U.S. patent application number 09/725938 was filed with the patent office on 2001-04-19 for extracts of hypericum perforatum and formulations containing them.
Invention is credited to Bombardelli, Ezio, Gabetta, Bruno, Morazzoni, Paolo.
Application Number | 20010000326 09/725938 |
Document ID | / |
Family ID | 11380209 |
Filed Date | 2001-04-19 |
United States Patent
Application |
20010000326 |
Kind Code |
A1 |
Bombardelli, Ezio ; et
al. |
April 19, 2001 |
Extracts of hypericum perforatum and formulations containing
them
Abstract
The invention relates to a method for extracting Hypericum
perforatum (St.-Johns-wort) by fractioning water-alcohol, alcohol,
or acetone extracts of the plant with esters of water-immiscible
C.sub.1-C.sub.5 alcohols. The extracts have high activity and are
stable over time. The invention also relates to formulations
containing the extract.
Inventors: |
Bombardelli, Ezio; (Milano,
IT) ; Gabetta, Bruno; (Milano, IT) ;
Morazzoni, Paolo; (Milano, IT) |
Correspondence
Address: |
PENNIE & EDMONDS LLP
1155 Avenue of the Americas
New York
NY
10036-2711
US
|
Family ID: |
11380209 |
Appl. No.: |
09/725938 |
Filed: |
November 30, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09725938 |
Nov 30, 2000 |
|
|
|
PCT/EP99/03881 |
Jun 4, 1999 |
|
|
|
Current U.S.
Class: |
424/730 ;
424/439; 424/456; 424/464; 424/725 |
Current CPC
Class: |
A61P 25/24 20180101;
C07C 45/78 20130101; A61K 36/38 20130101; C07C 45/78 20130101; C07C
49/743 20130101 |
Class at
Publication: |
424/730 ; 424;
424/439; 424/456; 424/464; 424/725 |
International
Class: |
A61K 009/20 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 10, 1998 |
IT |
MI98A001311 |
Claims
What is claimed is:
1. A method of preparing stable extracts of Hypericum perforatum
comprising: extracting flowering tops of Hypericum perforatum with
alcohol or acetone solvent to provide a first extract solution;
filtering the first extract solution; concentrating the first
extract solution to provide a concentrate; diluting the concentrate
with a water or a water-alcohol solvent to provide an aqueous
solution; extracting the aqueous solution with one or more
aliphatic ester solvents to provide an ester extract; filtering the
ester extract; and evaporating the solvents from the ester extract
to provide a stable extract of Hypericum perforatum.
2. The method of claim 1, further comprising solubilizing the
stable extract of Hypericum perforatum in a solvent comprising an
acid in aqueous ethanol to provide an aqueous ethanol solution and
evaporating the solvents from the aqueous ethanol solution.
3. The method of claim 2, wherein the organic solvents are
evaporated from the aqueous ethanol solution at a temperature of
less than 40.degree.C., the organic acid is selected from the group
consisting of citric acid, malic acid, acetylaspartic acid,
phosphoric acid, and mixtures thereof, and the aqueous ethanol
solution is 95 percent ethanol.
4. The method of claim 1, wherein the alcohol or acetone solvent is
one or more of methanol or ethanol.
5. The method of claim 1, wherein the alcohol or acetone solvent is
acetone.
6. The method of claim 1, wherein the flowering tops are present at
a weight relative to the volume of alcohol or acetone solvent to
provide a ratio of from 1:2 to 1:20.
7. The method of claim 1, wherein the concentrate is diluted with
an equal volume of water or water-alcohol solvent.
8. The method of claim 1, wherein the concentrate is diluted with a
water-alcohol solvent having an alcohol to water volume ratio of
from 1:2 to 1:5.
9. The method of claim 1, wherein the water-alcohol solution
comprises one or more of methanol or ethanol.
