CH642681A5 - METHOD FOR PRODUCING NEW POLYSACCHARIDES WITH ANTI-CARCINOGENIC EFFECT. - Google Patents

Description

The present invention relates to a process for the preparation of new polysaccharides with an anticarcinogenic effect, using a strain of a microorganism which is capable of producing an anticarcinogenic polysaccharide and which belongs to the genus Polyporus from the class Basidiomycetes.

According to the invention, any strain of such a type can be used, which belongs to the genus Polyporus from the class Basidiomycetes and is capable of producing an anticarcinogenic polysaccharide.

In order to explain the present invention, a strain of Polyporus tuberaster (Fr.) was used in the embodiments of the present invention, which was obtained by cultivating a fruiting body (organ or tissue) of a fungus species which is found in the National Park "Izumidake", Miyagi Prefecture, Japan, was collected in the fall of 1973. This species was identified with the following books: "Colored Illustrations of Fungi of Japan", published by Rokuya Imazeki and Tsugio Hongo (published by Hoiku-Sha, Osaka, Japan) in 1965; "Mycological flora of Japan", published by Seiya Ito (published by Yoken-Do, Tokyo, Japan), Vol. 2, No. 4, 1955; and Canadian Journal of Botany 29, 147-157, 1951.

The characteristics of this strain are as follows. The fruiting body is approximately 5.08 to 15.24 cm (2 to 6 inches) in diameter and approximately the same height. The cap of the fruiting body is hemispherical at first, but soon results in a structure that is slightly curved on the upper side and essentially flat on its lower side. This cap is light brown or cha-mois colored and sticks to the sclerotium (from which the entire fruiting body grows) through stubby stems. The cap is soft and cheese-like in texture and covered with fine chamois-colored scales when it ripens. There is a layer of tubes on the lower side of the cap, the tubes near the stalk end of the mushroom being shorter and flatter. The sclerotium of this stem is not spherical or oval, but is flattened in shape. The spores are colorless (but appear massively as white), oval and smooth, and their size is 1.2-4 p ..

Based on these characteristic features and with reference to the literature references mentioned above, this strain was identified as Polyporus tuberaster (Fr.). This strain is on August 30, 1978 at the Fermentation Research Institute, Agency of Industriai Science and Technology, Ministry of International Trade and Industry, 1-3, Higashi 1-chome, Yatabe-machi Tsubuka-gun, Ibaraki-ken 305 Japan No. FERM-P 4641 and deposited on July 9, 1979 at American Type Culture Collection, 12301 Parklaw Drive, Rockville, Maryland 20852, USA, under No. ATCC 20544.

The process according to the invention consists in that an aqueous extract of a fruiting body and / or a mycelium of a strain, which is capable of producing an anticarcinogenic polysaccharide and belongs to the genus Polyporus from the class of the Basidiomycetes, or from the filtered broth of a liquid culture medium, in to which the said strain has been incubated isolates the polysaccharide.

Collecting such a fruiting body in a sufficiently large amount is not easy in nature. It is therefore advantageous to vaccinate the stem to get a large amount of fruiting bodies. The incubation can be carried out in the media suitable for the incubation of fungi of the Basidiomycetes class, e.g. on wood flour or sawdust. For example, 200 g of sawdust, 100 g of rice bran and 300 ml of water can be mixed thoroughly to obtain a culture medium, which is then filled into a suitable container and sterilized. The strain grown separately in a slant culture medium is incubated on the culture medium obtained according to the above information in a known manner at 25 to 27 ° C. for about 1 month until the mycelium grows sufficiently. The culture medium is then covered with earth or sand and left to stand for 1 or 2 months so that the fruiting body can grow on it. The fruiting body thus grown is collected and used as the starting material for the present invention.

