Physiologically active polysaccharides, production and uses thereof

• US 4,313,934 A
• Filed: 04/17/1980
• Issued: 02/02/1982
• Est. Priority Date: 05/08/1979
• Status: Expired due to Term

• Current Assignee: Kirin Beer Kabushiki Kaisha
• Original Assignee: Kirin Beer Kabushiki Kaisha
• Inventors: Matsuki, Shigeru, Tanabe, Kozo, Kitamura, Kumpei
• Primary Examiner(s): Phillips, Delbert R.
• Application Number:
  US06/141,232
• Time in Patent Office: 656 Days
• Field of Search: 260/112 R, 260/112.5 R, 424/85, 424/177
• US Class Current: 530/371
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 36/064 Saccharomycetales, e.g. baker's yeast
• CPC Class Codes: A61K 36/07 Basidiomycota, e.g. Cryptococcus
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C12N 1/185 Saccharomyces isolates
• CPC Class Codes: C12P 1/02 by using fungi
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12R 2001/865 Saccharomyces cerevisiae

Abstract

A physiologically active polysaccharide is prepared by treating autolysis-insoluble matters of yeasts of Saccharomyces with a yeast cell wall-lytic enzyme and obtaining a water-soluble physiologically active substance in the resulting solution-fraction. The novel polysaccharide possesses specific physical and chemical properties, and provides a chemotherapeutic agent such as a carcinostatic agent which can be used in the treatment of implanted tumors in mice and rats and an interferon inducer.

Claims

1. A physiologically active polysaccharide which is derived from brewer'"'"'s yeast cell walls and possesses:

(1) an elemental analysis as follows by weight

space="preserve" listing-type="tabular">______________________________________ C 40.9% ±

1.2% H 6.0% ±

0.2% N 1.9% ±

0.1% O 50.7% ±

1.5% Ash 0.5% ±

0.05%;

______________________________________

(2) an average molecular weight determined by an ultrafiltration method of 140,000 to 220,000;

(3) no melting point since no melting point is generally observed in polysaccharides, the active polysaccharide turning brown at about 265°

C. and black at about 270°

C.;

(4) a specific rotation expressed by

[α

]_D²⁵ =+75.0 to +55.0 (C=1.0);

(5) an ultraviolet absorption spectrum as shown in FIG. 1, in which no specific absorption is observed;

(6) an infrared absorption spectrum as shown in FIG. 2;

(7) solubility in water and insolubility in methanol, ethanol, ether and acetone;

(8) a positive color reaction in anthrone reaction, Molisch'"'"'s reaction, ninhydrin reaction, biuret reaction and xanthoproteic reaction;

(9) a pH of a 1% aqueous solution thereof of 5.5 to 6.5;

(10) a white color;

(11) 75 to 86% of mannose and 14 to 25% of glucose, a small quantity of glucosamine being detected;

(12) a bond structure of the sugar in which mannose is linked by α

-bond since a sugar is liberated therefrom by an α

-mannanase; and

(13) constituent amino acids comprising 26 to 32% of serine, 16 to 20% of threonine, 13 to 17% of alanine, 7 to 9% of proline, 5 to 7% of glutamic acid, 5 to 7% of aspartic acid, 4 to 6% of valine, 3 to 5% of lysine, 3 to 4% of glycine, 2.5 to 3.5% of isoleucine, 1.5 to 2.5% of leucine, 0.5 to 1.5% of tyrosine, and 0.3 to 0.7% of phenylalanine.

2. A chemotherapeutic agent which can be used in the treatment of implanted tumors in mice and rats and which comprises an effective carcinostatic amount of the polysaccharide of claim 1 and a pharmacologically acceptable carrier.

3. A chemotherapeutic agent which can be used in the treatment of implanted tumors in mice and rats and which comprises the polysaccharide of claim 1 in an amount effective to increase an interferon titer and a pharmacologically acceptable carrier.

16. A method for increasing the interferon titer of a patient comprising administering to said patient the polysaccharide of claim 1 in an amount effective to increase the interferon titer.

