Method of removing pesticides from ginkgo biloba extracts and extracts obtained using said method

FIELD: chemistry.

SUBSTANCE: invention relates to a method of removing pesticides from Ginkgo biloba extracts. The method involves subjecting an extracted obtained by extracting Ginkgo biloba leaves with hexane, comprising the following steps: a) liquid-liquid extraction to obtain a medium polarity fraction containing ginkgo terpenes and pesticides not removable with hexane, and a high polarity fraction containing ginkgo flavone glycosides; b) crystallisation from the medium polarity fraction obtained at step a) to form an intermediate with ginkgolide content of not less than 50%; c) crystallisation from the residual solution obtained at step b) to obtain an intermediate product with bilobalide content of not less than 50%; d) mixing of the high polarity fraction obtained at step a) with the ginkgo terpene intermediate product and the bilobalide intermediate product obtained at steps b) and c).

EFFECT: invention enables to obtain a product with a given ratio of terpenes, in which residual content of pesticides is considerably low or pesticides are completely removed.

4 cl, 1 tbl, 5 ex

This invention relates to a method for removal of pesticides from extracts of Ginkgo biloba and the extracts obtained in the mentioned way.

The LEVEL of TECHNOLOGY

Leaf extracts of Ginkgo biloba long used in traditional Chinese medicine and in many Western countries due to the different pharmacological properties of the components mentioned extracts (vasodilator, immune-boosting, antioxidant and antiaggregatory properties).

Methods of preparation of leaf extracts of Ginkgo biloba are disclosed in US 5700468, EP 360556, EP 431535 and JP 09110713.

Pesticides, such as organic phosphates, the toxicity of which is well documented, used, or used for growing plants, which are mentioned extracts. Although many laws on health and agriculture have prohibited or restricted the use of such pesticides is not always easy to determine the source of plant material, and even in countries where pesticide use is controlled, it is impossible to eliminate illicit or illegal use of substances with pesticidal activity, the presence of which in the pharmaceutical or food standard extract is totally undesirable. Particularly insidious problem is the possible cross-contamination with pesticides closely spaced agriculture is ultor.

The problem of pollution in General is discussed in the publication Journal of AOAC International, Vol. 88(3), 2005, 729-735, which also describes a method for determining the content fosfororganicheskikh pesticides in the leaves of the Ginkgo gas chromatography.

In U.S. patent 5660832 described method of purification of plant extracts pesticides, including extraction of polar solvent at a specific pH with subsequent absorption and reabsorbtsiey on the polymers. In the case of leaves of Ginkgo biloba, extraction is carried out with a mixture of ethanol and water.

Pesticides are not only undesirable pollutants in extracts of Ginkgo biloba: for example, EP 1968625 describes a multi-stage method of preparation of an extract of Ginkgo biloba c low content of 4'-O-methylpyridoxine, also known as Ginkgo toxin, and/or biflavones. In this case, however, undesirable components naturally present in the plant.

Finally, EP 1868626 discloses a method of preparation of extracts of Ginkgo biloba, which has a low content of polycyclic aromatic hydrocarbons obtained from the use of fossil fuels near the cultures of Ginkgo. The described method is very complex, includes eleven stages, periods of prolonged cooking and the use of expensive reagents, such as polyamides, or toxic reagents, such as salts of lead.

Almost all known Mody include one or more stages removal of lipophilic components of Ginkgo extract (in particular, biflavones and ginkgolic). Mentioned extraction also reduces the presence of the content of any lipophilic pesticide, although pesticides with higher polarity remain in the extract, because their behavior is more similar to the behavior of hydrophilic components (such as flavone glycosides and terpenes).

Therefore, there is a need for a suitable method of purification, which reduces or removes pesticides or other pollutants from leaf extracts of Ginkgo biloba, to meet the limits established by the legislation, especially in the United States, controlling the residual content of any type of pesticide residues in products of plant origin for pharmaceutical, food or cosmetic use.

