Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE: LIGNAN COMPLEX DERIVED FROM FLAX SEED USED FOR
TREATMENT OF HYPERCHOLESTEROLEMIC
ATHEROSCLEROSIS
BACKGROUND OF THE INVENTION
1. FIELD OF THE INVENTION
This invention relates to the use of lignan complex isolated from flax for the
treatment of hypercholesterolemic atherosclerosis in humans and animals.
II. BACKGROUND ART
Hypercholesterolemia is a major risk factor for atherosclerosis (narrowing of
the artery due to deposition of fat in the arterial wall) and related
occlusive vascular
diseases such as heart attack, stroke and other peripheral vascular diseases.
Heart
disease is the number one killer. Hypercholesterolemic atherosclerosis has
been
reported to be associated with oxidative stress (increase in levels of
reactive oxygen
species (ROS), production of ROS by polymorphonuclear leukocytes as assessed
by
chemiluminescence (PMNL-CL), and a decrease in the antioxidant reserve)
(References 1-4, see list of References at the end of this description).
Pretreatment
with antioxidants (vitamin E, probucol, garlic, purpurogallin,
secoisolariciresinol
diglucoside) prevents the effects of hypercholesterolemia (References 1, 2, 4-
6). Flax
(especially flaxseed) is a rich source of a-linolenic acid and the richest
source of plant
lignans. Flaxseed has been shown to be effective in preventing the development
of
hypercholesterolemic atherosclerosis without lowering serum levels of
cholesterol
(Reference 7). Using flaxseed which has very low a-linolenic acid has shown
that the
antiatherogenic activity of flaxseed is not due to a-linolenic acid but may be
due to
the lignan component of flaxmeal (Reference 8).
Presently, the treatment of hypercholesterolemia and hypercholesterolemic
atherosclerosis is to reduce hypercholesterolemia by using various lipid
lowering
agents such as bile acid sequestrant (cholestyramine, colestipol), nicotinic
acid,
HMG-CoA reductase inhibitor (lovastatin, pravastatin, simvastatin, fluvastatin
and
atorvastatin) and gemfibrozil. Recently probucol which has both antioxidant
and lipid
lowering activity and vitamin E which has antioxidant activity have been used
to
prevent atherosclerosis and restenosis.
Drugs used for lowering serum lipids and for treatment of atherosclerosis
(heart attack and stroke) have many side effects and are expensive. Fibric
acid
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derivatives (gemfibrozil) produces gall stones, myopathy and hepatomegaly.
Nicotinic
acid produces gastrointestinal symptoms, flushing, hyperglycemia, hepatic
dysfunction, elevated uric acid, abnormal glucose tolerance, and skin rash.
Bile acid
sequestrant (cholestyramine, colestipol) produces gastrointestinal symptoms,
and high
serum levels of very low density-lipoprotein (VLDL). HMG-CoA reductase
inhibitors
(statin) produce gastrointestinal symptoms, myopathy and hepatotoxicity.
Probucol
produces diarrhea and decreases the serum levels of HDL (good cholesterol).
Prasad, U.S. Pat. No. 5,846,944, describes the use of secoisolariciresinol
diglucoside (SDG), isolated from flaxseed, for reducing hypercholesterolemic
atheroscleorsis and reducing serum cholesterol. However, isolating SDG from
flaxseed is a relatively expensive procedure.
In Westcott et al., U.S. Pat. No. 6,264,853, a new lignan complex is described
which has been isolated from flaxseed. This lignan complex typically contains
SDG
(35%), cinnamic acid glycosides and hydroxymethyl glutaric acid. Only a simple
procedure is required to isolate this lignan complex from flaxseed.
In Prasad, U.S. Pat. No. 6,673,773, a method for preventing
hypercholesterolemic atheroschlerosis is disclosed.
One purpose of the present invention, at least in preferred forms, is to
provide
a niethod of treating hypercholesterolemic atheroschlerosis in subjects that
already
have symptoms of the disease.
SUMMARY OF THE INVENTION
One exemplary aspect of the invention provides a method of treating
hypercholesterolemic atherosclerosis in a mammal (human or animal) exhibiting
symptoms thereof. The method comprises administering to the subject an
effective
amount of a lignan complex derived from flax and containing about 34 to 38% by
weight of secoisolariciresinol diglucoside (SDG), and preferably about 15 to
21 % by
weight cinnamic acid glucosides and about 9.6 to 11% by weight
hydroxymethylglutaric acid. The cinnamic acid glucosides preferably include
couinaric acid glucoside and ferulic acid glucoside.
