o~ saline or the polysaccharide was made intraperitoneally after one day of transplantation and and the subsequent administration was repeated 9 times every other day. An observation was made of the solid cancer transplanted on mice, and one day from the last administration _ g _ . , .. .. . . .... , . .. . . . .. j . . . .. . . . - . - . - - - -l~Z757~

(i.e. 10th day) the average increase of body weight was measured and then anatomized to check side effects and to determine the weight of tumors removed from the control group as well as those removed from the polysaccharide-treated groups. ~he "inhibition ratio (IR)" indicated in the following tables was calculated according to the formula: -IR (%) = (C - ~)/C x 100 wherein a represents an average weight of the tumors removed from each of the control group mice and T represents an ave-rage weight of tumors removed from the polysaccharide-treated group mice.
It has been confirmed that the polysaccharide of this invention exhibits strong anticarcinogenic activity against both Sarcome-180 solid cancer and Ehrlich carcinoma.
~ he invention will be further explained in the following Fxamples with reference partly to the drawing~
wherein Figs. 1 and 2 are infrared spectra of polysaccha-rides according to this invention.

Example .. ..
A strain (FE~-P 4641, A~CC 20544) of Polyporus tuberaster (Fr.) was incubated in the following liquid cul-ture medium-Glucose 30 g Polypeptone 5 g Yeast extract (Difco) 1 g Potassium phosphate (Primary) 1 g Magnesium sulfate (7 H20) 0.5 g ... . . ..

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Water 1 liter 5 6 - 5.8 (without Initial pH adjustment) The incubation was conducted by charging 100 ml of the above culture medium to each of 500 ml Erlenmeyer flasks. lhe flasks were stopped with cotton, sterilized for 30 minutes at 120C. After cooling there was inoculated in a conventional manner with said strain which had been cultured separately in a slant culture medium containing 2 % glucose, 0.5 % Ebios and 1.5 ~o agar. After 7 days shaking incubation at 27C., the contents of the flasks were used for the subsequent incubation. Twenty liters of the liquid culture medium described above in a 30 liter stainless steel ~ar fermenter were sterilized at 120C. for 30 minutes and cooled. Then, the content of the flasks obtained as above was inoculated in the culture medium in said jar fermenter. The medium was subjected to aerobic inocubation with stirring (250 r.p.m.) for 3days at 27C.
and with an aeration rate of 0.5 liter/liter/min. ~he cul-tured broth thus obtained was filtered to obtain 3000 g. of wet mycelium and 17 liters of filtered broth. The mycelium was washed with one liter of water and washing liauid was combined with the filtered broth. The washed mycelium was mixed with 6 liters of water and the mixture was heated at 120C. for 30 minutes Ln a closed vessel. Then the mixture was allowed to cool to room temperature and then filtered followed by concentration of the filtrate ~liquid extract) to 1 liter. The concentrated liquid extract was dialyzed with water and then freeze-dried to yield 12.08 g. of greyish white powdery substance. ~he infrared spectrum of this ..

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substance (as K~r tablet) was as shown in Fig. 1.
The filtered broth (18 liters) was concentrated to 2 liters and dialyzed with water and freeze-dried to yield 23.60 g. of a slightly brownish powdery substance.
Upon hydrolysis in the manner as explained before it was confirmed that each of these polysaccharides i9 com-posed of galactose, glucose, mannose, fucose and xylose.
The anticarcinogenic activities of these poly-saccharide sub~tances were as shown in the following Tables 1 and 2.