Description of the INVENTION

Now it is established that the pesticide content of extracts of Ginkgo biloba can be reduced by a method including:

a) liquid-liquid extraction with obtaining fractions of medium polarity, deterpenated Ginkgo and pesticides, do not remove the hexane, and the high polarity fraction containing flavone glycosides of Ginkgo;

b) crystallization of fraction average polarity obtained in stage a), with formation of an intermediate product with ginkgolide content not less than 50%;

(C) crystallization of the residual solution obtained in stage b), with images is of an intermediate product with content bilobalide not less than 50%;

d) mixing the high polarity fraction obtained in stage a), with an intermediate product of Ginkgo terpenes and intermediate product bilobalide obtained at stages b) and C).

The method according to the invention, in addition to removing or significantly reducing residual pesticide, also allows the content of Ginkgo terpenes and their relative value will be monitored.

The invention therefore also relates to the extracts obtained from the method described above, with a given ratio of Ginkgo terpenes.

According to the invention, stage a) gives one fraction enriched with Ginkgo terpenes, and the fraction enriched flavonovie glycosides of Ginkgo, the extraction solvent is not miscible with water, selected from esters, chlorinated hydrocarbons, mixtures of hydrocarbons and alcohols, ketones and mixtures of ketones and alcohols, preferably₂-C₈esters, such as ethyl acetate and tert-butyl acetate or a mixture of toluene-butanol.

Terpenes Ginkgo crystallized in mixtures of water and C₁-C₅alcohols, whereas crystallization of enriched terpene fraction obtained in stage C)is carried out in water or in a mixture of water and C₁-C₃the alcohols. The method according to the invention removes pesticides average polarity, which are not removed from sinkhole.

Invented the e is illustrated in more detail in the examples below.

Example 1: Purification of the extract obtained according to example I of U.S. patent 5700468 and EP 0 360 556 (Indena)

1) 100 kg of the crushed leaves is extracted 4 times with 400 l of 60% (wt./wt.) acetone at a temperature of approximately 50°C.

2) the combined filtrates are extracted in countercurrent with 500 l of hexane.

3) fat-free solution was concentrated in vacuo to a volume of 200 l, leave in continuation of the night at 2°C and then centrifuged to separate the fraction of biflavones.

4) the Aqueous solution is extracted in countercurrent with 400 l 8/2.about. a mixture of n-butanol/toluene.

5) Organic solution was concentrated in vacuo to a small volume, with the addition of water. Concentrate absorb water to produce a solution with approximately 30% of dry residue.

6) the Solution is extracted in countercurrent with approximately 65 l of ethyl acetate. The aqueous phase is concentrated to siropa-like consistency and dried in vacuum to obtain an intermediate product, enriched flavonovie glycosides of Ginkgo and devoid of terpenes (1,95 kg).

7) an ethyl acetate phase was concentrated in vacuo to siropa-like consistency and absorb the remainder with 3 l of 75% aqueous ethanol.

8) the Solution is extracted in countercurrent with 20 l of hexane, removing the combined organic phase.

9) Aqueous-alcoholic solution was concentrated in vacuo to siropa-like the consistency and add 0.9 liters of 50% aqueous ethanol.

The resulting solution was heated at the boil under reflux with stirring, then cooled to room temperature and left to crystallize for 4 days.

10) the Solution is then filtered and the solid is washed with 50% EtOH and dried in vacuum to obtain 95 g of purified ginkgolides.

11) the mother Liquor was concentrated in vacuo to remove ethanol and the residue is then absorbed with 1.9 l of water and heated to 90°C under stirring for 30 minutes. The aqueous solution is separated, and the residue absorb another 3 times with 1.9 l of water at 90°C, and the aqueous solution is regenerated each time.

12) Aqueous solutions are combined and extracted in countercurrent with 14 l of hexane.

13) an Aqueous solution concentrated to a volume of 0.3 l, then add 0.1 l 95° EtOH, and the solution is heated to 50°C. with stirring, then cooled to room temperature and left to crystallize for 3 days.

14) the Solution is filtered and the solid is washed with 20% EtOH and dried in vacuum to obtain 103 g of peeled bilobalide.

15) Products obtained in paragraphs 6, 10 and 14, is mixed with the receipt of 2.15 kg of Ginkgo biloba extract with the following characteristics: flavone glycosides of Ginkgo 26,0%; bilobalid 3,2%; ginkgolides a 3.5%; ginkgolide acid <5,0 ppm; each pesticide <10 h/bn

Example 2: Purification of the extract, p is obtained according to example II of U.S. patent 5700468 and EP 0 360 556 (Indena)

1) 100 kg of the crushed leaves is extracted 4 times with 400 l of 50% (wt./wt.) methanol at a temperature of approximately 35°C.