According to another exemplary aspect of the invention, there is provided a
composition comprising a lignan complex derived from flax capable of reducing
serum cholesterol, raising high-density lipoprotein cholesterol, reducing
hypercholesterolemic artherosclerosis, or any combination thereof, when orally
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admisistered to a mammal at a daily does of 20-60 mg per kg body weight of the
mammal.
Yet another exemplary embodiment of the invention provides a process
comprising obtaining a lignan complex comprising secoisolariciresinol
diglucloside,
cinnamic acid glycosides and hyroxymethyl glutaric acid from a plant; wherein
a
component of the lignan complex has a molecular weight of at least 30,000; and
placing the lignan complex in a composition suitable for oral administration
to a
mammal.
Yet another exemplary embodiment of the invention provides a composition
comprising: a lignan complex obtained from flax and comprising
secoisolariciresinol
diglucoside (SDG), cinnamic acid glucosides, and hydroxymethylglutaric acid;
wherein a component of the lignan complex has a molecular weight of at least
30,000;
wherein the lignan complex is effective in at least one of reducing serum
cholesterol,
raising high-density lipoprotein cholesterol, and reducing
hypercholesterolemic
artherosclerosis when orally administered to a rabbit at a daily dose of 40
mg/kg of
body weight of the rabbit for a period of two months.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a representative photograph of intimal surface of aorta from rabbits
of
four experimental groups of slowing of progression study showing Sudan IV -
stainable lipid deposit. Note marked brick red lipid deposits in groups II,
III and N.
Fig. 2 is a graph showing the extent of atherosclerosis in aortas of the four
experimental groups referred to above.
Fig. 3 shows photographs of the intimal surfaces of aortas of rabbits from 4
experimental groups of a regression study showing Sudan IV - stainable lipid
deposits.
Fig. 4 is a graph showing the extent of atherosclerosis in the aortas of the
four
experimental groups referred to above.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The effectiveness of lignan complex from flax for the prevention of
atherosclerosis in subjects that show no symptoms of the disease has
previously been
demonstrated by the inventor of the present invention in U.S. Patent No.
6,673,773,
the disclosure of which is incorporated herein by reference.
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The inventor has now established that lignan complex is not only effective in
preventing the development of hypercholesterolemic atheroschlerosis in healthy
mammals, but can actually slow the progression of the disease once it has
developed
in a subject
Lignan complex from flax, and a method for its extraction, is described in
U.S.
patent 6,264,853, issued July 24, 2001, the disclosure of which is
incorporated herein
by reference. The process may comprise obtaining an aqueous aliphatic alcohol
extract of commercial flax, e.g. flaxseed or flax meal. The extract is then
subjected to
ultrafiltration, whereby low molecular weight species remain with the filtrate
and
higher molecular weight species are retained. By proper selection of the
filtration
medium, it is possible to retain a lignan complex in substantially pure form
comprising secoisolariciresinol diglucoside (SDG), cinnamic acid glucosides
(CAG),
and hydroxymethylglutaric acid (HMGA). The ultrafiltration is preferably
carried out
for a size exclusion of 30,000 Daltons or greater, generally in the range of
30,000 to
100,000 Daltons, and more preferably 30,000 to 50,000 Daltons.
The complex used according to the present invention typically contains 34 to
38% by weight of secoisolariciresinol diglucoside (SDG), and optionally 15 to
21%
by weight cinnamic acid glucosides (CAG), and 9.6 to 11% by weight
hydroxymethylglutaric acid (HMGA). The CAG component typically includes
coumaric acid glucoside andlor ferulic acid glucoside, normally present in the
coniplex in amounts of about 9.5 to 16.0% by weight coumaric acid glucoside
and 4.5
to 5.0% by weight ferulic acid glucoside.
The complex typically contains about 59 to 70% by weight of the three stated
ingredients, the balance comprising protein, ash and water of crystallization.
In the present invention, the complex is preferably employed at a purity of
95% by weight or more.
