U.S. patent application number 12/160368 was filed with the patent office on 2010-09-09 for water insoluble helychrisum extract, process for preparing the same and uses thereof.
This patent application is currently assigned to ABOCA S.P.A. SOCIETA' AGRICOLA. Invention is credited to Michele Bonanomi, Caterina Ghiara, Valentino Mercati, Bruno Silvestrini.
Application Number | 20100227012 12/160368 |
Document ID | / |
Family ID | 38105880 |
Filed Date | 2010-09-09 |
United States Patent
Application |
20100227012 |
Kind Code |
A1 |
Bonanomi; Michele ; et
al. |
September 9, 2010 |
WATER INSOLUBLE HELYCHRISUM EXTRACT, PROCESS FOR PREPARING THE SAME
AND USES THEREOF
Abstract
The present invention relates to a water insoluble helychrisum
extract, a process for preparing the same and its use for preparing
pharmaceutical compositions for oral and/or topical administration.
Furthermore, the present invention relates to the pharmaceutical
compositions including said extract.
Inventors: |
Bonanomi; Michele; (Roma,
IT) ; Silvestrini; Bruno; (Roma, IT) ; Ghiara;
Caterina; (Sansepolcro (arezzo), IT) ; Mercati;
Valentino; (Sansepolcro (arezzo), IT) |
Correspondence
Address: |
PEARNE & GORDON LLP
1801 EAST 9TH STREET, SUITE 1200
CLEVELAND
OH
44114-3108
US
|
Assignee: |
ABOCA S.P.A. SOCIETA'
AGRICOLA
I-52037 SANSEPOLCRO (AREZZO)
IT
|
Family ID: |
38105880 |
Appl. No.: |
12/160368 |
Filed: |
December 12, 2006 |
PCT Filed: |
December 12, 2006 |
PCT NO: |
PCT/IB2006/003921 |
371 Date: |
July 9, 2008 |
Current U.S.
Class: |
424/764 |
Current CPC
Class: |
A61P 37/08 20180101;
A61P 29/00 20180101; A61P 37/00 20180101; A61K 36/28 20130101 |
Class at
Publication: |
424/764 |
International
Class: |
A61K 36/28 20060101
A61K036/28; A61P 29/00 20060101 A61P029/00; A61P 37/00 20060101
A61P037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 17, 2006 |
IT |
MI2006A000063 |
Claims
1-22. (canceled)
23. Water insoluble helychrisum extract, characterized by a
flavonoid content, expressed as isoquercitrin, from 2% to 15% by
weight, based on the total weight of the extract.
24. Extract according to claim 23, wherein said flavonoid content
is from 2.5% to 10% by weight, based on the total weight of the
extract; preferably, from about 3% to about 8% by weight; more
preferably, from about 3.8% to about 4.2% by weight.
25. Extract according to claim 23, further characterized by a
content of caffeil-quinic derivatives, expressed as chlorogenic
acid, from 0.5% to 5% by weight, based on the total weight of the
extract; preferably, from about 2.8% to 3.5% by weight; more
preferably, from 2.9% to 3.4% by weight.
26. Extract according to claim 23, further characterized by a water
solubility, expressed by weight of extract/water volume, <10
mg/ml; preferably, lower than 8 mg/10 ml; more preferably, lower
than 5 mg/10 ml.
27. Extract according to claim 23, further characterized by a
protection activity from the proteinic denaturation, expressed as
EC.sub.50, at a concentration of 0.2 mg/ml.
28. Extract according to claim 23, further characterized by the
HPLC-MSD chromatographic profile of the chromatogram c) reported in
the TAB. 1 and/or the HPLC-DAD chromatographic profile, at 220 nm,
of the chromatogram c) reported in the TAB. 2 and/or the HPLC-DAD
chromatographic profile, at 370 nm, of the chromatogram c) reported
in the TAB. 3.
29. Process for the preparation of a water insoluble helychrisum
extract according to claim 23, including the following steps: 1)
extracting the drug with a water/organic solvent mixture in order
to give the hydro-organic fluid helychrisum extract; 2) evaporating
the organic solvent from said hydro-organic fluid extract, to
precipitate a water insoluble fraction from the remaining aqueous
fraction; 3) separating said water insoluble fraction from said
aqueous fraction, to obtain a water insoluble solid extract.
30. Process according to claim 29, wherein, in the step 1), said
water/organic solvent mixture has a water content, based on the
total weight of the mixture, from 15% (v/v) to 85% (v/v);
preferably, from 20% (v/v) to 65% (v/v); more preferably,
approximately equal to 30% (v/v); preferably, said mixture consists
of 70.degree. ethanol.
31. Process according to claim 29, wherein, in the passage 1), said
organic solvent is a partly or completely water soluble solvent
selected from acetone and an alcohol containing one to three carbon
atoms, or their mixtures.
32. Process according to claim 31, wherein said alcohol is selected
from methanol, ethanol, n-propanol, isopropanol; preferably,
ethanol.
33. Process according to claim 29, wherein, in the passage 2), said
evaporation of the organic solvent is carried out under a reduced
pressure; preferably, at a residual pressure from 600 mbar to 150
mbar.
34. Process according to claim 33, wherein said evaporation is
continued until the initial volume of the fluid extract coming from
the passage 1) has been reduced at a value from about 1/3 to about
1/7 of said initial volume; preferably, from about 1/4 to about 1/6
of the initial volume; more preferably, at about 1/5 of the initial
volume.
35. Process according to claim 29, wherein, in the passage 3), said
separation of the water insoluble fraction from said aqueous
fraction is preferably carried out by centrifugation or by
filtration; more preferably, by centrifugation.