2) the combined filtrates are concentrated to 100 l in vacuum at 40°C. the concentrate absorb with 100 l of methanol and the resulting suspension filtered.

3) the Filtered solution is extracted 3 times with 100 l of 9:2 mixture of toluene/n-butanol.

4) the combined organic phases are washed with counter image by using 50 l of 50% methanol.

5) Water-methanol solution was concentrated in vacuo to an aqueous solution and after filtration of extracted using a 8:2 mixture of n-butanol/toluene.

6) the Organic layer is washed counter with water and concentrated in vacuo to a small volume, with the addition of water. The solution absorb water to produce a solution with 20% of dry residue.

7) the resulting solution is extracted in countercurrent with 90 l of a 1:1 mixture of n-butanol/toluene. The aqueous phase is concentrated to siropa-like consistency and dried in vacuum to obtain an intermediate product, enriched flavonovie glycosides of Ginkgo and devoid of terpenes (2,05 kg).

8) a Mixture of n-butanol/toluene was concentrated in vacuo to siropa-like consistency and absorb the remainder with 3.5 liters of 60% aqueous ethanol.

9) the resulting solution is extracted in countercurrent with 20 l of hexane, removing obyedinenny the organic phase.

10) Aqueous-alcoholic solution was concentrated in vacuo to siropa-like consistency and add 1.2 litres of 40% aqueous ethanol.

The resulting solution was heated at the boil under reflux with stirring for 45 minutes, then cooled to room temperature and left to crystallize in the next 3-4 days.

11) the Solution is then filtered and the solid is washed with 40% EtOH and dried in vacuum to obtain 97 g of purified ginkgolides.

12) the mother Liquor was concentrated in vacuo to remove ethanol and the residue is then absorbed using 2 l of water and heated at 90°C under stirring for 30 minutes. The aqueous solution is separated and the residue absorb another 3 times with 2 l of water at 90°C, and the aqueous solution is regenerated each time.

13) Aqueous solutions are combined and extracted in countercurrent with 17 l of hexane.

14) Aqueous solutions are concentrated to a volume of 0.4 l and add 0.15 l 95° EtOH; the resulting solution was heated at 50-55°C under stirring for 30 minutes, then cooled to room temperature and left to crystallize in the next 3-4 days.

15) the Solution is filtered and the solid is washed with 20% EtOH and dried in vacuum at 60°C to obtain 110 g of peeled bilobalide.

16) Products obtained in paragraphs 7, 11 and 15, is mixed with obtaining 2.26 kg of extract of Ginkgo biloba, analogion the Guo extract, obtained in example 1.

Example 3: Purification of the extract obtained in example IEP 431535 (Schwabe)

1) 100 kg of the crushed leaves is extracted twice with 750 kg of 60% (wt./wt.) acetone for 30 min at a temperature of 57-59°C.

2) the combined extracts are concentrated to an aqueous solution of 30-40% dry residue, add an equal volume of water and the mixture is left under stirring for one hour at 12°C.

3) Enriched in alkyl phenol precipitate was separated by centrifugation and removed.

4) 30% ammonium sulfate is added to the transparent solution with stirring and the solution is twice extracted with a half volume 6:4 mixture of methyl ethyl ketone/acetone.

5) the combined solution mixture of methyl ethyl ketone/acetone was concentrated in vacuo to a dry residue in 50-70% and then diluted with water and 95% ethanol to obtain a 50% ethanol solution with 10% dry residue.

6) an Aqueous solution of hydroxyacetate lead added under stirring until then, until the color changes from brown to light brown. The precipitate is removed by centrifugation.

7) spin-on solution is extracted 3 times with 1/3 volume of hexane.

8) fat-free solution was concentrated in vacuo to an aqueous solution and add 20% ammonium sulfate and twice extracted with a half volume 6:4 mixture of methyl ethyl ketone/ethanol. United organizes the e phases are washed with 20% ammonium sulfate, removing any water and the formed solid substance.