4. A process for preparation of a physiologically active polysaccharide, which comprises causing a yeast cell wall-lytic enzyme to act on an autolysis-insoluble matter of a yeast of Saccharomyces, and obtaining a water-soluble physiologically active polysaccharide in the resulting solution-fraction.

5. The process as set forth in claim 4, in which the yeast is selected from the group consisting of brewer'"'"'s yeasts and baker'"'"'s yeasts.

6. The process as set forth in claim 4, in which the yeast cell wall-lytic enzyme is an enzyme produced by a bacterium selected from the group consisting of the bacteria of Arthrobacter and Oerskovia.

7. The process as set forth in claim 6, in which the yeast cell wall-lytic enzyme is an enzyme produced by the bacteria of Arthrobacter.

8. The process as set forth in claim 7, in which the bacterium is that of Arthrobacter which is ATCC 21606 bacterium.

9. The process as set forth in claim 4, in which the process comprises adding 15 to 200 units of the enzyme per gram of the yeast-autolysis insoluble matter to a suspension containing 5 to 30% of the insoluble matter, and carrying out reaction at a pH of 6.0 to 9.0 and at a temperature of 20°

to 50°

C.

10. The process as set forth in claim 4, in which the yeast-autolysis insoluble matter has been contacted, at room temperature for 5 to 60 minutes, with an alkaline aqueous solution having a pH of 12 or higher.

11. The process as set forth in claim 4 which comprises, after termination of the enzymatic reaction, adjusting the pH of the reaction mixture to 3.0 to 6.0, removing any insoluble matters and obtaining a solution of the desired physiologically active polysaccharide.

12. The process as set forth in claim 11, in which a water-miscible organic non-solvent is added to the solution of the desired physiologically active polysaccharide to obtain the physiologically active polysaccharide as precipitates.

13. The process as set forth in claim 12, in which the resulting physiologically active polysaccharide is purified by subjecting the polysaccharide at least once to a purification treatment which comprises dissolving the polysaccharide in water and adding a water-miscible organic nonsolvent to the resulting solution to precipitate the polysaccharide.

14. The process as set forth in claim 13, in which at least one of the purification treatments is carried out under an alkaline condition of pH 11 or higher.

15. The process as set forth in claim 14, in which the resulting physiologically active polysaccharide possesses:

(1) an elemental analysis as follows by weight

space="preserve" listing-type="tabular">______________________________________ C 40.9% ±

1.2% H 6.0% ±

0.2% N 1.9% ±

0.1% O 50.7% ±

1.5% Ash 0.5% ±

0.05%;

______________________________________

(2) an average molecular weight determined by an ultrafiltration method of 140,000 to 220,000;

(3) no melting point since no melting point is generally observed in polysaccharides, the active polysaccharide turning brown at about 265°

C. and black at about 270°

C.;

(4) a specific rotation expressed by

[α

]_D²⁵ =+75.0 to +55.0 (C=1.0);

(5) an ultraviolet absorption spectrum as shown in FIG. 1, in which no specific absorption is observed;

(6) an infrared absorption spectrum as shown in FIG. 2;

(7) solubility in water and insolubility in methanol, ethanol, ether and acetone;

(8) a positive color reaction in anthrone reaction, Molisch'"'"'s reaction, ninhydrin reaction, biuret reaction and xanthoproteic reaction;

(9) a pH of a 1% aqueous solution thereof of 5.5 to 6.5;

(10) a white color;

(11) 75 to 86% of mannose and 14 to 25% of glucose, a small quantity of glucosamine being detected;

(12) a bond structure of the sugar in which mannose is linked by α

-bond since a sugar is liberated therefrom by an α

-mannanase; and

(13) constituent amino acids comprising 26 to 32% of serine, 16 to 20% of threonine, 13 to 17% of alanine, 7 to 9% of proline, 5 to 7% of glutamic acid, 5 to 7% of aspartic acid, 4 to 6% of valine, 3 to 5% of lysine, 3 to 4% of glycine, 2.5 to 3.5% of isoleucine, 1.5 to 2.5% of leucine, 0.5 to 1.5% of tyrosine, and 0.3 to 0.7% of phenylalanine.