Drug – bio-affecting and body treating compositions – Plant material or plant extract of undetermined constitution... – Containing or obtained from ginkgo
Reexamination Certificate
2000-05-25
2001-12-11
Lilling, Herbert J. (Department: 1651)
Drug, bio-affecting and body treating compositions
Plant material or plant extract of undetermined constitution...
Containing or obtained from ginkgo
C514S027000
Reexamination Certificate
active
06328999
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to extracts of ginkgo biloba leaves with reduced 4′-O-methyl pyridoxine and biflavones content, a process for their preparation and medicaments comprising said extracts, which do not exhibit undesirable side effects initiated by 4′-O-methyl pyridoxine and biflavones even at high dosages.
2. Present State of the Art
Extracts of Ginkgo biloba leaves have been used therapeutically for 30 years. The Commitee E of the Federal Health Office (Bundesgesundheitsamt) in Berlin has acknowledged that medicaments comprising defined extracts are effective in the symptomatic treatment of performance disturbances caused by cerebral insufficiency, peripheral occlusive disease, vertigo and tinnitus (BAnz No. 133, Jul. 19, 1994). These extracts are characterized by a content of 22 to 27% flavone glycosides, 5 to 7% terpene lactones, 2.8 to 3.4% thereof being ginkgolides A, B and C, and 2.6 to 3.2% bilobalide, and by a maximum content of 5 ppm ginkgolic acids (alkyl phenol compounds). Such extracts of Ginkgo biloba leaves and methods for their preparation are known from DE 39 40 091 (EP-B-0 431 535) and DE 39 40 092 (EP-30 B-0 431 536).
The occurence of 4′-methoxy pyridoxine (4′-O-methyl pyridoxine) in ginkgo biloba seeds has been described by Wada et al., Chem. Pharm. Bull. 33 (1985), 3555-3557. This compound causes symptoms of poisoning like convulsions and unconsciousness (“Gin-nan food poisoning” and “Gin-nan sitotoxism”), which may occur after consumption of larger amounts of ginkgo seeds. Therefore, the discoverer of this compound named it “ginkgotoxin”. In a later work the same research group found out that 4′-O-methyl pyridoxine is absent in ginkgo leaves (Wada et al., Biol. Pharm. Bull. 16 (1993), 210-212). Contrary to this statement is a recently issued publication reporting on the isolation and identification of 4′-O-methyl pyridoxine from ginkgo leaves (Arenz et al., Planta Medica 62 (1996), 548-551). In this work commercial preparations with extracts of ginkgo biloba leaves have been analyzed with respect to their anti-vitamin B
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(4′-O-methyl pyridoxine) content and concentrations of between about 4 and 10 &mgr;g/ml of liquid medicinal form have been found. This corresponds to a concentration of about 100 to 250 ppm in the dry extracts processed to preparations.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Ginkgo extracts used for therapy comprise additionally up to 1000 ppm biflavones. Our own investigations have surprisingly revealed that in addition to biological activities known from literature these compounds also show immunotoxicologic effects. In an popliteal lymph node assay in mice we have observed that the ethyl acetate soluble fraction of an ethanol-water total extract of ginkgo leaves causes a lymph node reaction; see example 1. Testing of the subfractions obtained by chromatographic separation revealed that the inununotoxicologic effect merely occurred after application of the subfraction which contained more than 70% biflavones. The biflavones found in ginkgo are the following compounds: sciadopitysin, ginkgetin, isoginkgetin, sequoiaflavone and bilobetin.
