Bioactive concentrate, its producing method and certain drug compositions containing also chondroitin sulphate

A bioactive concentrate in a 10% aqueous solution comprising as active principle 7% to 9% of an anti-hyaluronidase and anti-inflammatory mucopolysaccharide polymer and 1% to 3% of chondroitin sulfate, all of these being said active principle having a yellow to light brown color, a pH value ranging from 4 to 6, and an antihyaluronidase activity ranging between 450 and 750 I.U./ml, said bioactive concentrate prepared by a process which comprises the steps of:

• (a) extracting at a temperature of 0 to 4 degrees centigrade an organic raw material selected from the group consisting of bovine or sheep cartilaginous connective tissue, umbilical cord, young animal tendons, bowels, and testicles, and sea animals with an aqueous acid solution that contains 0.5% phenol and which has a pH of 2 to 4, in a first phase in a ratio of 1:3.5 to 4 of raw organic material to aqueous acid solution to obtain a first extract and in a second phase in a ratio of 1:1.5 of raw organic material to aqueous acid solution to obtain a second extract, said first phase and said second phase each taking place for 24 hours, stirring for 30 minutes every 3 hours;
• (b) combining the first extract and the second extract to obtain a combined aqueous extract;
• (c) adjusting the pH of the combined aqueous extract to 4.5 to 5 with calcium hydroxide solution, being then allowed to settle for 8 hours at a temperature of 0 to 4 degrees centigrade;
• (d) filtering the combined aqueous extract and concentrating the filtrate by vacuum evaporation at an internal temperature of up to 45 degrees centigrade until reaching a rate of concentration of 7:1;
• (e) passing the combined aqueous extract concentrated during step (d) through an anion exchanger column to obtain an aqueous solution;
• (f) removing lipids from the aqueous solution by solvent extraction in a ratio of 1:4 solution to extraction solvent in 6 to 10 cycles until the lipids remaining in the aqueous solution do not exceed 1% to 5%, the last defatting cycle carried out with n-hexane in the same ratio;
• (g) vacuum-concentrating the aqueous solution until reaching a volume of 70% of the aqueous solution before vacuum-concentration;
• (h) filtering the solution to obtain a filtrate and a protein precipitate and adding 96.degree. ethyl alcohol for deproteinization to the filtrate until an alcohol concentration of 50% is reached;
• (i) maintaining the aqueous alcoholic solution formed in step (h) at 0 to 5 degrees centigrade for 12 hours, then filtering the aqueous alcohol solution and concentrating the aqueous solution to remove the alcohol until reaching a volume of 70% as against the solution subject to deproteinization;
• (j) gradient-treating the protein precipitate formed during step (h) with ammonium sulfate to obtain a low molecular weight fraction of the protein precipitate and combining the low molecular weight protein precipitate with the aqueous solution concentrated during step (i);
• (k) removing any histaminic impurities from the combined low molecular weight protein precipitate and aqueous solution by successive treatment with trisodium phosphate, calcium chloride and sodium hydroxide until reaching a pH of 8.5, filtering the combined protein precipitate and aqueous solution, and passing the filtrate through a cationic exchanger column adjusting the pH of the resulting final solution to 4 to 6 to obtain the bioactive concentrate;
• and
• (l) adding chondroitin sulfate in an amount of 1% to 3% per 7% to 9% of the antihyaluronidase and anti-inflammatory mucopolysaccharide polymer.