As noted above, the lignan complex has been found effective for slowing the
progression of atherosclerosis and preventing the progression of
atherosclerosis that
occurs after removal of high cholesterol diet (normalizing the serum
cholesterol), and
hence may be useful for the treatment of related diseases, such as coronary
artery
disease, stroke, intermittent claudication, restenosis following coronary
artery
percutaneous intervention (PCI), coronary bypass grafts closure, and other
peripheral
vascular disease, as well as reducing complications associated with such
diseases.
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The lignan complex may be administered orally in any suitable form, e.g. as a
medicament for suitable for treating atherosclerosis in a mammal, preferably
mixed
with a pharmaceutically-acceptable diluent or carrier, optionally prepared,
for
example, as a tablet, capsule or food. Suitable dosages may be determined by
trail
5 and experimentation, but dosages of 20-60 mg per kg body weight of the
mammal are
normally most effective, particularly when the mammal is a rabbit.
The invention is further illustrated by the following non-limiting Examples.
EXAMPLES
Methods:
The studies below were conducted in New Zealand white female rabbits
weighing between 1.2 and 1.5 g. (6 to 8 wks old) after a one-week period of
acclimatization. The rabbits were housed individually under constant climate
conditions (temperature 20 to 22 C, relative air humidity 50 5 l0). The
rabbits were
housed under a 12-hour light and 12-hour dark cycle. The studies were approved
by
the Ethics Committee of the University of Saskatchewan, Saskatchewan, Canada,
and
the animal care was according to approved standard for Laboratory Animal Care.
Food and water were provided ad libitum.
Experimental Protocol
(i) For the study of the slowing of progression of atherosclerosis:
Rabbits in this study were divided into 4 groups.
Group I (n=6): control (regular diet).
Group II (n=5): 0.25% cholesterol diet for 2 months.
Group III (n=6): 0.25% cholesterol diet for 4 months.
Group IV (n=6): 0.25% cholesterol diet for 2 months, followed
by 0.25% cholesterol diet and lignan complex (40
mg/kg body weight per day, orally) for an additional
two months.
(ii) For the study of the regression of atherosclerosis:
Rabbits in this study were assigned to 4 groups.
Group I (n=6): control (regular diet)
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Group II (n=5): 0.25% cholesterol diet for 2 months.
Group III (n=1 1): 0.25% cholesterol diet for 2 months followed
by regular diet for 4 months.
Group IV (n=12): 0.25% cholesterol diet for 2 months
followed by regular diet and lignan complex (40 mg/kg
body weight per day, orally) for additional 4 months.
At the end of the protocol, rabbits were anesthetized with pentobarbital
sodium (40 mg/kg, intravenously) and the aortas were removed for the
assessment of
atherosclerotic changes. The assessment of atherosclerotic changes in the
aorta was
made by using Herxheimer's solution containing Sudan IV for lipid staining
(1).
Photographs of the stained intimal surface of the aorta were taken and color
slides
were prepared. The slides were projected on a Caramate Projector screen with a
grid
in mm2. The extent of atherosclerosis was expressed as a percentage of total
intimal
surface area.
Statistical Analysis:
Results are expressed as mean SEM. The Kruskal-Wallis test was used to
determine the differences in the atherosclerotic changes in the four groups.
The
Mann-Whitney U test was used to determine the significance of differences
between
any two groups. Type-I error for multiple comparison was controlled by the
Bonferroni correction.
Results:
Slowing of Progression of Atherosclerosis
This study was conducted to see if lignan complex would slow the progression
of atherosclerosis. Representative photographs of the atherosclerotic changes
in the
intimal surface of the aortas of the four groups are shown in Fig. 1 and the
results are
summarized in Fig. 2 (the results are expressed as mean + SEM).
The original photographs for Fig. 1 included brick red areas showing lipid
deposits for Groups II, III and IV. In the black and white photographs of the
accompanying drawings, the lipid deposits appear as grey areas.
Fig. 2 shows the extent of atherosclerosis in the aortas of the four
experimental
groups. The results are expressed as mean + SEM. There was no atherosclerosis
in
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the control group (the small value shown in the figure is just to locate the
control
group). The abbreviations shown in Fig. 2 represent the following:
Chol. = cholesterol
mo. = months
Lig. = lignan.
The numerical values in brackes above the bars in Fig. 2 shows the percentage
increase in the respective groups compared to the group fed 0.25% cholesterol
for 2
months. The numerical values in brackets below the bar shows the percentage
decrease in the group compared to the group fed 0.25% cholesterol for four
months.