The technical problem underlying the present invention is to provide an extract of ginkgo biloba leaves being virtually free of 4′-O-methyl pyridoxine and alkyl phenol compounds (ginkgolic acids, ginkgol), having the technically feasible minimum biflavone content and the flavonol glycosides and terpene lactones, which are the acknowledged activity-determining ingredients, in concentrations demanded by the Commitee E (BAnz No. 133, Jul. 19, 1994). The extract according to the present invention contains:
20 to 30% by weight flavonol glycosides
a total of 2.5 to 4.5% by weight of ginkgolides A, B, C and J
2.0 to 4.0% by weight bilobalide
below 10 ppm, preferably below 1 ppm alkyl phenol compounds (ginkgolic acids, ginkgol)
below 10% by weight proanthocyanidins
below 50 ppm 4′-O-methyl pyridoxine
below 100 ppm biflavones
The present invention further relates to a method for the preparation of such an extract of ginkgo biloba leaves.
This technical problem is solved in that in the known method for the preparation of ginkgo extracts two additional process steps are introduced:
1. treatment with cation exchanger to remove 4′-O-methyl pyridoxine
2. adsorption of the biflavones on activated charcoal
These measures are practiced in the process according to claim
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of DE 39 40 091 preferably before or after step g). Accordingly, after the filtration of the solution obtained in step f) said solution is passed through a cation exchange column and the column is eluted with aqueous ethanol or aqueous methanol. The obtained eluate is treated with activated charcoal. For the elution of the cation exchanger an aqueous solution containing 20 to 80% by weight, preferably 50% by weight ethanol or methanol can be used. The sequence of the two steps according to the present invention is interchangeable without altering the result. Thereafter work is continued according to example 3, below, and the solution is extracted with an aliphatic or cycloaliphatic solvent to remove the alkyl phenol compounds.
Step c) of the method serves to remove lipophilic components, which are poorly soluble in water, including the major amount of biflavones.
The preferred method for the preparation of an extract of the present invention is therefore characterized by the number and order of the following steps:
a) extraction of ginkgo biloba leaves with aqueous acetone, an aqueous alkanol having 1 to 3 carbon atoms or absolute methanol,
b) separating the organic solvent by concentrating the solvent up to a content of not more than 10% by weight, wherein water can be added in the last distillation steps,
c) diluting the remaining aqueous solution with water to a content of solids of 5 to 25% by weight, cooling to a temperature of below 25° C. and allowing it to stand until a precipitate forms,
d) a treatment of the remaining aqueous solution with ammonium sulfate and thereafter at least one extraction with methyl ethyl ketone or a mixture of methyl ethyl ketone and acetone,
e) concentrating the obtained extract and diluting with an ethanol-water mixture to a solution containing each 50% by weight water and ethanol and 10% by weight of solids,
f) treatment of the solution with a lead compound or an insoluble polyamide,
g) filtration of the solution and passage through a strongly acidic cation exchanger, elution of the cation exchanger with aqueous ethanol or aqueous methanol,
h) treating the eluate with activated charcoal,
i) extraction of the filtered solution with an aliphatic or cycloaliphatic solvent having a boiling point of 60 to 100° C.,
j) concentrating the remaining aqueous-alcoholic solution, subsequently treating with ammonium sulfate and extraction with methyl ethyl ketone and ethanol,
k) concentrating the obtained organic phase to a content of solids of 50 to 70% by weight, and
l) drying the concentrate under vacuum to a dry extract having a water content of below 5 %.
In a further embodiment of the present invention, steps g) and h) are interchanged. In a further embodiment of the present invention, steps g) and h) of the method are performed 20 only after step i).
In the known methods for enrichment of flavonol glycosides and terpene lactones by adsorption resins (EP-B-0 360 556, JP-A-04182434) the two steps according to the present invention are performed with the methanolic-aqueous solution of the extracts from the desorption step (cf EP-B-0 360 556, example III, claim
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) and the ethanolic-aqueous solution according to JP-A-04182434, respectively. Examples of useful cation exchangers are acidic cation exchangers. The most preferred cation exchanger is a strongly acidic cation exchanger like Merck I.
The extract prepared according to the present invention demonstrates in pharmacological test models circulation promoting-, ischemia damages preventing-, radical scavenger-, and thrombocyte aggregati
Dr. Willmar Schwabe GmbH & Co.
Lilling Herbert J.
Workman & Nydegger & Seeley
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