U.S. patent application number 12/504956 was filed with the patent office on 2010-01-21 for compositions and methods for treating hypercholesterolemia using ortanique peel extract.
This patent application is currently assigned to University of the West Indies, a Regional Institution, established by Royal Charter. Invention is credited to Helen N. Asemota, Curtis O. Green.
Application Number | 20100015255 12/504956 |
Document ID | / |
Family ID | 41530496 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100015255 |
Kind Code |
A1 |
Green; Curtis O. ; et
al. |
January 21, 2010 |
COMPOSITIONS AND METHODS FOR TREATING HYPERCHOLESTEROLEMIA USING
ORTANIQUE PEEL EXTRACT
Abstract
The invention relates to compositions and methods for treating
hypercholesterolemia, including methods for increasing HDL
cholesterol levels, using a composition comprising an extract from
the Ortanique peel. The invention also relates to compositions and
methods for treating hypercholesterolemia, including methods for
increasing HDL cholesterol levels, using a composition comprising
substantially similar amounts of tetramethylscutellarein and
nobiletin by weight and/or a composition comprising substantially
similar amounts of tetramethylscutellarein, nobiletin, and
tangeretin by weight.
Inventors: |
Green; Curtis O.; (St.
Catherine, JM) ; Asemota; Helen N.; (Kingston,
JM) |
Correspondence
Address: |
WILMERHALE/BOSTON
60 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
University of the West Indies, a
Regional Institution, established by Royal Charter
St. Augustine
TT
|
Family ID: |
41530496 |
Appl. No.: |
12/504956 |
Filed: |
July 17, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61081916 |
Jul 18, 2008 |
|
|
|
Current U.S.
Class: |
424/736 ;
514/456 |
Current CPC
Class: |
A61P 3/06 20180101; A61K
31/352 20130101 |
Class at
Publication: |
424/736 ;
514/456 |
International
Class: |
A61K 36/752 20060101
A61K036/752; A61K 31/352 20060101 A61K031/352; A61P 3/06 20060101
A61P003/06 |
Claims
1. A method for reducing LDL cholesterol level in a subject
comprising administering to a subject in need thereof an effective
amount of a composition comprising a polar solvent soluble extract
from Ortanique peel, wherein the reduction of LDL cholesterol level
is observed relative to the subject before administration of the
composition.
2. The method of claim 1, wherein the polar solvent is
methanol.
3. The method of claim 1, wherein administration of the composition
to the subject also results in an increase in HDL cholesterol level
in the subject relative to the subject before administration of the
composition.
4. The method of claim 1, wherein the subject is a human.
5. The method of claim 1, wherein the composition is suitable for
oral administration.
6. The method of claim 1, wherein the extract comprises at least
about 60% polymethoxylated flavones by weight.
7. The method of claim 1, wherein the extract comprises six
different polymethoxylated flavones.
8. The method of claim 7, wherein the six polymethoxlylated
flavones are nobiletin, sinensetin, tangeretin,
heptamethoxyflavone, hexamethyl-o-quercetagetin, and
tetramethylscutellarein.
9. The method of claim 7, wherein the extract comprises about
15-21% nobiletin, about 14.5-20.5% tetramethylscutellarein, about
4-10% sinensetin, about 13-19% tangeretin, about 0.3-2.3%
hexamethyl-o-quercetagetin, and about 0.5-3.5% heptamethoxyflavone
by weight.
10. The method of claim 9, wherein the extract comprises about 18%
nobiletin, about 17.5% tetramethylscutellarein, about 7%
sinensetin, about 16% tangeretin, about 1.3%
hexamethyl-o-quercetagetin, and about 2% heptamethoxyflavone by
weight.
11. A method for increasing HDL cholesterol level in a subject
comprising administering to a subject in need thereof an effective
amount of a composition comprising a polar solvent soluble extract
from Ortanique peel, wherein the increase of HDL cholesterol level
is observed relative to the subject before administration of the
composition.
12. The method of claim 11, wherein the polar solvent is
methanol.
13. The method of claim 11, wherein the composition is suitable for
oral administration.
14. The method of claim 11, wherein the extract comprises at least
about 60% polymethoxylated flavones by weight.
15. The method of claim 11, wherein the extract comprises six
different polymethoxylated flavones.