36. Process according to claim 29, further including the passage
of: drying said water insoluble extract coming from the passage 3)
of said claim 29.
37. Process according to claim 36, wherein said drying passage is
carried out in such conditions to avoid the extract degradation;
preferably, said drying is carried out by freeze-drying.
38. Pharmaceutical composition including an effective quantity of a
water insoluble helychrisum extract according to claim 23.
39. Process for the preparation of a composition according to claim
38, including at least a step in which said extract is additioned
with pharmaceutically acceptable co-formulations.
40. Method for the treatment of inflammatory pathologies of the
human or animal body comprising the step of administering a
pharmaceutical composition comprising an effective dose of the
water insoluble helychrisum extract according to claim 23, to a
patient in need thereof.
41. Method according to claim 40, in which said administration is
an oral or topical administration.
42. Method for the treatment of painful pathologies of the human or
animal body, comprising the step of administering a pharmaceutical
composition comprising an effective dose of the water insoluble
helychrisum extract according to claim 23, to a patient in need
thereof.
43. Method for the treatment of allergic pathologies of the human
or animal body, comprising the step of administering a
pharmaceutical composition comprising an effective dose of the
water insoluble helychrisum extract according to claim 23, to a
patient in need thereof.
Description
[0001] The object of the present invention is a water insoluble
helychrisum extract, a process for producing the same and the use
thereof for preparing pharmaceutical compositions for oral and/or
topical administration. Furthermore; the present invention relates
to the pharmaceutical compositions including said extract.
Helychrisum (Helichrysum italicum G. Don) is a perennial plant
belonging to the genus of the Asters, better known as Composites.
It is an aromatic plant originating from the Mediterranean region,
where it grows on dry sandy soils; its flowers are combined in
corymbs and have a typical golden yellow colour, from which the
name of the plant comes from (from the Greek Helios chrysos, golden
sun).
[0002] The "dry drug" of the helychrisum (hereinafter, "the drug")
consists of the dried flowered tops of the plant. By the term
"flowered tops of the plant" is understood to mean the flowers plus
the tender, non woody tops, of the branchlets, cut at about 5-15 cm
from the top, preferably at about 10 cm, depending on the
plant.
[0003] Amongst the chemical compounds found in the drug, there have
to be mentioned the flavonoids, such as, for example: pinocembrin,
apigenin, isoquercitrin, naringenin, luteolin, 7-glucoside,
gnaphaline; tiliroside;
4,2',4',6'-tetrahyroxychalcone-2'-O-glucoside;
naringin-4'-O-glucoside; kampferol-3-O-glucoside. Other types of
chemical compounds found in the tops and the flowers of helychrisum
are: phthalides (5-methoxy-7-hydroxy-phthalyde and
5,7-dimethoxyphthalide); triterpenes (ursolic acid, uvaol,
.alpha.-amirine); acetophenone derivatives; caffeil-quinic
derivatives; .beta.-sitosterol; nerols and neril acetate (these
latter comprise the 30-500 of the essential oils existing within
the plant in a varying percentage, between 0.05% and 0.20% by
weight, based on the weight of the drug, depending on the species
and the season).
[0004] The essential oils are generally obtained by steam
distillation of the flowered tops of the plant.
[0005] The known patent art mainly describes the use of helychrisum
essential oils.
[0006] In the US 2005/0003028 patent, the use both of a decoction
of a drug mixture, containing the 2% of helychrisum flowers, and an
alcoholic extract of a 3:1 mixture of Marian thistle and
helychrisum flowers; in both cases, the addition of helychrisum to
the preparation is motivated for the detoxifying properties and
assistance for the bile secretion. In the US 2004/0258783 patent
application, the anti-wrinkle action exerted by the essential oil
extracted by steam distillation from the flowered tops is
claimed.
[0007] In the U.S. Pat. No. 5,785,972 patent, a composition
containing commercial helychrisum oil (together with honey,
colloidal silver, water soluble lecithin), to be used in the
treatment of burns and sores is claimed.
[0008] In the WO 02/07744 patent application, an association of at
least two essential oils (one of which of helychrisum), to be used
for improving the resistance to chemotherapeutics in patients under
therapy for viral hepatites or tumours, is claimed.
[0009] In the WO 03/015522 patent application, the use of an
acaricidal composition consisting of a mixture of essential oils
(among which the helychrisum) is claimed. In the FR 2830198 patent,
the use of a composition containing helychrisum oil, together with
other essential oils for the topical treatment of viral infections,
immunodeficiencies and other pathologies, such as the cystic
fibrosis, is claimed.
[0010] In the FR 2845594 patent, the use of a composition
containing helychriSum oil, together with other essential oils for
the use in cosmetics or dermatology, is claimed.
[0011] In the FR 2774585 patent, the use of a composition
containing helychrisum oil, together with other essential oils for
stopping the hair loss and fighting for the formation of cuticles
(dandruff), is claimed.
[0012] In the KR 2003/046949 patent application, the extraction of
the oily fraction of helychrisum angustifolium and the
anti-inflammatory activity of a cosmetic preparation are
claimed.
[0013] The preparations obtained by the helychrisum drug more
generally used at present, are; the decoction, the fluid extract,
the syrup, the total dry extract.
[0014] By the term "decoction", is understood to mean a liquid
which is usually prepared extemporarily at the time of use by
placing an opportune drug quantity in cold water, boiling the water
and filtering away the coarse residue remained in suspension before
consuming the extraction liquid.