10. The method of claim 1, wherein the aqueous solution and one or
more aliphatic ester solvents are present in a volume ratio from
1:0.5 to 1:2.
11. The method of claim 1, wherein the aliphatic ester solvent is
selected from the group consisting of ethyl acetate, methyl
acetate, butyl acetate, and mixtures thereof.
12. The method of claim 11, wherein the aliphatic ester solvent is
ethyl acetate.
13. A stable extract of Hypericum perforatum obtainable by the
process of claim 1.
14. The extract of claim 13, wherein the extract has an IC.sub.50
for inhibition of serotonin uptake of less than 0.32 .mu.g/ml or an
IC.sub.50 for inhibition of dopamine uptake of less than 2.72
.mu.g/ml.
15. The extract of claim 13, wherein the hyperforin content is from
10 to 50 percent by weight of the extract, the total hypericin
content is greater than 0.5 percent by weight of the extract, and
the dimeric flavones content is from 1 to 2 percent by weight.
16. The extract of claim 13, wherein the hyperforin content is from
10 to 50 percent by weight of the extract, the total hypericin
content is from 0.5 to 1.2 percent by weight of the extract, and
the dimeric flavones content is from 1 to 2 percent by weight.
17. A pharmaceutical composition comprising the extract of
Hypericum perforatum of claim 12 and a pharmaceutically acceptable
excipient or carrier.
18. The pharmaceutical composition of claim 17, wherein the
composition is formulated to be a ready-to-use solution, a
soft-gelatin capsule, a hard-gelatin capsule, or a tablet and the
extract of Hypericum perforatum is present in an amount of from 10
to 100 mg.
19. The pharmaceutical composition of claim 17, wherein the
composition is formulated to be a controlled-release tablet and the
extract of Hypericum perforatum is present in an amount of from 10
to 300 mg.
20. A method of treating or preventing depression and anxiety in
humans and animals which comprises administering to a human or
animal a therapeutically effective amount of the extract of claim
1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
1. The application is a continuation of the U.S. national stage of
international application PCT/EP99/03881 filed Jun. 4, 1999, the
content of which is expressly incorporated herein by reference
thereto.
FIELD OF THE INVENTION
2. The present invention relates to a method for preparing an
extract of Hypericum perforatum (St. Johns wort) by fractioning
water-alcohol, alcohol, or acetone extracts of the plant with
esters of water-immiscible C.sub.1-C.sub.5 alcohols. The invention
also relates to formulations containing the extract.
BACKGROUND OF THE INVENTION
3. Flowering tops of Hypericum perforatum contain a number of
classes of structurally different substances that can act directly
or indirectly on the central nervous system. Hypericum perforatum
is known to contain active compounds such as hypericin, hyperforin,
and dimeric flavones that exert antidepressive and anxiolytic
activities on animals and humans. The mechanisms of action of these
compounds are different and include for example anti-MAO action,
action on serotonin release, and activity on benzodiazepine
receptors.
4. The activity of hypericin has been extensively discussed in the
literature and includes conflicting reports on the activity of
hypericin. The controversial antidepressive activity of hypericin,
however, was recently confirmed in pharmacological models in vivo.
It has been proven that hypericin is active when administered in
the presence of dimeric procyanidins contained in the extracts of
Hypericum perforatum (45.sup.th Annual Congress of the Society for
Medicinal Plant Research, Sep. 7th-12th, 1997, Regensburg, Germany,
V. Butterwecke et al., Abstract No. 011).
5. Hyperforin has recently been the subject of numerous studies
that have established its role as antidepressant. Studies carried
out by the Applicant have proven that hyperforin has
serotonin-mimetic activity.
6. Other components of Hypericum perforatum that are considered
important are dimeric flavones derived from apigenin which are
considered to be natural benzodiazepines, as reported in "Naturally
Occurring Benzodiazepines Structure, Distribution and Function", I.
Izquierdo and J. Medicine Eds., 1993, page 33.
