Colorimetric assay for bioactive polysaccharide

• US 5,512,488 A
• Filed: 05/05/1994
• Issued: 04/30/1996
• Est. Priority Date: 05/05/1994
• Status: Expired due to Fees

• Current Assignee: Aloe Technologies, LLC
• Original Assignee: Carrington Laboratories Incorporated
• Inventors: Eberendu, Alexis N. R., McAnalley, Bill H.
• Primary Examiner(s): McMahon, Timothy M.
• Assistant Examiner(s): BHAT, NINA NMN
• Application Number:
  US08/238,377
• Time in Patent Office: 726 Days
• Field of Search: 436/94, 436/164, 436/165, 436/169, 435/101, 435/4, 435/31, 435/32, 422/56
• US Class Current: 436/94
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

An assay for the qualitative and quantitative determination of a bioactive polysaccharide, such as acemannan, in a product, such as an aloe vera-containing product. The method uses a complexing agent to react with the bioactive polysaccharide present, if any, in the product to give a color change. The detected color change is compared with standard color changes generated by reacting the complexing agent with different known amounts of the bioactive polysaccharide, wherein differences between the color changes compared are dependent upon the presence or amount of the bioactive polysaccharide in the product. The assay can be confirmed by detecting a second color change of a different aliquot of the product with a different complexing agent.

Claims

1. A method of determining and confirming the presence or amount of a bioactive polysaccharide in a product, said method comprising the steps of:

bringing into contact a first aliquot of said product with a first calorimetric complexing agent in a first suitable medium to form a first reaction mixture;

allowing a first color change to occur in said first reaction mixture;

detecting said first color change in said first reaction mixture;

comparing the first detected color change with first standard color changes generated by reacting said first calorimetric complexing agent with different known amounts of said bioactive polysaccharide, wherein differences between said first color changes compared are dependent upon the presence or amount of said bioactive polysaccharide in said product;

separately bringing into contact a second aliquot of said product with a second calorimetric complexing agent in a second suitable medium to form a second reaction mixture;

allowing a second color change to occur in said second reaction mixture;

detecting said second color change in said second reaction mixture; and

comparing the detected second color change with second standard color changes generated by reacting said second calorimetric complexing agent with different known amounts of said bioactive polysaccharide, wherein differences between said second color changes compared are dependent upon the presence or amount of said bioactive polysaccharide in said sample and wherein said second color change confirms the determination of the presence or amount of said bioactive polysaccharide in said product by said first calorimetric complexing agent.

8. The method of claim 1, wherein said suitable medium comprises a basic solution or an aqueous buffer solution.

9. The method of claim 8, wherein said aqueous buffer solution has a pH in the range of from about 4 to about 14.

10. The method of claim 8, wherein said aqueous buffer solution has a pH in the range of from about 6 to about 14.

11. The method of claim 8, wherein said aqueous buffer solution has a pH in the range of from about 12 to about 14.

2. A method of determining the presence or amount of a bioactive aloe vera polysaccharide in a product, said method comprising the steps of:

bringing into contact an aliquot of said product with a colorimetric complexing agent in a suitable medium to form a reaction mixture;

allowing color change to occur in said reaction mixture;

detecting said color change in said reaction mixture; and

comparing the detected color change with standard color changes generated by reacting said colorimetric complexing agent with different known amounts of said bioactive aloe vera polysaccharide, wherein differences between the color changes compared are dependent upon the presence or amount of said bioactive aloe vera polysaccharide in said product.

3. The method of claim 2, wherein said product comprises a beverage.

4. The method of claim 2, wherein said bioactive aloe vera polysaccharide comprises polymeric mannan derivative.

5. The method of claim 2, wherein said bioactive aloe vera polysaccharide comprises acemannan.

6. The method of claim 2, wherein said colorimetric complexing agent is Congo Red.

7. The method of claim 2, wherein said colorimetric complexing agent is selected from the group consisting of Crystal Violet and Toluidine Blue.

12. The method of claim 2, wherein said amount of said bioactive aloe vera polysaccharide is below about 600 mg per kilogram of said product.

13. The method of claim 2, wherein said amount of said bioactive aloe vera polysaccharide is below about 300 mg per kilogram of said product.

14. The method of claim 2 further comprising the steps of:

separately bringing into contact a second aliquot of said product with a second colorimetric complexing agent in a second suitable medium to form a second reaction mixture;

allowing second color change to occur in said second reaction mixture;

detecting said second color change in said second reaction mixture; and

comparing the detected second color change with second standard color changes generated by reacting said second colorimetric complexing agent with different known amounts of said bioactive aloe vera polysaccharide, wherein differences between the second color changes compared are dependent upon the presence or amount of said bioactive aloe vera polysaccharide in said product and wherein said second color change confirms the determination of the presence or amount of said bioactive aloe vera polysaccharide in said product by said colorimetric complexing agent.