[0015] By the term "fluid extract" (hereinafter "fluid helychrisum
extract") is understood to mean the liquid, more or less thick,
which is obtained by macerating the drug with a proper quantity of
solvent mixture, normally hydro alcoholic (alcohol at 60-70.degree.
or alcohol at 95.degree.) or hydro glyceric, for a time period
varying depending on the kind of drug and, subsequently, filtering
the macerated matter in order to eliminate the insoluble residue of
the drug.
[0016] By the term "syrup" is understood to mean a liquid which is
obtained by properly diluting the above fluid extract (normally
with water) and adding flavours, sugar and excipients of a
different nature.
[0017] By the term "total dry extract" (hereinafter, "dry
helychrisum extract") is understood to mean the solid residual
which is obtained by completely removing, by evaporation, the
solvent mixture (hydro alcoholic or hydro glyceric) from the above
fluid extract, operating at low temperatures. The total dry
helychrisum extract can be also prepared in a freeze-dried form
(hereinafter, freeze-dried helychrisum extract", or "extracted
freeze-dried helychrisum" in the enclosed Tables).
[0018] Unfortunately, also the above helychrisum preparations, even
if they are known for their good tolerability, are affected by a
series of non negligible drawbacks, typical of the
phyto-therapeutic preparations just described.
[0019] For example, the mixture of the extracted active substances
(responsible for the pharmacological activity/s ascribed to the
drug) can result too much diluted (as in the case of the fluid
extract and/or the syrup) or, even not reproducible, nor
standardizable as for the dosage (as in case of decoction).
[0020] Furthermore, the extraction methods above described are not
able to selectively extract only the pharmacologically beneficial
active substances, but, at the same time, they extract a
significant quantity of substances at least pharmacologically
inert, if not potentially or really toxic.
[0021] Therefore, for the purpose of the optimization of the
pharmacological activity of the helychrisum and the standardization
of the doSages, in particular for the treatment of the acute and
chronic pathologies, it should be useful to be able to provide
pharmaceutical compositions including a therapeutically effective
quantity of helychrisum, characterized by a high quantity of
pharmacologically beneficial active substances (as a standardized
title) and a low quantity of pharmacologically inactive, if not
even toxic, substances. Likewise, it should be useful to be able to
provide pharmaceutical compositions wherein the quantity of mixture
including the beneficial active substances of the helychrisum
(dose) is, beside being standardized in the components thereof,
substantially lower to the one normally administered with the
traditional preparations above described.
[0022] Compositions having the features above described are not
known.
[0023] Therefore, there remains the need of being able to provide
pharmaceutical compositions based on helychrisum extract, including
a therapeutically effective quantity of the same, containing the
greatest possible quantity of pharmacologically beneficial active
substances and the lowest possible quantity of pharmacologically
inactive, if not toxic, substances.
[0024] An object of the present invention is to give an adequate
answer to the need above described.
[0025] These and other objects, which will result evident from the
following detailed description, have been attained by the
Applicant, which has unexpectedly found that a water insoluble
fraction of the above fluid helychrisum extract is able to solve
the problem above described.
[0026] By easiness, hereinafter said water insoluble fraction will
be shown by the term "water insoluble helychrisum extract" (or
"water insoluble helychrisum fraction" in the enclosed Tables).
[0027] It is an object of the present invention a water insoluble
helychrisum extract, as reported in the appended independent
claim.
[0028] Another object of the present invention is a process for
preparing said water insoluble helychrisum extract, as reported in
the appended independent claim.
[0029] Another object of the present invention is then a
pharmaceutical composition including the above water insoluble
helychrisum extract, as reported in the appended independent
claim.
[0030] Another object of the present invention is the use of said
water insoluble helychrisum extract for the preparation of
pharmaceutical compositions, as reported in the appended
independent claim.
[0031] Further objects of the present invention are the use of said
extract as a medicament and/or for the preparation of
pharmaceutical compositions for the treatment of pathological
states in humans and animals, as reported in the appended
claims.
[0032] Preferred embodiments of the present invention are reported
in the appended dependent claims.
[0033] The present invention is shown in detail in the following
description. Said invention is further illustrated also with the
help of the enclosed Tables 1 to 3, wherein: [0034] Table 1 shows,
from the top downwards, the comparison between the chromatographic
profiles of, respectively: a) freeze-dried helychrisum extract; b)
water soluble helychrisum fraction (namely the fraction of the
freeze-dried helychrisum extract soluble in water); c) water
insoluble helychrisum fraction (namely the water insoluble
helychrisum extract according to the present invention), wherein
the chromatograms have been obtained with the HPLC-MSD method
described in the following experimental section (Example 4) [0035]
Table 2 shows, from the top downwards, the comparison between the
chromatographic profiles of, respectively: a) freeze-dried
helychrisum extract; b) water soluble helychrisum fraction (namely
the fraction of the freeze-dried helychrisum extract soluble in
water); c) water insoluble helychrisum fraction (namely the water
insoluble helychrisum extract according to the present invention),
wherein the chromatograms have been obtained with a detector
adjusted to 220 nm, with the HPLC-DAD method described in the
following experimental section (Example 4) [0036] Table 3 shows,
from the top downwards, the comparison between the chromatographic
profiles of, respectively: a) freeze-dried helychrisum extract; b)
water soluble helychrisum fraction (namely the fraction of the
freeze-dried helychrisum extract soluble in water); c) water
insoluble helychrisum fraction (namely the water insoluble
helychrisum extract according to the present invention), wherein
the chromatograms have been obtained with a detector adjusted to
370 nm, with the HPLC-DAD method described in the following
experimental section (Example 4).