7. These components, and particularly hyperforin, are not stable
under typical extraction conditions and storage conditions. WO
97/13489 (Schwabe) discloses that the hyperforin content of a
water-alcohol extract of St.-John's-wort falls to almost zero after
only a few weeks. According to WO 97/13489, in order to obtain
stable extracts with a constant hyperforin content it is necessary
to perform the extraction, purification and storage in the presence
of antioxidants such as vitamin C and the esters thereof,
sulfurated amino acids, and the like.
8. EP 0599307 (Schwabe) discloses the removal of hypericin,
responsible for undesired photo-sensitizing effects, by means of
polyvinylpyrrolidone and other chemicals. The patent describes
extracts obtained without using antioxidants and having a
hyperforin content of at least 5%.
9. Comparative pharmacological and clinical data between
conventional methanolic extracts or extracts prepared according to
the monograph of Commission E with the extracts prepared according
to EP 0599307 and WO 97113489 are not available. It is recognized,
however, that conventional extract of Hypericum perforatum contain
large amounts of flavonoids, which are potent radical scavengers
and therefore natural stabilizing agents for easy-to-oxidize
substances, together with other substances which can significantly
contribute to the activity of the extract.
SUMMARY OF THE INVENTION
10. The invention relates to a method of preparing stable extracts
of Hypericum perforatum. The method includes the steps of
extracting flowering tops of Hypericum perforatum with alcohol or
acetone solvent to provide a first extract solution; filtering the
first extract solution; concentrating the first extract solution to
provide a concentrate; diluting the concentrate with a water or a
water-alcohol solvent to provide an aqueous solution; extracting
the aqueous solution with one or more aliphatic ester solvents to
provide an ester extract; filtering the ester extract; and g)
evaporating the solvents from the ester extract to provide a stable
extract of Hypericum perforatum.
11. The method may further include the step of solubilizing the
stable extract of Hypericum perforatum in a solvent comprising an
acid in aqueous ethanol to provide an aqueous ethanol solution and
evaporating the solvents from the aqueous ethanol solution at a
temperature of less than 40.degree. C. The organic acid may be
citric acid, malic acid, acetylaspartic acid, phosphoric acid, or a
mixture thereof. The aqueous ethanol solution may be 95 percent
ethanol.
12. The alcohol or acetone solvent used in step (a) may be one or
more of methanol or ethanol or it may be acetone. The ratio of the
weight of the flowering tops to the alcohol or acetone solvent in
step (a) may be from 1:2 to 1:20.
13. The concentrate in step (d) may be diluted with an equal volume
of water or water-alcohol solvent. The concentrate may be diluted
with a water-alcohol solvent having an alcohol to water volume
ratio of from 1:2 to 1:5. The water-alcohol solution in step (d)
may contain one or more of methanol or ethanol
14. The volume ratio of the aqueous solution to the one or more
aliphatic ester solvents in step (e) may be from 1:0.5 to 1:2. The
aliphatic ester solvent in step (e) maybe ethyl acetate, methyl
acetate, butyl acetate, or mixtures thereof. The aliphatic ester
solvent may be ethyl acetate.
15. The invention also relates to an extract of Hypericum
perforatum obtained by the process of the invention. The extract
may have an IC.sub.50 for inhibition of serotonin uptake of less
than 0.32 .mu.g/ml or an lC.sub.50 for inhibition of dopamine
uptake of less than 2.72 .mu.g/ml. The hyperforin content of the
extract may be from 10 to 50 percent by weight of the extract, the
total hypericin content of the extract may be greater than 0.5
percent by weight of the extract, and dimeric flavones content of
the extract may be from 1 to 2 percent by weight. In another
embodiment the hyperforin content is from 10 to 50 percent by
weight of the extract, the total hypericin content is from 0.5 to
1.2 percent by weight of the extract, and dimeric flavones content
is from 1 to 2 percent by weight.