The mycelium which can be used for the present invention can be grown by conventional breeding methods, e.g. as a solid culture or as a liquid culture. If it is a solid culture, it can be, for example, an agar, gelatin, starch, wood flour, pulp or another conventional solid culture medium or a combination thereof. Such a liquid culture medium, which contains various nutrients as are best known for the cultivation of microorganisms, is suitable as a liquid culture. Thus the liquid growth medium, a carbon source e.g. Glucose, maltose, lactose, sucrose, starch, molasses etc., a nitrogen source of organic or inorganic nature, e.g. Peptone, yeast, yeast extract, corn grain liquid, urea, ammonium salts, etc., and one or more organic and / or inorganic salts such as e.g. Phosphates, magnesium salts, etc. contain. If desired, other growth materials such as e.g. Vitamins to be added. These materials, which are to be used for solid and liquid culture media, are generally known per se for the cultivation of mushrooms, so that no further explanation is required here in this regard. Cultivation in liquid medium can be carried out in a conventional manner, e.g. as a fixed culture, shake culture or submerged culture. For economic and other reasons, a liquid culture and especially the submerged culture is cheaper than a solid culture.

When performing the liquid culture, the following conditions may apply:

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Initial pH 2 to 9

Incubation temperature 15 to 35 ° C

Incubation period 2 to 20 days

In the case of a submerged culture, the medium must be aerated at 0.1 to 2.0 L / L / min with stirring at 30 to 500 revolutions per minute.

The mycelium grown in solid or liquid culture medium is collected in a conventional manner and then used as the starting material for the present invention.

For example, in the case of a liquid culture, the mycelium can be collected by subjecting the resulting liquid culture medium to a conventional separation process, e.g. centrifugation, filtration, etc. The filtrate obtained in this way is referred to as filtered broth, which can also be used as a starting material for the present invention.

The fruit body and / or the mycelium collected in the above manner is then extracted with an aqueous solvent. The fruiting body and / or the mycelium as such can be extracted directly. If desired, the fruiting body and / or the mycelium can be pretreated before extraction, e.g. by washing with water, air drying, shrinking and pulverizing or extracting with a non-polar solvent.

The aqueous solvent to be used for the extraction consists of water or a mixture of water and at least one water-soluble material, e.g. an acid, base, salt or organic solvent.

When performing the extraction, the pretreated or untreated fruit body or the mycelium is mixed with the aqueous solvent. The temperature of the solvent is not particularly important, but should be kept below 120 ° C. Preferred temperature ranges, however, are between 50 ° C and 100 ° C. The temperature to be used will be adapted to the economic circumstances. The extraction takes place for a sufficiently long time to effect the desired extraction. In general, the extraction time will be shorter at higher temperatures. Within the preferred temperature range mentioned above, the extraction is preferably carried out within 30 minutes to 10 hours. The extraction is preferably carried out by shaking in a container which consists of glass or of glass-lined, enamelled or stainless steel. The amount used can also vary within a wide range, but is generally 10 to 100 times the amount by weight (based on dry basis) of the fruiting body and / or the mycelium. A pulverized fruiting body or pulverized mycelium is preferably used for the extraction. After the extraction, the mycelium or the fruiting body and other solid constituents are removed from the aqueous extract in a known manner, for example by filtration or centrifugation. The aqueous extract is then concentrated, for example by vacuum evaporation or the like, generally to a third to a tenth of the original volume.

The extract obtained in this way is then purified in the manner described below, and the desired polysaccharide can be precipitated and recovered. The term “aqueous extract” used in the present description means a filtrate or a centrifugate which has been obtained from the extract after the mycelium and / or the fruiting body and other solid materials have been removed.

The filtered broth can preferably be used as such for the purification process described below, whereby the intended polysaccharide is precipitated and recovered. The term "filtered broth or filter broth" used in the present description means a filtrate which contains the active substance, i.e. the polysaccharide, and by removing the mycelium and other solid components from the culture broth, i.e. the culture medium in which the strain has been incubated in a liquid culture medium in the manner described above. The filter broth is concentrated, for example, by evaporation in vacuo or the like and can then be treated further. In general, the filtered broth will be concentrated to a third to a tenth of the original volume. The filtered, concentrated broth is then cleaned.

The aqueous extract and the filtered broth can be cleaned separately. But you can combine the aqueous extract and the filter broth and then clean this mixture.

Cleaning can be done using one of the following methods.

A. Precipitation of the desired substance by adding a highly polar organic solvent, e.g.

a lower alcohol or ketone, e.g. Methanol, ethanol, propanol, butanol or acetone etc., or by salting out by adding water-soluble inorganic salts such as e.g. Ammonium sulfate, sodium chloride, potassium chloride, etc.