[0037] As it has been previously mentioned, the Applicant has
unexpectedly found that the therapeutic properties of the
Helichrysum italicum are substantially referable/ascribable to a
water insoluble fraction of the fluid helychrisum extract (that is,
that fraction previously defined as "water insoluble helychrisum
extract", obtainable from the drug (or from its fluid extract, or
from its total dried/freeze-dried extract) by means of a particular
and original extraction process.
[0038] According to a preferred embodiment of the present
invention, the water insoluble helychrisum extract is obtainable by
means of an extraction process from the drug including the
following steps:
1) extracting the drug with a water/organic solvent mixture in
order to give the hydro-organic fluid tract of helychrisum; 2)
evaporating the organic solvent from said fluid extract, to give
the precipitation of a water insoluble fraction from the remaining
aqueous fraction; 3) separating said water insoluble fraction from
said aqueous fraction, to give a water insoluble solid extract.
[0039] When one wishes to start from the total dried/freeze-dried
extract, instead of the drug or the fluid extract, then the steps
1) and 2) should be replaced by the preparation of a suspension of
said total dried/freeze-dried extract in a proper quantity of
water.
[0040] In the step 1), the water/organic solvent mixture has a
water content, relative to the total volume of the mixture, from
15% (v/v) to 85% (v/v); preferably, from 20% (v/v) to 65% (v/v);
more preferably, approximately equal to 30% (v/v).
[0041] The organic solvent employed in said mixture is a partly or
completely water soluble solvent, preferably selected from acetone
and an alcohol containing from one to three carbon atoms (methanol,
ethanol, n-propanol, isopropanol), or their mixtures; more
preferably, said organic solvent is ethanol.
[0042] In a preferred embodiment of the invention, said
water/organic solvent mixture consists of 70.degree. ethanol.
[0043] In this case, in particular, the ethanol is produced from
biological wheat and consists of 66.12 parts (by weight) of alcohol
against 33.88 parts (by weight) of water.
[0044] In the step 2), the evaporation of said organic solvent is
preferably carried out under a reduced pressure; preferably, at a
residual pressure from 600 mbar to 150 mbar.
[0045] Preferably, the evaporation is then continued until the
initial volume of the fluid extract coming from the passage 1) has
been reduced at a value from about 1/3 to about 1/7 of said initial
volume; preferably, from about 1/4 to about 1/6 of the initial
volume; more preferably, at about 1/5 of the initial volume.
[0046] In the step 3), the separation of said water insoluble
fraction from the aqueous fraction is preferably carried out by
centrifugation or filtration; more preferably, by
centrifugation.
[0047] In a preferred embodiment of the invention, said extraction
process further includes the step of:
4) drying the water insoluble extract coming from the above passage
3).
[0048] In the step 4), the drying is carried out in such conditions
to avoid the degradation of said extract; preferably, said drying
is carried out by freeze-drying; preferably, of an aqueous
suspension of the extract itself.
[0049] The above step 1) (that is the preparation of the
hydro-organic fluid extract of helychrisum) has the object of
extracting the greater possible number of pharmacologically active
substances existing in the drug.
[0050] In a preferred embodiment of the invention, said step
includes at least one of the following steps (preferably all of
them):
1a) extracting (by maceration and percolation) the drug, using a
drug/solvent mixture (D/S) weight ratio (w/w) from 1:1 to 1:25;
preferably, from 1:5 to 1:20; more preferably, from 1:10 to 1:15;
still more preferably, of about 1:13; 1b) extracting the drug a a
temperature from 20.degree. C. to the reflux temperature of the
organic solvent used, or the water/organic solvent mixture;
preferably, at a temperature of 60.degree. C.; 1c) extracting the
drug in two steps: namely, by carrying out a first extraction,
preferably with about 60% of the solvent mixture, followed by a
second extraction, with the remainder about 40%, and joining
together the two extracts; 1d) filtering the end fluid extract for
the purpose of clarifying the same.
[0051] The above step 2) (that is the partial evaporation of the
hydro-organic solvent mixture) has the purpose of completely
removing the organic solvent from the fluid extract deriving from
the step 1) and partly concentrating the water until the water
insoluble fraction (namely, the water insoluble helychrisum
extract), which contains the mixture of the pharmacologically
beneficial active substances of the drug, has precipitated.
[0052] In a preferred embodiment of the invention, said step 2)
includes at least one of the following steps (preferably all of
them):
2a) evaporating the organic solvent under a reduced pressure;
preferably at a residual pressure from 600 mbar to 150 mbar; 2b)
carrying out said evaporation at a outer temperature from
40.degree. C. to 130.degree. C.; preferably, from 50.degree. C. to
70.degree. C.; more preferably, at about 60.degree. C.
[0053] The above step 3) (namely the separation of the water
insoluble extract of helychrisum from the aqueous step) has the
putpose of isolating and washing the helychrisum extract from the
residual aqueous fraction, which includes a series of non
pharmacologically active water soluble products.
[0054] In a preferred embodiment of the invention, said step 3)
includes at least one of the following steps (preferably all of
them):
3a) centrifuging the solid/water phase mixture resulting from the
passage 2), for example by means of a horizontal centrifuge; 3b)
collecting the solid (that is the water insoluble extract of
helychrisum); 3c) washing said solid with water; for example, using
a solid/water weight ratio (w/w) from 1:1 to 1:10; preferably, from
1:1 to 1:5.
[0055] The above additional, possible, passage 4) has the purpose
of transforming the isolated and washed water insoluble helychrisum
extract in a powder utilizable for preparing and standardizing the
desired different pharmaceutical formulations based on
helychrisum.