9) Clear solution is then concentrated to a small volume, with the addition of water.

Concentrate absorb water to form a solution with about 10% of dry residue.

10) the Solution is extracted in countercurrent with approximately 100 l of tert-butyl acetate. The aqueous phase is concentrated to siropa-like consistency and dried in vacuum to obtain an intermediate product, enriched flavonovie glycosides of Ginkgo and devoid of terpenes (1,88 kg).

11) Butylacetate phase was concentrated in vacuo to siropa-like consistency and add 0.8 l of 60% aqueous ethanol.

The resulting solution was heated at the boil under reflux with stirring for 25 minutes, then cooled to room temperature and left to crystallize for 2 days.

12) the Solution is then filtered and the solid is washed with 60% EtOH and dried in vacuum to obtain 98 g of purified ginkgolides.

13) the mother Liquor was concentrated in vacuo to remove ethanol and the residue is then absorbed with 1.9 l of water and heated at 90°C With stirring for 30 minutes. The aqueous solution is separated and the residue absorb another 3 times with 1.9 l of water at 90°C, and the aqueous solution is regenerated each time.

14) the combined aqueous solutions are concentrated to the volume of 0.6 l and heated to 70°C, under stirring for 60 minutes, then cooled to room temperature and left to crystallize for 1 day.

15) the Obtained solution is filtered and the solid is washed with water and dried in vacuum to obtain 99 g of peeled bilobalide.

16) Products obtained in paragraphs 10, 12 and 15, is mixed with the receipt of 2.08 kg of extract of Ginkgo biloba, similar to the extract obtained in example 1.

Example 4: Purification of the extract obtained by JP 09110713 (Nippon Green wave)

1) 100 kg of the crushed leaves is extracted 3 times with 500 l of 70% ethanol at 50°C for 30 hours.

2) the combined filtrates are concentrated to 100 l in vacuum and the concentrate absorb with 100 l of water.

The resulting suspension is filtered.

3) the Filtered solution was injected into the chromatographic column containing 100 l NR resin (Mitsubishi)to adsorb the extract. The column is then washed with 200 l of water and the extract is obtained by elution with 200 l of 70% ethanol.

4) Water-ethanol solution was concentrated in vacuo to a small solution, with the addition of water. Concentrate absorb with 20% ethanol to obtain a solution with 20% of dry residue.

5) the resulting solution is extracted in countercurrent with 70 l of methylene chloride. Water-ethanol phase to concentrate siropa-like consistency and dried in vacuum to obtain n megalocnus product, enriched flavonovie glycosides of Ginkgo and devoid of terpenes (2.4 kg).

6) Chlormethiazole phase was concentrated in vacuo to siropa-like consistency and absorb the remainder with 4 l of 75% aqueous methanol.

7) the resulting solution was extracted 10 times with 2 l of hexane, removing the combined hexane phases.

8) Aqueous-alcoholic solution was concentrated in vacuo to siropa-like consistency and add 1 l of 50% aqueous methanol.

The resulting solution was heated at the boil under reflux with stirring for 30 minutes, then cooled to room temperature and left to crystallize for 5 days.

9) the Solution is then filtered and the solid is washed with 50% MeOH and dried in vacuum to obtain 105 g of purified ginkgolides.

10) the mother Liquor was concentrated in vacuo to remove methanol and the residue is then absorbed with 2.1 liters of water and heated at 90°C With stirring for 30 minutes. The aqueous solution is separated and the residue absorb another 3 times with 2 l of water at 90°C, and the aqueous solution is regenerated each time.

11) the combined aqueous solutions are concentrated to a volume of 0.4 l and add 0.1 l Meon; the resulting solution was heated at 50°C under stirring for 30 minutes, then cooled to room temperature and left to crystallize for 3 suto is.

12) the Obtained solution is filtered and the solid is washed with 20% MeOH and dried in vacuum to obtain 102 g of peeled bilobalide.

13) Products obtained in paragraphs 5, 9 and 12, is mixed with the receipt of 2.6 kg of extract of Ginkgo biloba, similar to the extract obtained in example 1.

