Method for extracting and separating ginkgolides

1. A method for extracting and separating ginkgolides, comprising:

A. extracting:

crushing ginkgo leaves, adding an organic solvent for extraction to produce an extracted solution,

concentrating the extracted solution to produce a concentrated extracted solution,

adding an anti-oxidization protection agent to the concentrated extracted solution, adjusting a pH to 4 to 5 with a pH adjusting agent, which is followed by concentrating and refrigerating to form a refrigerated concentrated solution;

wherein in the step A, the organic solvent for extraction is ethanol, acetone, or ethyl acetate, with a concentration of 50 to 80% v/v and an amount in liters that is in a range of from 5 to 12 times a number equal to a mass in kilograms of the ginkgo leaves;

B. further extracting:

extracting the refrigerated concentrated solution 2 to 3 times with n-hexane or petroleum ether firstly to produce a first aqueous phase and a first organic extracting phase,

extracting the first aqueous phase 4 to 5 times with a lipid-soluble solvent to produce a second aqueous phase and a second organic extracting phase,

extracting the second aqueous phase 4 to 5 times with a water saturated sec-butyl alcohol-ethyl acetate mixed solvent or water saturated n-butyl-ethyl acetate mixed solvent to produce a third aqueous phase and a third organic extracting phase,

combining the second and the third organic phase extracted solutions, and concentrating under reduced pressure to produce a extracted concentrated solution;

C. passing through a column:

passing the extracted concentrated solution through a polyamide resin column, sequentially eluting with 15 BV of water, 3 to 5 BV of 20% to 40% v/v ethanol and 2 to 3 BV of 60% to 90% v/v ethanol, controlling a flow velocity of an eluant to 2 to 3 BV/h;

concentrating the eluant under reduced pressure, and drying to produce a dry substance;

D. separating crystals out:

adding the dry substance to boiling water, dissolving by agitating, cooling, extracting a supernate 4 to 5 times with ethyl acetate, ethyl formate or acetone in a volume equal to that of the supernate, combining extracted solutions, concentrating under reduced pressure, drying by distillation, adding an amount of 30% to 50% v/v ethanol in liters that in a range of from 5 to 8 times a number equal to a mass in kilograms of the dried substance, dissolving by heating and agitating, filtering, refrigerating, separating crystals out, filtering to obtain a filtrate I for later use, washing the crystals with 30% to 50% v/v ethanol, and drying under reduced pressure to obtain crystals I;

concentrating the filtrate I until the alcohol content is 10 to 30% v/v, refrigerating, separating crystals out, filtering to obtain a filtrate II for later use; and washing with 30 to 50% v/v ethanol, and drying under reduced pressure to obtain crystals II;

concentrating the filtrate II, adding 0.1% to 0.5% active carbon for adsorption, filtering to obtain a filtrate, concentrating the filtrate until the alcohol content is 10% to 30% v/v, refrigerating, separating crystals out, filtering to obtain a filtrate III for later use, washing the crystals with 30% to 50% v/v ethanol, and drying under reduced pressure to obtain crystals III;

concentrating the filtrate III, passing through an active-carbon-silica gel column, eluting with 30% to 50% v/v ethanol firstly, then eluting with 70% to 90% v/v ethanol, collecting eluants, concentrating until the alcohol content is 10% to 30% v/v, refrigerating and separating crystals out, filtering the crystals out to obtain a filtrate IV for later use; and washing the crystals with 30% ethanol, and drying under reduced pressure to obtain crystals IV;

concentrating the filtrate IV, refrigerating, separating crystals out, filtering, washing the crystals with 30% v/v ethanol, and drying under reduced pressure to obtain crystals V; and

E. mixing the crystals:

uniformly mixing the crystals I, II, III, IV and V, and crushing to obtain the ginkgolides.

2. The method for extracting and separating ginkgolides according to claim 1 , wherein a manner of the extracting in the A is reflux extraction or decoction extraction.