[0056] In a preferred embodiment of the invention, said step 4)
includes:
4a) suspending the solid helychrisum extract in water; 4b) freezing
said suspension; 4c) freeze-drying said frozen suspension; 4d)
milling the obtained freeze-dried product for the purpose of
rendering homogeneous the particle size of solid freeze-dried
helychrisum extract.
[0057] The above freeze-drying process is carried out using
equipments and working conditions known to a skilled in the
art.
[0058] The solid helychrisum extract can also be freeze-dried in
the presence of proper excipients, such as, for example:
stabilizers, preservatives, flavourings, sweeteners.
[0059] In an embodiment of the invention, the Italic helychrisum
used for the preparation of the pharmacologically active water
insoluble extract, has been cultivated in a calcareous and
half-arid land typical of the Tuscany-Emilia Apennine. The
cultivation, arranged at 600 metres above the sea-level, was
exposed to South-South-West, with an optimum exposition to the
sun's rays and a slope about 15-200. The harvesting has been
carried out in July, at the beginning of the blossom time, cutting
the tender tops of the branches for 10 cm about and avoiding the
lower wooden portion. The fresh harvest has been immediately dried
in air furnaces at a temperature of 40-45.degree. C. The dried drug
thus obtained has shown a flavonoid content, expressed as
isoquercitrin, from 0.42% to 0.50% by weight, based on the total
weight of the drug.
[0060] Furthermore, a content in caffeil-quinic derivatives,
expressed as chlorogenic acid (monoester of the quinic acid with
the caffeic acid), from 2.5% to 3.1% by weight, based on the total
weight of the drug, has been determined.
[0061] As pointed out in the enclosed Table 1 (TAB. 1--chromatogram
a)), the chromatographic profile of the total freeze-dried
helychrisum extract, obtained from the drug by the HPLC-MSD method,
described hereinafter in the Example 4, is characterized by the
presence of a series of signals/products having widely varying
retention times (from few minutes to about 40 minutes, until a
series of signals having retention times lower than about 60
minutes).
[0062] The chromatographic profile of the water soluble fraction of
the helychrisum extract (TAB. 1--chromatogram b)) is, on the
contrary, characterized by the presence of the series of signals
having retention times lower than about 40 minutes.
[0063] In turn, the chromatographic profile of the water insoluble
extract of helychrisum (TAB. 1--chromatogram C)) is, on the
contrary, characterized by the presence of the series of signals
having retention times higher than about 60 minutes.
[0064] The same type of situation is also substantially confirmed
by the chromatograms of the enclosed Tables 2 and 3, from which it
is evident that the (active) substances, forming the water
insoluble helychrisum extract are characterized by retention times
higher than about 40 minutes (differently from the total
freeze-dried total and the water soluble fraction).
[0065] The water insoluble helychrisum extract corresponds to about
2.3%-2.5% by weight with respect to the total weight of the dry
drug and about 13%-15% by weight with respect to the total weight
of the total dry extract of the same.
[0066] The water insoluble helychrisum extract is characterized by
a water solubility (at a room temperature of 25.degree. C.)<10
mg of extract/10 ml of water; preferably, lower than 8 mg/10 ml;
more preferably, lower than 5 mg/10 ml.
[0067] The solubility study of said extract in other solvents has
allowed to individuate the following solubility profile (expressed
as mg of water insoluble extract/ml of solvent):
ethanol 95.degree. about 10 mg/0.2 ml; ethyl acetate about 10
mg/0.5 ml; ethyl ether<10 mg/l chloroform about 10 mg/0.5 ml;
dichloromethane about 10 mg/l ml; hexane<10 mg/2.5 ml; toluene
about 10 mg/l ml; acetone about 10 mg/0.8 ml.
[0068] The water insoluble helychrisum extract, obtained according
to the extraction method of the present invention, has been
additionally characterized by chemical and biological methods, for
the purpose of standardizing the same not only from the
constituents point of view but also the pharmacological
activity:
[0069] As for the qualitative characterization of the extract,
chromatographic methods have been developed by means of HPLC/DAD
and HPLC/MSD, as described hereinafter in the Example 4, which have
allowed to acquire the characteristic chromatographic profile
(fingerprint) of the extract itself.
[0070] As already above reported, the water insoluble helychrisum
extract resulted characterized by the chromatographic profiles
(fingerprint) c) disclosed in enclosed TAB. 1-3.
[0071] As for the quantitative characterization of said extract;
the same has been carried out through the determination of two
important classes of constituents, the flavonoids and the
caffeic-quinic derivatives.
[0072] The spectrophotometric methods employed for carrying out
such quantitative determinations are described in detail in the
following experimental section (Examples 2 and 3,
respectively).
[0073] The water insoluble helychrisum extract resulted
characterized by a flavonoid content, expressed as isoquercitrin,
from 2% to 15% by weight, with respect to the total weight of the
extract; preferably, from 2.5% to 10% by weight; more preferably,
from about 3% to about 8% by weight.
[0074] On average, said flavonoid content is from about 3.5% to
5.5% by weight; preferably, around 4% eight. Therefore, in the
water insoluble extract, the flavonoids are resulted more
concentrated with respect to their starting concentration in the
dry drug.
[0075] Furthermore, the water insoluble helychrisum extract is
resulted characterized by a content of caffeil-quinic derivatives,
expressed as chlorogenic acid, from 0.5% to 5% by weight, with
respect to the total weight of the extract; preferably, from 2.86%
to 3.5% by weight; more preferably, from 2.9% to 3.4% by weight; on
average, of about 3% by weight.