16. The invention also relates to pharmaceutical compositions
containing the extract of Hypericum perforatum prepared by the
process of the invention and a pharmaceutically acceptable
excipient or carrier. The pharmaceutical composition may be
formulated to be a ready-to-use solution, a soft-gelatin capsule, a
hard-gelatin capsule, a tablet, or a controlled-release tablet.
When the pharmaceutical composition is formulated to be a
ready-to-use solution, a soft-gelatin capsule, a hard-gelatin
capsule, or a tablet and the extract of Hypericum perforatum may be
present in an amount of from 10 to 100 mg. When the pharmaceutical
composition is formulated to be a controlled-release tablet and the
extract of Hypericum perforatum may be present in an amount of from
10 to 300 mg.
17. The invention further relates to a method of treating or
preventing depression and anxiety in humans and animals by
administering to a human or animal a therapeutically effective
amount of the extract prepared by the process of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
18. It has now been found that stable, highly active extracts of
Hypericum perforatum, containing the main compounds responsible for
pharmacological activity, in particular hypericin, hyperforin,
flavonoids, and xanthones, can be prepared by a process that
comprises:
19. a) extracting the flowering tops of Hypericum perforatum with
alcohol or acetone;
20. b) filtering the extracts and concentrating the extract;
21. c) diluting the concentrate from step (b) with water or a
water/alcohol mixture;
22. d) extracting the aqueous mixture from step (c) with aliphatic
esters;
23. e) filtering and evaporating the ester extracts from step (d)
to dryness; and, optionally,
24. f) solubilizing the dry residue from (e) in a solution of an
organic acid in aqueous ethanol and evaporating the solvent at a
temperature below 40.degree. C.
25. Preferably the first extraction (step a) is carried out with
methanol or ethanol; a weight/volume ratio for the flowering tops
to solvent ranging from 1:2 to 1:20, preferably from 1:2 to 1:10;
and a temperatures ranging from room temperature to the reflux
temperature of the solvent, preferably between room temperature and
40.degree. C.
26. Aliphatic esters for use in step (d) are preferably ethyl
acetate, methyl acetate, and butyl acetate.
27. The extraction (step d) is carried out after treating the
concentrated alcohol or acetone extract with an equal volume of
water or with alcohol/water mixtures having alcohol:water volume
ratios ranging from 1:2 to 1:5. The volume ratio of aqueous mixture
to ester is not critical and can range within wide limits, but
typically ranges from 1:0.5 to 1:2. The extraction is preferably
carried out repeatedly, generally at least three times, using fresh
aliquots of solvent.
28. Optional step (f) is effected by dissolving the concentrate
from (e) in a solution of an organic acid such as citric, malic,
acetylaspartic, or phosphoric acids in 95% ethanol.
29. The resulting extract of the invention, analyzed according to
the procedure described in M. Brolis et al., J. of Chromatography,
825, (1998), 9-16, contains hyperforin in amounts ranging from 5 to
20% by weight when using spontaneous vegetable biomasses and in
amounts from 10 to 50% by weight when using selected vegetable
biomasses. In both cases, the total hypericin content is higher
than 0.5% and dimeric flavones are present in amounts from 1 to 2%
by weight. The content of hyperforin, hypericin, and dimeric
flavones shows wide variability depending on the time at which the
plant is collected, the seed content in the flowering tops, and the
amount of stems present in the biomass.
30. This extract, compared to the total extract, surprisingly has
high activity in various pharmacological models used for evaluating
antidepressive and anxiolytic effects and is stable over time
without further treatment. The process of the invention provides
stable extracts with no need for further processing. The extracts,
however, should be shielded from light to avoid
photo-degradation.
31. Table I shows the activity of the extract of the invention
compared to other extracts and compounds on the inhibition of
serotonin (5-HT) and dopamine (DA) uptake.