In Fig. 2:
* p<0.05, 0.25% cholesterol (2 months) vs. 0.25% cholesterol (4 months), or
0.25% cholesterol (2 months) + 0.25% cholesterol and lignan complex (2
months).
t p<0.25, 0.25% cholesterol (4 months) vs. 0.25% cholesterol (2 months) +
0.25% cholesterol and lignan complex (2 months).
The intimal surface of the aorta of Groups II, III and IV were covered with
atherosclerotic plaques to the extent of 37.76 7.96% and 76.6 9.04% and
52.95 f
10.29%, respectively. As expected 0.25% cholesterol diet for four months
produced a
103% greater extent of atherosclerosis than a similar diet for two months. The
atherosclerotic process progressed with hypercholesterolemic diet. The rabbits
in
group IV, which received 0.25% cholesterol and lignan complex for two months
following a 0.25% cholesterol diet for two months, had a lesser extent of
atherosclerotic plaques compared to Group III that received 0.25% cholesterol
for 4
months (52.95 10.3% vs. 76.59 9.04%). Lignan complex group (Group IV) had
40 /o greater atherosclerosis as compared to Group II, but 31 % less than that
of Group
III. Lignan slowed the progression of atherosclerosis by 31%. These results
suggest
that lignan complex slows the progression of atherosclerosis.
Reg,resssion of Atherosclerosis
This study was conducted to determine if lignan complex can produce
regression of already-developed atherosclerosis. The representative
photographs of
the atherosclerotic changes in the intimal surface of the aortas from the four
groups
are shown in Fig. 3 and the results are summarized in Fig. 4 (the results are
expressed
as mean SEM).
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The original photographs for Fig. 3 included brick red areas showing lipid
deposits for Groups II, III and IV. In the black and white photographs of the
accompanying drawings, the lipid deposits appear as grey areas.
The results shown in Fig. 4 are expressed as mean + SEM. The abbreviations
used in Fig. 4 represent the following:
Chol. = cholesterol
mo. = months
Lig. - lignan
Reg. = regular
T = increase
decrease.
The numerical values in brackets above the bars in Fig. 4 show the percentage
clianges in the respective groups compared to the group fed 0.25% cholesterol
for 2
months. The numerical values in brackets below the bar shows the percentage
change
in the group compared to the group fed 0.25% cholesterol for two months and a
regular diet for four months.
In Fig. 4:
* p<0.05, 0.25% cholesterol (2 months) vs. 0.25% cholesterol (2 months) +
regular diet (4 months), or 0.25% cholesterol (2 months) + regular diet and
Lignan complex (4 months)
#p<0.05, 0.25% cholesterol (2 months) + regular diet (4 months) vs. 0.25%
cholesterol (2 months) + regular diet and lignan complex (4 months).
The atherosclerotic plaques were absent in the group of rabbits (Group I) on a
regular diet. It was found that 37.75 7.96% of the intimal surface of the
aortas of
rabbits fed on 0.25% cholesterol for two months was covered with
atherosclerotic
plaques. The extent of atherosclerosis in group of rabbits on a regular diet
for 4
months following 0.25% cholesterol diet was 57.0% greater than those on 0.25%
cholesterol diet for 2 months (atherosclerosis 59.29 6.71 vs. 37.76
7.96%). This
shows that the atherosclerotic process continued even on regular diet for 4
months
following a 0.25% cholesterol diet. The extent of atherosclerosis in the group
of
rabbits on lignan complex and a regular diet (Group IV) was not significantly
different from those of group I1(40.26 4.42 vs. 37.76 7.96), but was 32.1%
lower
compared to Group III. These results suggest that the lignan complex did not
produce
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regression of already present atherosclerotic lesions, however it completely
prevented
the progression of atherosclerosis that developed after removal of high
cholesterol
diet (normalization of serum lipids).
Conclusion
The results suggest that lignan complex is very effective in slowing the
progression (28-31 %) of atherosclerosis. It did not produce regression of
atherosclerosis. However, it completely prevented the further development of
atherosclerosis that occurs after removal of a high cholesterol diet.
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References
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4. Prasad K. Reduction of serum cholesterol and hypercholesterolemic
atherosclerosis in rabbits by secoisolariciresinol diglucoside isolated from
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