[0076] The water insoluble helychrisum extract has also been
characterized by determining its protection activity from the
proteinic denaturation, expressed as EC.sub.50.
[0077] Said extract has proven to be standardized to inhibit a 50%
proteinic denaturation when used at a concentration of 0.2
mg/ml.
[0078] The pharmacological activities ascribed to the traditionally
used extracts of the helychrisum, have resulted
referable/ascribable to the mixture of compounds contained in the
water insoluble helychrisum extract obtained through the innovative
extraction/separation process of the present invention.
[0079] The anti-inflammatory/pain-killing activity of said extract
has been documented, at a pre-clinical level, using the in vitro
test of the proteinic denaturation and of the Writhing in the
mouse.
[0080] The anti-allergic/anti-asthmatic activity has further been
documented on the bronchospasm and the plasma extravasation induced
by ovalbumin in guinea pigs sensitized to this compound.
[0081] The studies have confirmed that the pharmacological activity
of the water insoluble helychrisum extract, obtained by the
extraction/separation process of the present invention, is the one
known of the whole drug. Moreover, the pharmacological activity of
said extract has resulted greater and better than the one
ascribable to the drug, thus allowing to prepare pharmaceutical
compositions with a reduced and standardized dosage which allow the
repeatability of the dosages and the treatments.
[0082] Advantageously, the water insoluble helychrisum extract has
shown to possess a good anti-inflammatory activity.
[0083] Furthermore, said extract has a good pain-killing
activity.
[0084] Furthermore, said extract has a good anti-allergic
activity.
[0085] Therefore, the water insoluble extract of the present
invention has proved to be particularly useful for a use as a
medicament; in particular for preparing pharmaceutical compositions
to be used in a medical field in the therapy of pathologic states
in human and animal, as above pointed out.
[0086] Therefore, it is also a subject of the present invention a
process for the preparation of a pharmaceutical composition
containing a therapeutically effective quantity of water insoluble
helychrisum extract; said process includes at least a step in which
said extract is additioned with opportune pharmacologically
acceptable co-formulations.
[0087] Said pharmaceutical compositions can be used both for oral
and topical administration (including aerosol).
[0088] Said pharmaceutical compositions are preferably formulated
in admixture with appropriate excipients, such as vehicles,
lubricants, dispersants, flavourings, sweeteners, stabilizers,
preservatives, antioxidants, additives, such as amino acids,
vitamins, enzymes commonly used in the pharmaceutical formulation
art.
[0089] By mere way of absolutely not limiting example, amongst the
particularly preferred excipients and additives there may be
mentioned starch, flavours, such as those of mandarin, grapefruit,
strawberry, bilberry, all fruits, sucrose, glucose, ascorbic acid,
glutamine, arginine, inulin.
[0090] Particularly preferred compositions of the present invention
are those for oral administration (including the sublingual
one).
[0091] Typical preferred formulation forms are, for example,
capsules, beads, syrups, solutions or suspensions ready-to drink,
powders or granulates in sachets (to be suspended or dissolved in
water or in non-carbonated and non-alcoholic beverages at the
moment of use) or analogous forms, tablets, effervescent
formulations:
[0092] The compositions of the present invention can also be
formulated in a coated, lacquered, encapsulated or
microencapsulated form, such that to result gastro-resistant.
[0093] Said compositions can also be formulated in a
controlled-release form, so as to selectively deliver the active
substances in the intestinal tract, in particular within the
colon.
[0094] Other preferred compositions of the invention are those for
topical administration.
[0095] Said compositions can be prepared in form of pomades,
ointments, gels; or they can be opportunely formulated and
incorporated in transdermal vehicles, such as for example
plasters.
[0096] The compositions of the present invention are prepared in a
traditional way by using, depending on the kind of formulation that
one wishes to carry out, preparative techniques known to the
pharmaceutical artisan skilled in the art.
[0097] The compositions of the present invention have proved to be
particularly useful for the prevention and/or the treatment of
different pathologies, both for topical use (for example for the
treatment of eczema and atopic dermatitis, irritative contact
dermatitis, allergic contact dermatitis), and for systemic use (for
example chronic rheumatic affections, inflammatory pathologies,
acute pain from fractures consequences, asthma and allergic
rhinitis).
[0098] The present invention is described hereinafter, by mere way
of non limiting example, by means of some experimental
examples.
EXAMPLE 1
[0099] Preparation of the water insoluble extract of italic
helychrisum, starting from the drug.
[0100] 178 kg of dry drug of helychrisum were extracted with
70.degree. ethanol with the D/S weight ratio 1:13. A first
extraction with the 600 of the solvent mixture was carried out over
5 h at 60.degree. C., then the hydro alcoholic extract was removed
and the drug extracted a second time with the remaining 40% of the
solvent mixture for 3 h at 60.degree. C. The extraction was carried
out by percolation/maceration.
[0101] The pooled hydro alcoholic extracts were filtered (weight of
the fluid extract=1830 kg) and concentrated under vacuum
(60.degree. C., 600 mbar) until 380 kg of a concentrated aqueous
suspension were obtained.
[0102] The concentrated aqueous suspension was subjected to
centrifugation through a horizontal centrifuge. In this way, the
precipitate was separated from the remaining water phase in form of
a black-coloured thick, solid mass.
[0103] 6 kg of wet solid precipitate and 370 kg of water phase were
thus obtained.
[0104] The wet precipitate obtained was washed with 6 l of
ultra-neat water, then subjected to freeze-drying giving 4.2 kg of
water insoluble helychrisum extract, equal to about 2.4% by weight,
based on the initial weight of the drug employed.