B. Removal of acids, ions and low molecular weight substances by dialysis, gel filtration using dextran or polyacrylamide gel, e.g. Sephadex, Biogel, etc., ion exchange resin treatment using, for example, various commercially available anion and cation exchange resins such as Amberlite, Dowex, Duo-lite, etc., ultrafiltration or a combination thereof, so as to obtain a substantially pure solution from which the desired active substance is obtained.

C. Treatment for removal of free proteins, for example by the Sevag method, trifluorotrichloromethane method, protease treatment, etc.

The above three methods are well known. If desired, two or more of them can be combined. In general, the liquid extract or the filter broth, after being concentrated, is subjected to ion exchange resin treatment, dialysis, gel filtration, ultrafiltration or a combination of these measures in order to decolorize, deacidify and remove the low molecular weight substances. The desired active substance can be obtained from such a purified solution by suitable measures, for example by freeze-drying. If desired, the above-mentioned measure (s) can be repeated in order to achieve the desired degree of cleaning.

The substance thus obtained has the following properties:

1. The substance is a white or grayish white powder and cannot be dialysed.

2. Molic reaction and anthrone reaction are positive

3. Iodine reaction, Elson-Morgan reaction and biuret reaction are negative.

4. Ninhydrin reaction is weakly positive or negative.

5. The substance is in water and in an aqueous solution of a substance which is soluble in water, e.g.

an organic solvent, an acid, a basic salt and the like, but on the other hand in an organic solvent, e.g. Petroleum ether, ethyl ether, benzene, acetone, ethanol, methanol etc., insoluble.

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6. This substance does not have a sharp melting point and chars when heated excessively.

7. The hydrolyzate obtained by dissolving the substance in aqueous 0.1 N sulfuric acid and heating the solution containing 0.5% by weight of this substance for 5 hours at 100 ° C. contains at least one of the following substances: galactose, glucose, Mannose, fucose and xylose, with different types of sugar being produced depending on the type of cleaning and the extent of cleaning.

Due to the above properties, this substance can be regarded as a high molecular polysaccharide composed of one of the above sugars and having a molecular weight of 8,000 or more.

For the reactions mentioned under 2., 3. and 4. above, see:

- MR. Christians, Fundamentals of Organic Chemistry, Sauerländer Verlag, Aarau (Switzerland) 1970, page 268 / Ninhy-drin;

- L.F. Fieser and M. Fieser, Organic Chemistry, D.C. Heath and Co., Boston 1950, 2nd edition, pages 437 and 460 / ninhydrin and biuret;

- G.A. Hill and L. Kelly, Organic Chemistry, The Blaki-ston Co., Philadelphia, Toronto 1945, pp. 316,458,459, 480 / Biuret, Ninhydrin, Molisch;

- E. Stahl, thin layer chromatography, Springer-Verlag, Berlin, Heidelberg, New York 1967, pages 147, 312, 824, 834 / iodine reaction, Elson-Morgan;

- The Merck Index, Merck and Co. Inc., Rahway (NJ, USA) 1960, pages 86 and 721 / Anthron and Ninhydrin, respectively.

Anthron reacts with glucose and similar sugars or with hydroxymethyl furfural, thereby forming dyes of the formula:

CH

0

In contrast, 2-deoxy sugar does not react [Pharm. Week-blad 88 (1953), 144].

The polysaccharide obtainable according to the invention has no antimicrobial activity against bacteria, fungi and yeasts, such as e.g. Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Aspergillus niger, Candida albicans, etc. Thus, this substance is fundamentally different from the antibacterial, cancer-preventing agents obtained from the species belonging to the genus Actinomycetes.

The polysaccharide shows no cytotoxicity and no side effects, as they usually have to be accepted with the known means, e.g. a decrease in the number of leukocytes, anemia of the liver and other organs, atrophy of the spleen, loss of body and loss of appetite. The acute toxicity (LDso) of the polysaccharides in the mouse is over 2500 mg / kg with intraperitoneal injection.

The polysaccharides obtainable according to the invention have an anticarcinogenic effect. This anticarcinogenic activity is confirmed by the following test.