1TABLE I Effect Hypericum perforatum Extract Prepared According to
Substance .sup.3H DA .sup.3H-5 HT Alcoholic extract 4.05 .+-. 0.93
28.0 .+-. 1.7 Hexane extract 0.86 .+-. 0.02 3.08 .+-. 0.62 Example
1 0.32 .+-. 0.04 2.72 .+-. 1.1 Hyperforin 1.54 .+-. 0.23 4.75 .+-.
0.79 Hypericin >50 >50 Pseudo-hypericin 1.40 .+-. 0.13 27.0
.+-. 1.1020
32. The data in Table I shows that the extract of the invention has
a potency several times higher than hyperforin and other known
extracts.
33. The extract of the invention showed a higher activity in in
vivo tests than known products, such as alcoholic, methanolic, and
hexane extracts, with or without hypericin. Moreover, the extract
has proved to be more reproducible and to exhibit greater stability
over time. The in vivo tests used to verify the antidepressive
effect were the escape deficit development test and the inhibition
of the ethanol consumption in Sardinia alcohol preferring rats,
according to procedure known in literature.
34. In the escape deficit development test, the extract of the
invention surprisingly showed a higher activity than known extracts
and an activity comparable with that of known medicaments, such as
imipramine. In the escape deficit development test rats are
fastened and subjected to mild, short, unavoidable electric shocks
for 50 min (pre-test). Twenty-four hours later, animals are tested
for their ability to avoid the same stimuli on their tails, in a
situation in which escape is impossible. On the average a rat makes
26 escapes out of 30 stimuli (naive controls), whereas an animal
subjected to pre-test only makes 1-3 escapes (ED controls).
Hyporeactivity induced by the pre-test does not take place in rats
pre-treated for 1-3 weeks with antidepressants such as imipramine
or fluoxetine. The St.-John's-wort extracts orally administered to
rats one hour before exposure to the unavoidable stress cause an
increase in reactivity to the escape test, which is further
enhanced when pre-treatment is effected for 1-2 weeks. Table II
summarizes the antidepressive effect of Hypericum perforatum
extracts and fractions thereof in rats in the escape test with a 2
week pre-treatment.
2TABLE II Antidepressive Effect of Hypericum perforatum Extracts
and Fractions Substance Dose (mg/kg) Number of escapes Hypericum
alcoholic extract 1000 16.6 .+-. 2.8 Hexane extract 600 17.2 .+-.
1.6 Example 1 200 23.3 .+-. 0.4 Example 1 100 18.3 .+-. 0.2 Example
1 50 13.3 .+-. 0.4 ED Controls -- 1.6 .+-. 0.1 Naive controls --
24.1 .+-. 0.1 Statistical analysis: Kruskal-Wallis non parametric
ANOVA KW = 13.462 p = 0.0012 Hypericum alcoholic extract vs naive p
< 0.01 Hypericum extracted as in Example 1, 200 mg/kg n.s. vs
naive Naive vs AND p < 0.01
35. In the test of the reduction of alcohol consumption in Sardinia
rats according to procedures known in literature (which is an index
of depression and anxiety), the extracts of the invention, after
three days administration, induced a 75% decrease in alcohol
consumption in favor of water compared with controls, whereas the
reduction in alcohol consumption after treatment with methanolic or
hexane extracts was 30 and 40%, respectively.
36. The extract of the invention can be included in formulations
for oral use, such as ready-to-use solutions, soft- or hard-gelatin
capsules, tablets, and controlled-release tablets. The dosage of
extract in the formulations range from 10 to 100 mg per dose in the
usual formulations and up to 300 mg in the controlled-release
formulations, in this case the preferred dose is 300 mg per dose
daily.
EXAMPLES
37. The examples reported hereinbelow illustrate the preferred
embodiments of the present invention in greater detail but should
not be construed to limit the invention in any way.