[0105] The water phase was, in turn, subjected to freeze-drying
giving 26 kg of a water soluble residue, or fraction, equal to
about 14.6% by weight, based on the initial weight of the drug
employed.
[0106] The freeze-dried water insoluble extract was milled by a
cryo-mill to give 3 kg of a powder which was stored at a
temperature of +4.degree. C., in a sealed and under vacuum
aluminium sachet. The freeze-dried water insoluble extract resulted
characterized by a content of flavonoids, expressed as
isoquercitrin, equal to 3.78% by weight, based on the total weight
of the extract, and a content of caffeic-quinic derivatives,
expressed as chlorogenic acid, equal to 3.36% by weight, based on
the total weight of the extract.
[0107] The chromatographic profiles of the water insoluble extract
thus obtained are those respectively reported in the chromatograms
c) of the enclosed TAB. 1-3.
EXAMPLE 2
[0108] Spectrophotometric method for the determination of the
flavonoid content (expressed as isoquercitrin) in the drug and the
water insoluble helychrisum extract (modification of the method
described in Eur. Ph, v Ed., monograph of Sambuci flos)
[0109] About 0.60 g of pulverized drug (or about 0.30 g of water
insoluble helychrisum extract) were treated with 1 ml of aqueous
solution (5 g/l) of hexamethyltetramine, additioned with 20 ml of
acetone and 2 ml of a hydrochloric acid solution (70 g of conc.
hydrochloric acid in 100 ml of water); the suspension was heated
until boiling for 30 minutes.
[0110] A filtration through cotton in a 100 ml volumetric flask is
carried out. The residue and the cotton were treated in two stages
each time with 20 ml of refluxed acetone over 10 minutes; the two
washes were added to the parent solution and, after cooling,
adjusted to volume (100 ml) with acetone (Solution A).
[0111] 20 ml of the solution A (10 ml in case of the water
insoluble extract) were added to 20 ml of water and extracted with
ethyl acetate (1.times.15 ml, 3.times.10 ml).
[0112] The pooled extract were washed with water (2.times.50 ml),
dried over dry sodium sulphate and adjusted to volume with ethyl
acetate in a 50 ml volumetric flask (Solution B).
[0113] 10 ml of the solution B were transferred in a 25 ml flask
and added with 1 ml of aluminium trichloride solution (at 20 in
methyl alcohol containing 5% of glacial acetic acid) and adjusted
to volume with the methanol solution (50 ml/l) of glacial acetic
acid. (Solution under examination). 10 ml of the solution B were
transferred in a 25 ml flask and adjusted to volume with the
methanol solution (50 ml/l) of glacial acetic acid. (Comparison
solution).
[0114] After 30 minutes, the absorbance of the solution under
examination was measured, at 425 nm, with respect to the comparison
solution.
[0115] Knowing that A.sub.1%, 1cm of the isoquercitrin is equal to
500 at 425 nm, the percent content of flavonoids, expressed as
isoquercitrin, was computed with the formula:
%=(A.times.V.times.F)/A.sub.1%, 1cm.times.p
wherein: p=weight of the sample expressed in grams A=absorbance of
the sample at 425 nm V=extraction volume (100 ml) F=dilution factor
(6.25)
EXAMPLE 3
[0116] Spectrophotometric method for the determination of the
caffeil-quinic acids content (expressed as chlorogenic acid) in the
drug and the water insoluble helychrisum extract (isolation of the
orthodiphenolic fraction in form of lead salt) (modification of the
method described in FU IX ed, monograph of the Cynara dry
extract).
[0117] About 0.50 g of pulverized drug (or 0.30 g in case of the
water insoluble helychrisum extract) were treated with 40 ml of
water until boiling. The suspension was heated until boiling,
filtered through cotton in a centrifuge tube. To the still hot
solution, 2 ml of a solution saturated with lead (PbII) acetate
were added. Cooling and centrifugation were performed, and the
clear supernatant solution was eliminated. The precipitate was
washed with water (5 ml), then again centrifuged removing the water
phase. The precipitate was dissolved in 70 ml of a 10% acetic acid
aqueous solution and heated to ebullition. The hot mixture was
filtered through cotton, 2 ml of 20% sulphuric acid were added. The
lead sulphate precipitate was separated by centrifugation, the
clear solution was trans-ferred in a 100 ml volumetric flask. The
precipitate remained on the bottom of the centrifuge tube was
washed with 5 ml of 10% acetic acid; the washing solution, after
centrifugation, was transferred in the same flask, where it was
adjusted to an end volume of 100 ml with 10% acetic acid.
[0118] 1 ml of this solution was diluted to 25 ml with methanol:
the absorbance of the solution at 325 nm was read, against a
solution obtained by diluting 1 ml of 10% acetic acid to 25 ml with
methanol.
[0119] Knowing that A.sub.1%, 1cm of the chlorogenic acid at 325 nm
is equal to 485, the percent content of caffeic-quinic acids,
expressed as chlorogenic acid, was computed with the formula:
%=(A.times.V.times.F)/A.sub.1%, 1cm.times.p
wherein: A=absorbance of the sample at 425 nm. F=dilution factor
(25) p=weight of the sample expressed in grams V=extraction volume
(100 ml)
EXAMPLE 4
[0120] HPLC/DAD and HPLC/MSD methods for the acquisition of
chromatographic profile of the helychrisum extracts (applicable,
respectively, over: drug, freeze-dried extract, water insoluble
extract and water soluble fraction)
[0121] The study of the HPLC chromatographic profiles was carried
out by acquiring the chromatograms by means of two different
detectors (DAD: Photo Diode Array; MSD: Ion Trap, Agilent, mod SL)
with different analytical methodologies in the two cases.