A mouse from the IRC-JCL strain with a weight of 20 + 2 g was inoculated intraperitoneally with solid Sarcoma 180 cancer cells or Ehrlich cancer cells. After a week, there was a sufficient increase in ascites fluid

observed in the mouse. The existing cancer cells were transplanted subcutaneously into the armpits of other mice in an amount of 4000000 cells per mouse. These mice were then divided into different groups, each group consisting of eight individual animals. The first group, namely the control group, was only given saline, while all the other animals were given the polysaccharide. The first administration of saline or polysaccharide was one day after the transplant, etc. intraperitoneally while the subsequent administrations were repeated nine times at one day intervals. The solid cancerous tumor transplanted into the mice was observed and 1 day after the last administration, i.e. on the tenth day, the average body weight gain was measured. The animals were then dissected to see the side effects and to determine the tumor weight from the control animals and from the animals treated with polysaccharide. The inhibition rate (IR) shown in the following tables was calculated using the following equation:

IR (%) = (C - T) / C x 100

where C is the average weight of the tumors taken from each animal in the control group and T is the average weight of the tumors removed from the mice treated with polysaccharide.

It was shown that the polysaccharides had a strong anticarcinogenic activity against both Sarcom 180 cancer and Ehrlich carcinoma.

The invention is illustrated by the following examples with reference to the accompanying drawings, in which FIGS. 1 and 2 show the infrared spectra of the polysaccharides.

example 1

A strain (FERM-P 4641, ATCC 20544) of Polyporus tuberaster (Fr.) was incubated in the following liquid culture medium:

Glucose 30 g

Polypeptone 5 g

Yeast extract (Difco) 1 g primary potassium phosphate 1 g

Magnesium sulfate (7 H2O) 0.5 g

Water 1 liter initial pH 5.6-5.8

(without setting)

The incubation was carried out by adding 100 ml of the above growth medium to a 500 ml Erlenmeyer flask. The flasks were plugged with cotton and sterilized at 120 ° C for 30 minutes. After cooling, inoculation was carried out in a conventional manner with the said strain, which had been grown separately in a slant culture medium which contained 2% glucose, 0.5% Ebios and 1.5% agar. After shaking at 27 ° C. for 7 days, the contents of the flask were used for the subsequent incubation. 201 of the liquid culture medium described above were sterilized in a 30 1 steel-free fermentation container for 30 minutes at 120 ° C. and then cooled. The contents of the flask mentioned above were then added to the culture medium in the fermentation tank. The medium was subjected to aerobic incubation with stirring (250 revolutions per minute) for 3 days at 27 ° C., with 0.5 liter / liter / min.

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ventilated. The culture broth thus obtained was filtered to give 3000 g of moist mycelium and 17 l of filtered broth. The mycelium was washed with 1 liter of water and the washing liquid was added to the filter broth. The washed mycelium was mixed with 6 liters of water and the mixture was heated to 120 ° C. in a closed container for 30 minutes. The mixture was then allowed to cool to room temperature and then filtered and the filtrate (liquid extract) was concentrated to a volume of 1 liter. The concentrated liquid extract was dialyzed with 10 water and then dried with freezing,

to obtain 12.08 g of a grayish-white, powdery substance. The infrared spectrum of this substance (as a KBr tablet) can be found in Fig. 1.

18 liters of the filter broth were concentrated to a volume of 2 liters and dialyzed with water and deep-frozen to obtain 23.60 g of a light brown powdery substance.

Hydrolysis in the manner described above showed that each of these polysaccharides consisted of galactose, glucose, mannose, fucose and xylose.

The anticarcinogenic effects of these polysaccharide substances can be found in Tables 1 and 2 below.

Table 1

Substance from the mycelium Substance from the filter broth

Dose Type of Complete IR Dose Type of Complete IR

mg / kg / day administration regression% mg / kg / day administration regression%

5 ip 0/8 63.8

25 ip 6/8 94.8

125 ip 5/8 92.8

5 ip 0/8 61.9

25 ip 5/8 91.8

125 ip 5/8 89.0

Cancer cells: Sarcoma-180 4x 106 cells / mouse.

Animal strain: ICR-ICL 20 g ± 2 g.

Treatment: Intraperitoneally administered 1 day after transplantation (control: saline) 10 times.

