High molecular polysaccharide MPS-80

• US 4,396,763 A
• Filed: 11/17/1981
• Issued: 08/02/1983
• Est. Priority Date: 01/14/1981
• Status: Expired due to Fees

• Current Assignee: Meiji Milk Products Co Ltd (Meiji Holdings Company Limited)
• Original Assignee: Meiji Milk Products Co Ltd (Meiji Holdings Company Limited)
• Inventors: Kanbe, Michio, Oda, Munehiro, Miyazawa, Kyushichi, Ebisawa, Naoyuki, Tsuchiya, Fumiyasu
• Primary Examiner(s): BROWN, JOHNNIE
• Application Number:
  US06/322,183
• Time in Patent Office: 623 Days
• Field of Search: 536/1.1, 536/123, 424/180
• US Class Current: 536/123
• CPC Class Codes: A61K 31/715 Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Abstract

New high molecular polysaccharide MPS-80 was obtained by culturing high molecular polysaccharide MPS-80-producing bacteria belonging to genera Lactobacillus or Streptococcus. The new high molecular polysaccharide MPS-80 has an excellent anti-tumor effect, and it is expected for the utilization as anti-tumor agent.

Claims

1. High molecular polysaccharide MPS-80 having the following physicochemical properties:

(1) elementary analysis;

C;

42.2%

H;

6.9%

O;

50.4%

(2) molecular weight;

(i) molecular weight determined by ultrafiltration method by means of Sepharose 2B as shown in FIG. 1;

said polysaccharide is fractionated near the void volume under conditions comprising 2.5×

40.5 cm column size, 5 g fraction, 2.5 mg (1 ml) sample loaded and 0.05 M phosphate buffer solutin (pH 6.0) used as developer;

(ii) molecular weight determined by ultracentrifugation wherein

a sample is dissolved in 0.2 M phosphate buffer solution (pH 7.3) and the resulting solution having a concentration of 0.1% is subjected to the ultracentrifugation (preset rotation rate;

51,200 RPM) to obtain a sedimentation constant of 7.98S (S;

Svedberg unit), the sedimentation pattern being single;

(3) melting point (decomposition point);

said polysaccharide begins to discolor at around 262°

C. and is blackened at 263°

-264°

C.

(4) specific rotatory power;

space="preserve" listing-type="equation">[α

].sub.D.sup.18 =+33.2 (C═

0.5%)

(5) ultraviolet ray absorption spectrum;

as shown in FIG. 2.

(6) infrared ray absorption spectrum;

as shown in FIG. 3.

(7) solubilities in solvents;

soluble in water but insoluble in methanol, ethanol, acetone and ether;

(8) color reactions;

(i) Molisch reaction;

+

(ii) Anthrone reaction;

+

(iii) Cysteine sulfuric acid reaction;

+

(iv) Aniline hydrochloric acid reaction;

-

(v) Carbazole sulfuric acid reaction;

-

(vi) Elson-Morgan reaction;

-

(vii) Biuret reaction;

-

(9) basicity, acidity or neutrality;

2. 1-0.5% aqueous solution of said polysaccharide has a neutral pH;

(10) color;

freeze-dried polysaccharide is in the form of white fibers;

(11) saccharide constituting the polysaccharide are glucose and galactose determined by examining said polysaccharide by GLC using 5% SE-52 (2 m column) as follows;

the sample was hydrolyzed with 2 N-H₂ SO₄ in boiling water for 4 hours, then neutralized with barium carbonate and subjected to the filtration;

the filtrate was demineralized with Amberlite IRA-410 and Amberlite IR-120B, concentrated to dryness and subjected to TMS treatment followed by GLC;

(12) proportion of saccharide constituting the polysaccharide;

a sample of said polysaccharide was hydrolyzed with 2N-H₂ SO₄ in boiling water for 4 hours, neutralized with barium carbonate and subjected to filtration;

the filtrate was examined to determine a proportion of saccharide constituting the polysaccharide by enzyme method;

the ratio of glucose to galactose was 2.2-1.9 to 1;

(13) C¹³ -NMR Spectrum (in D₂ O, TMS standard) (ppm);

as shown in FIG. 4;

(14) decomposition with enzymes;

said polysaccharide was dissolved in 0.05 M acetate buffer solution and subjected to reaction with various enzymes;

the decomposition by the enzymes was judged from increase in amount of reduced sugar by Somogyi-Nelson method;

Enzymes used and reaction conditions;

space="preserve" listing-type="tabular">______________________________________ a. Amylase (a product of Boehringer) pH 5.9, 37°

C., 4 hrs. b. Amylase (a product of Boehringer) pH 4.8, 30°

C., 4 hrs. c. Galactosidase (a product of Boehringer) pH 4.8, 30°

C., 4 hrs. d. Amyloglucosidase (a product of Boehringer) pH 4.8, 30°

C., 4 hrs. e. Galactosidase (a product of Boehringer) pH 4.8, 30°

C., 4 hrs. ______________________________________

no increase in amount of reduced sugar was recognized at all under above conditions a through e;

(15) LD₅₀

ddY w♀

mice (average body weight;

21.3 g;

each group consisted of 7 mice) were used;

a sample solution in physiological saline was administered intraperitoneally once in various amounts to the mice and they were observed for 10 days to determine LD₅₀ ;

as a result, LD₅₀ was not less than 200 mg/Kg-body weight; and

(16) MPS-80 is produced by cultivating a member selected from the group consisting of Lactobacillus Jugurti No. 851, FERM BP-66 and Streptococcus thermophilus No. 127, FERM BP-65.