HPLC/DAD Method
Equipment:
[0122] HPLC Agilent series 1100, fitted with vacuum degasser,
quaternary pump, self-sampler, thermostated compartment at
20.degree. C. for the housing of two columns, diode array detector
(DAD). Reverse phase analytical column Prodigy RP-18 (250
mm.times.4.6 mm, 100 .ANG., 5.mu., Phenomenex, USA) with a
pre-column RP-18 (4 mm.times.3 mm).
Preparation of the Solutions to be Examined:
[0123] drug: extracting 0.5 g of dry drug with two subsequent
portions of 25 ml of 50.degree. ethanol, over 30' in an ultrasonic
bath; centrifugation is carried out, the extracts are pooled and
adjusted to volume to 50 ml in a volumetric flask; [0124]
freeze-dried extract: extracting 0.1 g of extract with 10 ml of
50.degree. ethanol, over 30' in an ultrasonic bath; centrifugation
is carried out and the overlying solution is examined, as described
in the Process; [0125] water insoluble extract (or water insoluble
fraction): extracting 0.1 g with 10 ml of 50.degree. ethanol, over
30' in an ultrasonic bath, centrifugation is carried out and the
overlying solution is examined, as described in the Process. [0126]
water soluble fraction: extracting 0.1 g with 10 ml of 50.degree.
ethanol, over 30' in an ultrasonic bath, centrifugation is carried
out and the overlying solution is examined, as described in the
Process.
Process:
[0127] collecting 0.5 ml of the solution to be examined, filtering
over a cellulose acetate filter (0.45 .mu.m), injecting 20 .mu.l in
the apparatus. Flow rate: 1 ml/min Elution solvent: 0.2%
H.sub.3PO.sub.4 in water/methyl alcohol, in a gradient (Table no.
1)
TABLE-US-00001 TABLE no. 1 Gradient composition: minutes 0.2%
H.sub.3PO.sub.4 in water CH.sub.3OH 0 90% 10% 35 50% 50% 75 5%
95%
[0128] The chromatograms are acquired at 220, 265, 280 330 and 370
nm.
HPLC/MSD Method
Equipment:
[0129] HPLC Agilent series 1100, fitted with vacuum degasser,
binary pump, self-sampler thermostated at 10.degree. C.,
compartment thermostated at 20.degree. C. for the housing of two
columns, diode array detector (DAD) and ion trap mass detector,
model SL. Reverse phase analytical column Prodigy RP-18 (250
mm.times.4.6 mm, 100 .ANG., 5.mu., Phenomenex, USA) with a
pre-column RP-18 (4 mm.times.3 mm).
Preparation of the Solutions to be Examined:
[0130] drug: extracting 0.5 g of dry drug with two subsequent
portions of 25 ml of 60% methanol, over 30' in an ultrasonic bath;
centrifugation is carried out, the extracts are pooled and adjusted
to volume at 50 ml in a volumetric flask, always with 605 methanol;
[0131] freeze-dried extract: dissolving 10 mg in 10 ml of 60%
methanol over 30' in an ultrasonic bath; [0132] water insoluble
extract (or water insoluble fraction): dissolving 10 mg in 10 ml of
methanol, over 30' in an ultrasonic bath; [0133] water soluble
fraction: dissolving 10 mg in 10 ml of 0.60% methanol over 30' in
an ultrasonic bath.
Process:
[0134] collecting 0.5 ml of the solution to be examined, filtering
over a cellulose acetate filter (0.45 .mu.m), injecting 5 .mu.l in
the apparatus. Flow rate: 1 ml/min Elution solvent: 0.025% formic
acid in water/methyl alcohol, in a gradient (Table no. 2)
TABLE-US-00002 TABLE no. 2 Gradient composition: 0.025% HCO.sub.2H
in minutes water CH.sub.3OH 0 90% 10% 35 50% 50% 75 5% 95% 80 5%
95%
[0135] Before the introduction within the mass detector, the flow
is properly split to 0.6 ml/min. through a T-valve using, for the
different connections, capillary tubes with a same caliper but
different length. The analyses have been carried out using the ESI
source and selecting negative ions. In Table no. 3, the parameters
for the acquisition of the mass spectra during the chromatographic
elution are reported.
TABLE-US-00003 TABLE NO. 3 Acquisition modes of the mass spectra
Source ESI Nebulizer 50 Capillary 3500 Polarity Negative Dry gas 10
Skimmer -40.0 Target 30000 Dry Temp. 350 Cap. exit -90.8 MAT 100
Oct. RF 108.9 Oct 1DC -12.00 Scan 100/1500 Lens 1 5.0 Oct 2DC -1.70
Average 7 (RA = 2) Lens 2 60.0 Trap drive 55.00
[0136] For the water insoluble extract, a specific HPLC/MSD method
was further developed, capable of pointing out with a greater
accuracy the characteristic components. The methodology is
differentiated from the preceding one only for the used gradient,
whose composition is reported in the Table no. 4.
TABLE-US-00004 TABLE no. 4 Gradient composition 0.025% HCO.sub.2H
in minutes water CH.sub.3OH 0 30% 70% 75 5% 95% 80 5% 95%
Control of the Anti-Denaturant Activity
[0137] The test is carried out in vitro, using electrophoresis
techniques, and allows to correlate the anti-denaturant activity
exerted by the water insoluble helychrisum extract on plasma
proteins and expressed as EC.sub.50, to the anti-inflammatory
activity of vegetal extracts. In the same test, in fact, the FANS
are capable of inhibiting the denaturation of the proteins.
* * * * *