15. A method of determining the presence or amount of acemannan in an aloe vera-containing product, said method comprising the steps of:

bringing into contact an aliquot of said product with Congo Red in a suitable medium to form a reaction mixture;

allowing color change to occur in said reaction mixture;

detecting said color change in said reaction mixture; and

comparing the detected color change with standard color changes generated by reacting said Congo Red with different known amounts of said acemannan, wherein differences between the color changes compared are dependent upon the presence or amount of said acemannan in said product.

16. The method of claim 15, wherein said product comprises a beverage.

17. The method of claim 15, wherein said medium is an aqueous solution having a pH in the range of from about 4 to about 14.

18. The method of claim 15, wherein said medium is an aqueous solution having a pH in the range of from about 6 to about 14.

19. The method of claim 15, wherein said medium is an aqueous solution having a pH in the range of from about 12 to about 14.

20. The method of claim 15, wherein said amount of said acemannan is below about 600 mg per kilogram of said product.

21. The method of claim 15, wherein said amount of said acemannan is below about 300 mg per kilogram of said product.

22. A method of determining and confirming the presence or amount of acemannan in an aloe vera-containing product, said method comprising the steps of:

bringing into contact a first aliquot of said product with a first colorimetric complexing agent in a first suitable medium to form a first reaction mixture;

allowing a first color change to occur in said first reaction mixture;

detecting said first color change in said first reaction mixture;

comparing the first detected color change with first standard color changes generated by reacting said first colorimetric complexing agent with different known amounts of said acemannan, wherein differences between said first color changes compared are dependent upon the presence or amount of said acemannan in said product;

separately bringing into contact a second aliquot of said sample with a second colorimetric complexing agent in a second suitable medium to form a second reaction mixture;

allowing a second color change to occur in said second reaction mixture;

detecting said second color change in said second reaction mixture; and

comparing the detected second color change with second standard color changes generated by reacting said second colorimetric complexing agent with different known amounts of said acemannan, wherein differences between said second color changes compared are dependent upon the presence or amount of said acemannan in said product and wherein said second color change confirms the determination of the presence or amount of said acemannan in said product by said first colorimetric complexing agent.

23. The method of claim 22, wherein said product comprises a beverage.

24. The method of claim 22, wherein said first and second media are aqueous solutions having a pH in the range of from about 4 to about 14.

25. The method of claim 22, wherein said first and second media are aqueous solutions having a pH in the range of from about 6 to about 14.

26. The method of claim 22, wherein said first and second media are aqueous solutions having a pH in the range of from about 12 to about 14.

27. The method of claim 22, wherein said amount of said acemannan is below about 600 mg per kilogram of said product.

28. The method of claim 22, wherein said amount of said acemannan is below about 300 mg per kilogram of said product.

29. The method of claim 22, wherein said first colorimetric complexing agent is selected from the group consisting of Congo Red, Crystal Violet and Toluidine Blue.

30. The method of claim 22, wherein said second colorimetric complexing agent is selected from the group consisting of Congo Red, Crystal Violet and Toluidine Blue.

31. A method of determining and confirming the presence or amount of a bioactive aloe vera polysaccharide in a product, said method comprising the steps of:

bringing into contact a first aliquot of said product with a first colorimetric complexing agent in a first suitable medium to form a first reaction mixture;

allowing a first color change to occur in said first reaction mixture;

detecting said first color change in said first reaction mixture;

comparing the first detected color change with first standard color changes generated by reacting said first colorimetric complexing agent with different known amounts of said bioactive aloe vera polysaccharide, wherein differences between said first color changes compared are dependent upon the presence or amount of said bioactive aloe vera polysaccharide in said product;

separately bringing into contact a second aliquot of said sample with a second colorimetric complexing agent in a second suitable medium to form a second reaction mixture;

allowing a second color change to occur in said second reaction mixture;

detecting said second color change in said second reaction mixture; and

comparing the detected second color change with second standard color changes generated by reacting said second colorimetric complexing agent with different known amounts of said bioactive aloe vera polysaccharide, wherein differences between said second color changes compared are dependent upon the presence or amount of said bioactive aloe vera polysaccharide in said product and wherein said second color change confirms the determination of the presence or amount of said bioactive aloe vera polysaccharide in said product by said first colorimetric complexing agent.

32. The method of claim 31, wherein said product comprises a beverage.

33. The method of claim 31, wherein said first and second media are aqueous solutions having a pH in the range of from about 4 to about 14.

34. The method of claim 31, wherein said first and second media are aqueous solutions having a pH in the range of from about 6 to about 14.

35. The method of claim 31, wherein said first and second media are aqueous solutions having a pH in the range of from about 12 to about 14.

36. The method of claim 31, wherein said amount of said bioactive aloe vera polysaccharide is below about 600 mg per kilogram of said product.

37. The method of claim 31, wherein said amount of said bioactive aloe vera polysaccharide is below about 300 mg per kilogram of said product.

38. The method of claim 31, wherein said first colorimetric complexing agent is selected from the group consisting of Congo Red, Crystal Violet and Toluidine Blue.

39. The method of claim 31, wherein said second colorimetric complexing agent is selected from the group consisting of Congo Red, Crystal Violet and Toluidine Blue.