Example 5: Purification of purified extract, including pesticides, with a high content of terpenes

1) 1 kg of the leaf extract of Ginkgo biloba containing 24% Platonovich glycosides of Ginkgo, 12% of Ginkgo terpenes, 2 ppm ginkgolic acids and 315 h/bn chlorpyrifos and tricyclazole, suspended under stirring in 5 l of water.

2) the Suspension is extracted with 12 times with 5 l of ethyl acetate at room temperature. The aqueous phase is then concentrated to siropa-like consistency and dried in vacuum to obtain an intermediate product, enriched flavonovie glycosides of Ginkgo and devoid of terpenes (810 g).

3) combined an ethyl acetate phase was concentrated in vacuo to siropa-like consistency and absorb the remainder with 2 l of 50% aqueous ethanol.

The resulting solution was heated at the boil under reflux with stirring for 1 hour, then cooled to room temperature and left to crystallize for 2 days.

4) the Solution is then filtered and the solid is washed with 50% EtOH and dried in the vacuum p is the receiving 82 g of purified ginkgolides.

5) the mother Liquor was concentrated in vacuo to remove ethanol and the residue is then absorbed with 2.1 liters of water and heated at 90°C under stirring for 15 minutes. The aqueous solution is separated and the residue absorb 2 times with 2 l of water at 90°C, and the aqueous solution is regenerated each time.

6) Aqueous solutions are combined and concentrated in vacuo to siropa-like consistency; the residue is then absorbed with 400 ml of 10% EtOH. The resulting solution was heated at 50°C under stirring for 60 minutes, then cooled to room temperature and left to crystallize for 2 days.

7) the Solution is filtered and the solid is washed with 10% EtOH and dried in vacuum to obtain 70 g of purified bilobalide.

8) 41 g of purified ginkgolides and 35 g of peeled bilobalide mixed with the product obtained in paragraph 2, with the release of 885 g of an extract of Ginkgo biloba with the following characteristics: A. flavone glycosides of Ginkgo 27%; b. the Ginkgo terpenes, 6.8 percent; C. ginkgolide acid and 1.0 ppm; d. each pesticide < 10 h/bn

The described method removes pesticides and accepts that the content of terpenes in extracts of Ginkgo biloba will be regulated, and it is installed as required at the stage when mixed purified intermediate products. In practice, the ratio 24/6 (GFG/GT), considered the ideal in extracts, often the difference is between a natural relationship, meet in plants. For example, Chinese leaves often have a higher content of terpenes, and requires a stage of reduction of the titer of Ginkgo terpenes to obtain the corresponding extract.

The table below shows the pesticide content of the extracts obtained according to the procedures described above. For each example was performed two tests: first, follow the procedure disclosed in the patent, secondly, add processing terpene fraction.

All extracts were obtained from the same plant.

1. Method of removing pesticides from extracts of Ginkgo biloba, which is on the selfishness of exposure to the extract, obtained by extraction of the leaves of the Ginkgo biloba extract with hexane, the following stages:
a) liquid-liquid extraction with obtaining fractions of medium polarity, deterpenated Ginkgo and pesticides, do not remove the hexane, and the high polarity fraction containing flavone glycosides of Ginkgo, and the specified extraction is carried out is not miscible with water solvent selected from esters, chlorinated hydrocarbons, mixtures of hydrocarbons and alcohols, ketones and mixtures of ketones and alcohols;
b) crystallization of fraction average polarity obtained in stage a), with formation of an intermediate crystallized product with ginkgolide content of not less than 50% of the crystallized product;
c) crystallization of the residual solution obtained in stage b), with formation of an intermediate crystallized product with content bilobalide not less than 50% of the crystallized product;
d) mixing the high polarity fraction obtained in stage a), with an intermediate product of Ginkgo terpenes and intermediate product bilobalide obtained at stages b) and c).

2. The method according to claim 1, wherein said solvent is an ester having from 2 to 8 carbon atoms.

3. The method according to claim 1 or 2, in which the crystallization of Ginkgo terpenes in stage b) is carried out in mixtures of water and C_{1/sub> -C3of spirits.}

4. The method according to any one of claims 1 to 3, in which the crystallization bilobalide in stage c) is carried out in water or in a mixture of water and C₁-C₃the alcohols.