Methods for fucoidan purification from sea weed extracts
View Patent ↗Methods for purifying fucoidan in extracts from brown seaweed are disclosed. In particular, methods of purifying fucoidan in the extract to remove heavy metal ions, bacterial and endotoxin contaminants, and other impurities are disclosed. The methods include the use of a chelating agent, selective precipitation, and filtration.
1. A method for purifying fucoidan from a heterogeneous mixture, the method comprising:
(a) providing a source of fucoidan;
(b) removing heavy metal ions from said source by treating with a chelating agent immobilized on a solid support to produce a first fucoidan mixture;
(c) selectively precipitating fucoidan present in said first fucoidan mixture to remove contaminants;
(d) resuspending fucoidan-containing precipitate in aqueous solution to produce a second fucoidan mixture;
(e) repeating steps (c) and (d) one or more times; and
(f) filtering the aqueous solution comprising fucoidan to remove bacterial and endotoxin contaminants to yield purified fucoidan.
2. The method of claim 1 , wherein the fucoidan possesses from 5 to 25 percent by weight sulfur.
3. The method of claim 2 , wherein the fucoidan is of algal origin.
4. The method of claim 3 , wherein the fucoidan is from the genus Fucus or Laminari.
5. The method of claim 4 , wherein the fucoidan is from Fucus vesiculosis or Laminaria japonica.
6. The method of claim 1 , wherein the chelating agent is selected from the group consisting of ethylene diamine tetra acetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), 2,3-dimercaptopropanel-1-sulfonic acid (DMPS), and 2,3-dimercaptosuccinic acid (DMSA).
7. The method of claim 6 , wherein the chelating agent is EDTA.
8. The method of claim 1 , wherein the chelating agent immobilized on the solid support is an iminodiacetate chelating resin.
9. The method of claim 1 , wherein the fucoidan in said first fucoidan mixture is selectively precipitated with ethanol.
10. The method of claim 9 , wherein the concentration of ethanol is about 40% to 50% (v/v).
11. The method of claim 9 , wherein the pH is maintained between about pH 5.7 to about pH 6.0.
12. The method of claim 11 , wherein the pH is adjusted to about pH 5.95.
13. The method of claim 9 , wherein NaCl is added to the fucoidan mixture at a concentration of about 20-24 g/liter.
14. The method of claim 1 , comprising repeating steps (c) and (d) three times.
15. The method of claim 1 , further comprising lyophilization of the purified fucoidan.
16. A composition comprising purified fucoidan produced by the method of claim 1 , wherein said composition has non-detectable levels of arsenic, bromine, cerium, cobalt, lead, lithium, molybdenum, tin, tungsten and vanadium.
17. The composition of claim 16 , wherein the fucoidan is biologically active.
18. The composition of claim 17 , wherein the fucoidan has pro-coagulant activity.
19. The composition of claim 16 , further comprising a pharmaceutically acceptable excipient.
20. A method for treating a subject in need of enhanced blood coagulation comprising administering a therapeutically effective amount of the composition of claim 16 to said subject.
21. The method of claim 20 , wherein the fucoidan is administered at a dosage of about 0.01 mg/kg to about 100 mg/kg.
22. The method of claim 20 , wherein the subject has a bleeding disorder selected from the group consisting of a chronic or acute bleeding disorder, a congenital coagulation disorder caused by a blood factor deficiency, and an acquired coagulation disorder.
23. The method of claim 20 , wherein the subject is in need of improved hemostasis in an acute injury or wound setting.
24. The method of claim 20 , wherein the cause of the need for enhanced blood coagulation is prior administration of an anticoagulant, surgery, or other invasive procedure.
25. The method of claim 20 , wherein the cause of the need is as an anti-inflammatory agent, anti-cancer agent, anti-viral agent, or for mobilizing hematopoietic progenitor cells.