30th

Determination: The weight of the tumor of the fixed type. Inhibition rate (IR) = (C - T) / C x 100. C: average weight of the tumors removed from the control group.

T: Average weight of tumors removed from polysaccharide-treated mice.

Table 2

Substance from the mycelium Substance from the filter broth

Dose Type of Complete IR Dose Type of Complete IR

mg / kg / day administration regression% mg / kg / day administration regression%

5 25 125

IP ip ip

1/8 7/8 5/8

69.9 92.5 88.5

5 25 125

iP ip ip

1/8 7/8 5/8

67.3 92.3 87.2

Cancer cell: Ehrlich carcinoma 4 x 106 cells / mouse. Animal strain, treatment and determination was carried out as in Table 1.

Example 2

A mixture of 200 g of wood flour, 100 g of rice bran and 300 ml of water was thoroughly mixed together to form a growth medium, which was then placed in a cylindrical container of 5 cm in diameter and 20 cm in height which was made of a synthetic resin. The growth medium was sterilized at 120 ° C for 60 minutes and then allowed to cool to room temperature. The strain FERM-P 4641, ATCC 20544, which had been grown separately in a slant culture medium in the same manner as in Example 1, was incubated on the culture medium obtained as described above at 26 ° C for 1 month to allow sufficient growth of the mycelium. The culture medium was then embedded in soil and left to stand for 2 months. The trunk's fruiting body grew in the ground. The yield of fruiting bodies per container was approximately 180 g.

The fruiting body thus obtained was collected, air dried and pulverized. Then 100 g of the powdery fruiting body thus obtained were mixed with 3 l of water and the mixture was heated to boiling for 3 hours. The mixture was then filtered, using a moist

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ten filter cake and a filtrate (liquid extract) received. The moist filter cake was again mixed with 3 l of water, heated to boiling for 3 hours and then filtered off, giving a filtrate (liquid extract). Both extracts were combined and concentrated to a volume of 1 liter. The concentrated extract was dialyzed with water and then freeze-dried, yielding 5.40 g of a grayish-white, powdery substance (polysaccharide), which was composed of galactose, glucose, SS mannose, fucose and xylose. The anticarcinogenic effect of this substance on solid Sarcoma 180 was 95.5% (dose 25 mg / kg / day) and the complete regression was 7/8.

60 Example 3

The strain FERM-P 4641, ATCC 20544 was incubated in the same manner as in Example 1 in a fermentation container. Then 11 liters of the filter broth were cleaned in the same manner as described above. The filtered broth was concentrated to a volume of 1.5 liters by vacuum evaporation and the concentrate was dialyzed in running water for 24 hours. 1.6 liters of the dialysate was added by adding an aqueous

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Sodium hydroxide solution adjusted to pH 8.0 and then treated with DEAE Sephadex. A portion of the liquid passed through DEAE Sephadex was dried by freezing to give a polysaccharide powder (referred to as Sample A-1) which looked greyish white and consisted of galactose, glucose, mannose, fucose and xylose. The rest of said liquid, namely 1.5 liters, was mixed with 0.5 liters of ethanol, with a precipitate occurring, which was dissolved in 1 liter of water. The solution thus obtained was dialyzed in running water for 24 hours. The dialysate was adjusted to pH 3.5 with hydrochloric acid and then subjected to an SP-Sepha-dex treatment. Ethanol was added to the liquid that flowed through the SP-Sephadex to obtain a 25% alcohol concentration. This gave 15 a precipitate, which was dissolved in 1 liter of water and dialyzed again in running water for 24 hours. The dialysate was dried by freezing to obtain 3.0 g of a solid substance (polysaccharide) which was put in 1 liter of a solution containing 0.3 molar acetic acid and 0.3 molar 20 sodium chloride; a precipitate formed. The mixture was centrifuged to obtain a solid substance and a filtrate. The filtrate was dialyzed and the dialysate freeze-dried to obtain 0.18 g of an acid-soluble polysaccharide powder (referred to as Sample A-3 below), which was a slightly greyish-white in color and composed of mannose, fucose, xylose and galactose.

