Method of identifying natural substances capable of complexation

Patent metadata

Field	Value
Publication number	US-9029166-B2
Application number	US-201113220105-A
Country	US
Kind code	B2
Filing date	Aug 29, 2011
Priority date	Aug 31, 2010
Publication date	May 12, 2015
Grant date	May 12, 2015

Abstract

The present invention relates to a method of identifying a natural substance that is capable of complexation with Ni 2+ , Cu 2+ and/or Fe 2+ ions, wherein an extract containing natural substances is led over a stationary phase loaded with Ni 2+ , Cu 2+ and/or Fe 2+ ions, which is suitable for immobilized metal affinity chromatography (IMAC).

First claim

The invention claimed is:

1. A method of identifying a natural substance that is capable of complexation with Ni 2+ , Cu 2+ and/or Fe 2+ , comprising:
(a) providing an aqueous, alcoholic or aqueous-alcoholic extract comprising natural substances,
(b) providing a device comprising a stationary phase loaded with Ni 2+ , Cu 2+ and/or Fe 2+ ions, which is suitable for immobilized metal affinity chromatography (IMAC) (IMAC device),
(c) leading the extract comprising natural substances from (a) through the device from (b), so that the natural substance capable of complexation with Ni 2+ , Cu 2+ and/or Fe 2+ ions is adsorbed on the stationary phase of the IMAC device,
(d) optionally rinsing the stationary phase of the IMAC device from (c), loaded with the natural substance capable of complexation with Ni 2+ , Cu 2+ and/or Fe 2+ ions, with a solvent,
(e) desorption of the natural substance from the stationary phase of the IMAC device from (c), or optionally from (d) is by leading a solution containing one or more desorption agents through the IMAC device from step (c), or optionally from step (d),
(f) optionally separating the natural substance from the desorption agent, and
(g) identifying the natural substance capable of complexation with Ni 2+ , Cu 2+ and/or Fe 2+ ions,
wherein the extract comprising natural substances provided in (a) and used in (c) is a plant extract, obtained by extraction of one or more plant parts suitable for human consumption and wherein the natural substance to be identified is not a protein, and has a molecular weight of less than or equal to 1000 g/mol.

2. The method as claimed in claim 1 , wherein the extract comprising natural substances provided in (a) and used in (c) is a plant extract, which was obtained by extraction of one or more plant parts selected from the group consisting of stems, leaves, flowers, roots, seeds, and fruits.

3. The method as claimed in claim 1 , wherein the extract comprising natural substances provided in (a) and used in (c)
is an aqueous extract, or
an alcoholic extract with one or more C 1 -C 4 alcohols, or
an aqueous-alcoholic extract with one or more C 1 -C 4 alcohols.

4. The method as claimed in claim 1 , wherein the solvent used in (d) is selected from the group consisting of water, ethanol, methanol and mixtures thereof.

5. The method as claimed in claim 1 , wherein in (e), a solution of a desorption agent is used that comprises one or more compounds selected from the group consisting of EDTA, NaEDTA, Na 2 EDTA, CaNa 2 EDTA, oxalic acid, citric acid, phytic acid and ethylhexanoic acid.

6. The method as claimed in claim 1 , wherein the solution used in (e) is an aqueous, an ethanolic-aqueous or a methanolic-aqueous solution of a desorption agent.

7. The method as claimed in claim 1 , wherein in (f), the natural substance is separated from the desorption agent by means of extraction or by means of chromatography.

8. The method of claim 1 , wherein desorption of the natural substance from the stationary phase of the IMAC device comprises leading a solution containing one or more desorption agents through the IMAC device from (c) or optionally from step (d).

9. The method as claimed in claim 3 , wherein the extract comprising natural substances provided in (a) and used in (c)
is an aqueous extract, or
an ethanol, or
an aqueous-ethanolic extract.

10. The method as claimed in claim 2 , wherein the solvent used in (d) is selected from the group consisting of water, ethanol, methanol and mixtures thereof.

11. The method as claimed in claim 9 , wherein the solvent used in (d) is selected from the group consisting of water, ethanol, methanol and mixtures thereof.

12. The method as claimed in claim 4 , wherein in (e), a solution of a desorption agent is used that comprises one or more compounds selected from the group consisting of EDTA, NaEDTA and Na 2 EDTA.

13. The method as claimed in claim 5 , wherein in (e), a solution of a desorption agent is used that comprises one or more compounds selected from the group consisting of EDTA, NaEDTA and Na 2 EDTA.

14. The method as claimed in claim 5 , wherein the solution used in (e) is an aqueous, an ethanolic-aqueous or a methanolic-aqueous solution of a desorption agent.

15. The method as claimed in claim 6 , wherein the solution used in (e) is an aqueous, an ethanolic-aqueous or a methanolic-aqueous solution of a desorption agent.

16. The method as claimed in claim 7 , wherein in (f), the natural substance is separated from the desorption agent by means of SPE, liquid/liquid separation, precipitation or by means of gel chromatography, HPLC or HILIC (hydrophilic interaction chromatography; aqueous normal-phase chromatography).

Assignees

• Degenhardt Andreas
• Krammer Gerhard
• Schulze Nicole
• Symrise Ag

Inventors

• Degenhardt Andreas
• Krammer Gerhard
• Schulze Nicole

Classifications

• G01N33/0098

  Plants or trees (wood G01N33/46) · CPC title

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• G01N1/405

  by adsorption or absorption · CPC title

• G01N1/4077

  by other techniques involving separation of suspended solids · CPC title

• G01N2030/022

  characterised by the kind of separation mechanism · CPC title