Example 1
Preparation of a Dry Extract of Hypericum perforatum Standardized
in the Active Components
38. 4 Kg of Hypericum perforatum flowering tops were extracted with
4.times.15 L of methanol in an extraction apparatus of 25 L
capacity. The combined methanol extracts were concentrated under
vacuum to 2.5 L, the concentrate diluted with an equal volume of
water, and counter-extracted with 3.times.1.5 L of ethyl acetate.
The organic phase was filtered and concentrated to dryness under
vacuum and the resulting residue was dissolved in a solution of 2 g
of citric acid in 1.3 L of 95% ethanol. The organic phase was then
evaporated to dryness under vacuum at a temperature of less than
40.degree.C. to provide 0.32 kg of a brown-yellow extract
containing 20% of hyperforin, 0.9% of total hypericins (hypericin
plus pseudohypericin), and 1% of diapigenin.
Example 2
Preparation of an Extract of Hypericum perforatum Standardized in
the Active Components
39. 60 kg of Hypericum perforatum flowering tops were collected and
mechanically dried at a temperature less than 60.degree. C. and
were extracted under mild reflux with 4.times.20 L of acetone. The
combined extracts were filtered to remove biomass residues and
concentrated under vacuum to dryness to provide 3 kg of an extract
containing 0.4% of total hypericins and about 25% of hyperforin.
The extract was suspended in 10 L of a methanol/water mixture (3:7)
and counter-extracted with butyl acetate to completely extract the
polyphenols. Extraction of the polyphenols was monitored by thin
layer chromatography using a silica gel support and eluting with
ethyl acetate/methanol/H.sub.2O (100:13.5:10). The aqueous phase
was removed, and the organic phase dried (Na.sub.2SO.sub.4) and
concentrated to dryness under vacuum at a temperature of less than
40.degree.C. to provide 1.6 kg of dry extract containing 0.7% of
total hypericins, about 40% of hyperforin, and 1.4% of dimeric
flavones.
Example 3
Preparation of a Multicomponent Extract of Hypericum perforatum,
Standardized in the Active Components
40. 60 kg of Hypericum perforatum flowering tops were collected and
mechanically dried at a temperature of less than 60.degree. C. and
were continuously extracted with 98% methanol until all the
extractables were removed. The methanolic extract was then
concentrated to 30 L and the concentrate diluted with an equal
volume of water. Insolubles that separated during dilution were
removed by filtration and the resulting clear solution was
extracted with 3.times.30 L of water-saturated ethyl acetate, dried
(Na.sub.2SO.sub.4), and the solvent removed under vacuum to provide
3.8 kg of a brown extract containing 25% of hyperforin, 1.2% of
total hypericin, and 1.2% of dimeric diflavones.
Example 4
Solution Containing the Lipophilic Extract of Hypericum perforatum
Prepared According to Example 1
41.
3 Extract of Hypericum perforatum 10.0 g prepared according to
Example 1 Ammonium glycyrrhizinate 0.5 g Propylene glycol 35.0 g
70% Sorbitol solution 25.0 g Purified water q.s. to 100 ml
Example 5
Coated Tablets Containing the Extract of Hypericum perforatum
Prepared According to Example 2
42.
4 Extract of Hypericum perforatum 300.00 mg prepared according to
Example 2 Soy polysaccharides 54.75 mg Lactose 46.00 mg
Cross-linked sodium 40.00 mg carboxymethyl cellulose Silica 20.00
mg Polyvinylpyrrolidone 5.00 mg Talc 2.50 mg Magnesium stearate
1.75 mg Hydroxypropyl methylcellulose 10.00 mg Talc 8.50 mg
Titanium dioxide 5.00 mg Triacetin 2.00 mg Polysorbate 80 0.50 mg
Red iron oxide 1.00 mg
Example 6
Soft-gelatin capusles
43.
5 Extracts of Hypericum perforatum 100 mg of Example 1 Saccharose
monopalmitate 100 mg Polyethylene glycol 400 220 mg Glycerin 15 mg
Purified water 15 mg
* * * * *