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Example 2 A mixture of 200 g. of wooddust J 100 g. of ricebran and 300 ml of water was well mixed,to prepare a culture medium, which was packed in a cylindrical vessel (15 cm. dia. x 20.cm. height) made of a synthetic resin.
The cultured medium was sterilized at 120C. for 60 minute~
and allowed to cool to the room temperature. The strain ~ERM-P 4641, ATCC 20544 which had been cultured separately in a slant culture medium in the same manner as in Example 1 was incubated on the above prepared culture medium at 26C. for one month to sufficiently grow the mycelium.
~he culture medium was then embedded in soil and left to stand for.2 months so that the fruit body of the strain grows on the soil. The yield of the fruit body was about 180 g. per one vessel. . -The fruit body thus obtained was collected, air-dried and pulverized. Then 100 g. of the fruit body powder were mixed with 3 liters of water and the mixture was boiled, for 3 hours. The mixture was filtered to obtain a wet filter cake and filtrate (liquid extract). The wet filter cake was '' again added with 3 liters of water, boiled for 3 hours and then filtered to obtain a filtrate (liquid extract). The both extracts were combined together and concentrated to 1 liter. The concentrated extract was.subjected to dialysis , with water and then freeze-dried to obtain 5.40 g. of a greyish white powdery substance (polysaccharide), ,which was composed of galactose, glucose, mannose, fucose and xylose. The anticarcinogenic acti~ity of this substance against solid Sarcoma 180 was 95.5 % ~dose 25 mg/kg/day~
and the complete regression was 7/8.

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~27572 xample 3 The strain of ~ERM-P 4641, ATCC 20544 was incubated in a jar fermenter in the same manner as in Example . Then the filtered broth (11 liters) was subjected to the purifi-cation in the following manner. Thus the filtered broth was concentrated to 1.5 liter by vacuum evaporation and the concentrate was dialyzed for 24 hours in flowing water.
The dialyzate (1.6 liters) was àdjusted to pH 8.0 by aq. NaOH and subjected to DEAE Sephadex treatment. A part o~ the liquid passed through D~AE Sephadex was freeze-dried to obtain a polysaccharide powder (referred to as Sample A-l) which is greyish white in colour and composed of galactose, glucose, mannose, fucose and xylose. The rest of said liquid (1.5 liters) was added with 0.5 liter of ethanol to form a precipitate, which was dissolved in 1 liter of water and the solution was subjected to dialysis in running water for 24 hours. The dialyzate was adjusted to pH 3.5 by HCl and then subjected to SP-Sephadex treatment. The liquid passed through the SP-Sephadex was added with ethanol so as to be 25 ~ alcohol concentration to form a precipitate, which was dissolved in one liter of water and again subjected to dialysis in flowing water for 24 hours. The dialyzate was freeze-dried to obtain 3.0 g. of solid substance (poly-saccharide), which was added with 1 liter containing 0.3 M
acetic acid and 0.3 M ~raCl to form a precipitate. The mi~-ture was centrifuged to obtain a solid substance and filtrate.
The filtrate was dialyzed and the dialyzate was freeze-d~ied to obtain 0.18 g. of acid soluble polysaccharide powder (referred to as Sample A-3) which is slightly greyish white in colour and composed of mannose, fucose, xylose and galactose.

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Said solid substance resulting from the centrifugationwas dissolved in one liter of water and dialyzed in flowing water for 24 hours. The dialyzate was centri~uged to obtain a filtrate and solid precipitate. The filtrate was freeze-dried to obtain 1.27 g. of polysaccharide powder (referred to as Sample A-4) which is slightly greyish white in colour and composed of glucose. The solid precipitate was also freeze-dried to obtain alkali-soluble polysaccharide powder (referred to as Sample A-5) which is slightly greyish white in colour and composed o~ glucose, manno~e and xylose.
The infrared spectrum of Sample A-4 is as shown in Fig. 2.
The anticarcinogenic activities of the~e samples in mice were as follows: -Anticarcino~enic activit~ in mice (Sarcoma-180) bancer cell : Sarcoma-180 4 x 106 cells/mouse Animal strain : ICR-JC~ 20 g. + 2 g.
Treatment : Administered intraperitoneally after one day of transplantation. (control: saline) 7 times.
Determination : Solid type tumor weight.
IR (Inhibition ratio) IR = (C - T)/C x 100 C : Average weight of tumors removed from control group.
T : Average weight of tumors removed -from the polysaccharide treated group.