3. The method for extracting and separating ginkgolides according to claim 2 , wherein the reflux extraction adopts ethanol, acetone or ethyl acetate for extraction, wherein concentrations of different extracting solvents and extraction conditions are as follows:

ethanol: concentration 50% to 80% v/v, extraction temperature 75 to 85° C., extraction times 2 to 3 times with 1 to 2 h a time; acetone: concentration 50% to 80% v/v, extraction temperature 45 to 55° C., extraction times 2 to 3 times with 1 to 2 h a time; ethyl acetate: concentration 50% to 80% v/v, extraction temperature 55 to 65° C., extraction times 2 to 3 times with 1 to 2 h a time.

4. The method for extracting and separating ginkgolides according to claim 3 , wherein the reflux extraction adopts ethanol, acetone or ethyl acetate for extraction, wherein concentrations of different extracting solvents and extraction conditions are as follows:

ethanol: concentration 65% v/v, extraction temperature 75 to 85° C., extraction times 3 times with 1.5 h a time; acetone: concentration 50% v/v, extraction temperature 45 to 55° C., extraction times 3 times with 1.5 h a time; ethyl acetate: concentration 60% v/v, extraction temperature 55 to 65° C., extraction times 3 times with 1.5 h a time.

5. The method for extracting and separating ginkgolides according to claim 2 , wherein the decoction extraction adopts ethanol, acetone or ethyl acetate for extraction, wherein concentrations of different extracting solvents and extraction conditions are as follows:

ethanol: concentration 50% to 80% v/v, extraction temperature 80 to 90° C., extraction times 2 to 3 times with 1 to 2 h a time; acetone: concentration 50% to 80% v/v, extraction temperature 50 to 60° C., extraction times 2 to 3 times with 1 to 2 h a time; ethyl acetate: concentration 50% to 80% v/v, extraction temperature 60 to 65° C., extraction times 2 to 3 times with 1 to 2 h a time.

6. The method for extracting and separating ginkgolides according to claim 5 , wherein the decoction extraction adopts ethanol, acetone or ethyl acetate for extraction, wherein concentrations of different extracting solvents and extraction conditions are as follows:

ethanol: concentration 65% v/v, extraction temperature 80 to 90° C., extraction times 3 times with 1.5 h a time; acetone: concentration 50% v/v, extraction temperature 50 to 60° C., extraction times 3 times with 1.5 h a time; ethyl acetate: concentration 60% v/v, extraction temperature 60 to 65° C., extraction times 3 times with 1.5 h a time.

7. The method for extracting and separating ginkgolides according to claim 1 , wherein the anti-oxidization protection agent in the step A is neutral amino acid.

8. The method for extracting and separating ginkgolides according to claim 1 , wherein the anti-oxidization protection agent in the step A is at least one of serine, methionine, asparagine or threonine.

9. The method for extracting and separating ginkgolides according to claim 1 , wherein the anti-oxidization protection agent in the step A is methionine.

10. The method for extracting and separating ginkgolides according to claim 1 , wherein the pH adjusting agent in the step A is organic weak acid.

11. The method for extracting and separating ginkgolides according to claim 1 , wherein the pH adjusting agent in the step A is at least one of citric acid, malic acid or sorbic acid.

12. The method for extracting and separating ginkgolides according to claim 1 , wherein the pH adjusting agent in the step A is citric acid.

13. The method for extracting and separating ginkgolides according to claim 1 , wherein the lipid-soluble solvent in the step B is at least one ethyl acetate, ethyl formate, acetone or butanone.

14. The method for extracting and separating ginkgolides according to claim 1 , wherein the particle size of polyamide resin in the polyamide resin column in the step C of passing through the polyamide resin column is 30 to 60 mu.

15. The method for extracting and separating ginkgolides according to claim 1 , wherein the volume ratio of active carbon and silica gel in the active carbon-silica-gel column in the step D of separating crystals out is 1:1 to 1:3.