The solid substance obtained by centrifugation was dissolved in 1 liter of water and dialyzed in running water for 24 hours. The dialysate was centrifuged

to give a filtrate and a solid precipitate. The filtrate was freeze-dried to obtain 1.27 g of a polysaccharide powder (hereinafter referred to as Sample A-4) which looked slightly grayish white and was composed of glucose. The solid precipitate was also freeze-dried to obtain an alkali-soluble polysaccharide powder (hereinafter referred to as Sample A-5), which was a slightly greyish-white in color and was composed of glucose, mannose and xylose.

The infrared spectrum of sample A-4 can be found in Fig. 2.

The anticarcinogenic effects of these samples in mice were as follows:

Anti-carcinogenic effects in mice (Sarcoma-180)

Cancer cells: Sarcoma-180 4x 106 cells / mouse.

Animal strain: ICR-JCL 20 g ± 2 g.

Treatment: Intraperitoneally administered 1 day after transplantation (control: saline 7 times).

Determination: weight of the solid tumor.

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IR (inhibition rate).

IR = (C - T / C x 100.

C: Average weight of tumors removed from the control group.

T: Average weight of tumors removed from a polysaccharide-treated group.

Per

dose

Type of

Complete

Average blind

IR

be mg / kg /

Ad dige

± standard sample

%

Day handing

repression

Dev.

A-l

5

ÎP

3/8

0.41 + 0.42

2.29 ±

1.0482.1

50

ÌP

4/8

0.28 + 0.15

do.

87.8

A-3

2nd

ÌP

2/8

0.52 + 0.25

do.

77.3

20th

ÌP

3/8

0.41 + 0.27

do.

82.1

A-4

5

ip

6/8

0.20 + 0.19

do.

91.3

50

ÌP

1/8

0.54 + 0.32

do.

76.4

A-5

5

ÌP

4/8

0.27 + 0.21

do.

88.2

50

ÌP

3/8

0.40 + 0.38

do.

82.5

Anticarcinogenic activity in mice (Ehrlich carcinoma) Cancer cells: Ehrlich carcinoma 4 x 106 cells / mouse Animal strain: ICR-JCL 20 g ± 2 g

Treatment: Intraperitoneally administered 1 day after transplantation (saline control) 7 times

Determination: weight of the solid tumor

IR (inhibition ratio) = (C - T) / C x 100 C: average weight of the tumors removed from the control group

T: Average weight of tumors removed from the polysaccharide treated group

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Per

dose

Type of

Com

Average blind- IR

be mg / kg /

Admit plette

+ Standard search%

Day handing

Regression

Dev.

A-3

0.169

ÌP

2/8

0.69 + 0.64

1.74 ± 0.93 60.3

1,690

ÌP

5/8

0.30 + 0.28

do. 82.8

16.90

ÌP

3/8

0.51 + 0.46

do. 70.7

A-4

0.2

ÌP

1/8

0.93 + 0.65

do. 46.6

2.0

ÌP

5/8

0.20 ± 0.19

do. 88.5

20.0

ÌP

7/8

0.13 + 0.17

do. 92.5

A-5

0.2

ÌP

0/8

1.09 ± 0.67

do. 37.4

2.0

ÌP

6/8

0.21 ± 0.10

do. 87.9

20.0

ÌP

4/8

0.21 + 0.11

do. 87.9

2 sheets of drawings

Claims (4)

642 681 1. A process for the preparation of an anticarcinogenic effect polysaccharides, characterized in that from an aqueous extract of a fruiting body and / or a mycelium of a strain which is capable of producing an anticarcinogenic polysaccharide and belongs to the genus Polyporus from the class of the Basidiomycetes, or the polysaccharide is isolated from the filtered broth of a liquid culture medium in which said strain has been incubated. 2. The method according to claim 1, characterized in that the strain belongs to the type Polyporus tuberaster. 2nd PATENT CLAIMS 3. The method according to claim 2, characterized in that the strain Polyporus tuberaster Fr. FERM-P 4641, ATCC 20544 is. 4. Polysaccharides produced by the process according to claim 1 with anticarcinogenic activity, characterized in that they are positive in the molar and anthrone reaction, negative in the iodine, Elson-Morgan and biuret reaction and negative in the ninhydrin reaction. Reaction weakly positive or negative react that their hydrolyzate contains at least one of the carbohydrates galactose, glucose, mannose, fucose and xylose and that their molecular weight is 8000 or more.