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Sam- Dose lete + Standard Dev. / IR
ple mglkg/day Route re- / Control ' gr A-l 5 ip 3/8 0.41+0.42/2.29+1.04 82.1 " 4/8 0.28+0.15/ " 87.8 A-3 2 n . 2/8 0.52+0.25/ " 77.3 " 3/8 0.41+0.27/ " 82.1 :: . _ _ _ n 6/8 0.20+0.19/ " 91.3 " 1/8 0.54+0.32/ " 76.4 _, _ A-s 5 4/8 0.27+0.21/ " 88 2 5~ " 3/8 0.40+0.38/ " 82.5 Anticarcinogenic activity in mice (Ehrlich carcinoma) Cancer cell : Ehrlich carcinoma 4 x 106 cells/mouse Animal strain : ICR-JC~ 20 g. + 2 g.
.
Treatment : Administered intraperitoneally after one day of transplantation. (control: saline) 7 times.
Determination : Solid type tumor weight.
IR (Inhibition ratio) = (C - T)/C x 100 a Average weight of tumors removed from control group.
T : Average weight of tumors removed from the polysaccharide treated group.

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Co~- Average Sam- Dose plete + Standard D ~ IR
ple mg/kg/day gress. "-~ Control 0.169 ip 2/8 0.69+0.64/1.74+0.93 60.3 A-3 1~690 " 5/8 0.30+0.28/ " 82.8 16.90 " 3/8 0.51+0.46/ " 70.7 0.2 " 1/8 0.93+0.65/ " 46.6 ~_4 2.0 " 5/8 0.20+0.19/ " 88.5 20.0 " 7/8 0.13~0.17/ " 92.5 _ : 0.2 " 0/8 1.09+0.67/ " 37,4 A-5 2.0 " 6/8 0.21+0.10/ " 87 9 20.0 " 4/8 0.21+0.11/ " . 87.9 ._ _ _ ;~ . , , .

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Claims (8)

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

1. A polysaccharide having an anticarcinogenic activity prepared by forming a filtered broth from a culture medium or liquid extract of fruit body or mycelium of a strain which is capable of producing an anticarcino-genic polysaccharide and belongs to the genus Polyporus of the class Basidiomycetes, and recovering the poly-saccharide from the filtered broth or liquid extract in a purified form.

2. A polysaccharide according to Claim 1, wherein the strain is that of the species Polyporus tuberaster.

3. A polysaccharide according to Claim 2, wherein the strain is Polyporus tuberaster Fr. (FERM-P
4641, ATCC 20544).

4. A polysaccharide according to Claim 1, 2 or 3, which is positive in molisch and anthrone reactions, negative in iodine, Elson-Morgan and biuret reactions and weak positive or negative in ninhydrin reaction, the hydrolyzate of which contains at least one of galactose, glucose, mannose, fucose and xylose.

5. A process for producing a polysaccharide having an anticarcinogenic activity which comprises for-ming a filtered broth from a culture medium or liquid ex-tract of fruit body or mycelium of a strain which is capable of producing an anticarcinogenic polysaccharide and belongs to the genus Polyporus of the class Basidiomy-cetes, and recover- ......................................

ing the polysaccharide from the filtered broth or liquid extract in a purified form.

6. A process according to Claim 5 wherein the strain is that of the species Polyporus tuberaster.

7. A process according to Claim 6 wherein the strain is Polyporus tuberaster Fr. (FERM-P 4641, ATCC 20544).

8. A process according to any of Claims 5 - 7 wherein the polysaccharide is positive in molisch and anthrone reac-tions, negative in iodine, Elson-Morgan and biuret reactions and weak positive or negative in ninhydrin reaction, the hydrolyzate of said polysaccharide containing at least one of galactose, glucose, mannose, fucose and xylose.