16. The method of claim 15, wherein the six polymethoxlylated
flavones are nobiletin, sinensetin, tangeretin,
heptamethoxyflavone, hexamethyl-o-quercetagetin, and
tetramethylscutellarein.
17. The method of claim 16, wherein the extract comprises about
15-21 % nobiletin, about 14.5-20.5% tetramethylscutellarein, about
4-10% sinensetin, about 13-19% tangeretin, about 0.3-2.3%
hexamethyl-o-quercetagetin, and about 0.5-3.5% heptamethoxyflavone
by weight.
18. The method of claim 17, wherein the extract comprises about 18%
nobiletin, about 17.5% tetramethylscutellarein, about 7%
sinensetin, about 16% tangeretin, about 1.3%
hexamethyl-o-quercetagetin, and about 2% heptamethoxyflavone by
weight.
19. A method for reducing total cholesterol level in a subject
comprising administering to a subject in need thereof an effective
amount of a composition comprising a polar solvent soluble extract
from Ortanique peel, wherein the reduction of total cholesterol
level is observed relative to the subject before administration of
the composition.
20. The method of claim 19, wherein the polar solvent is
methanol.
21. The method of claim 19, wherein the composition is suitable for
oral administration.
22. The method of claim 19, wherein the extract comprises at least
60% polymethoxylated flavones by weight.
23. The method of claim 19, wherein the extract comprises six
different polymethoxylated flavones.
24. The method of claim 23, wherein the six polymethoxlylated
flavones are nobiletin, sinensetin, tangeretin,
heptamethoxyflavone, hexamethyl-o-quercetagetin, and
tetramethylscutellarein.
25. The method of claim 24, wherein the extract comprises about
15-21 % nobiletin, about 14.5-20.5% tetramethylscutellarein, about
4-10% sinensetin, about 13-19% tangeretin, about 0.3-2.3%
hexamethyl-o-quercetagetin, and about 0.5-3.5% heptamethoxyflavone
by weight.
26. The method of claim 25, wherein the extract comprises about 18%
nobiletin, about 17.5% tetramethylscutellarein, about 7%
sinensetin, about 16% tangeretin, about 1.3%
hexamethyl-o-quercetagetin, and about 2% heptamethoxyflavone by
weight.
27. A method for reducing triglyceride levels in a subject
comprising administering to a subject in need thereof an effective
amount of a composition comprising a polar solvent soluble extract
from Ortanique peel, wherein the reduction of triglyceride levels
is observed relative to the subject before administration of the
composition.
28. The method of claim 27, wherein the polar solvent is
methanol.
29. The method of claim 27, wherein the composition is suitable for
oral administration.
30. The method of claim 27, wherein the extract comprises at least
about 60% polymethoxylated flavones by weight.
31. The method of claim 27, wherein the extract comprises six
different polymethoxylated flavones.
32. The method of claim 31, wherein the six polymethoxlylated
flavones are nobiletin, sinensetin, tangeretin,
heptamethoxyflavone, hexamethyl-o-quercetagetin, and
tetramethylscutellarein.
33. The method of claim 32, wherein the extract comprises about
15-21% nobiletin, about 14.5-20.5% tetramethylscutellarein, about
4-10% sinensetin, about 13-19% tangeretin, about 0.3-2.3%
hexamethyl-o-quercetagetin, and about 0.5-3.5% heptamethoxyflavone
by weight.
34. The method of claim 33, wherein the extract comprises about 18%
nobiletin, about 17.5% tetramethylscutellarein, about 7%
sinensetin, about 16% tangeretin, about 1.3%
hexamethyl-o-quercetagetin, and about 2% heptamethoxyflavone by
weight.
35. A method for reducing LDL cholesterol level in a subject
comprising administering to a subject in need thereof an effective
amount of a composition comprising substantially similar amounts of
tetramethylscutellarein and nobiletin by weight, wherein the
reduction of total cholesterol level is observed relative to the
subject before administration of the composition.
36. The method of claim 35, wherein the composition comprises about
16-19% tetramethylscutellarein and about 16-19% nobiletin by
weight.
37. The method of claim 35, wherein the composition comprises about
17.5% tetramethylscutellarein and about 18% nobiletin by
weight.
38. The method of claim 35, wherein administration of the
composition to the subject also results in an increase in HDL
cholesterol level in the subject relative to the subject before
administration of the composition.
39. The method of claim 35, wherein administration of the
composition to the subject also results in a decrease in total
cholesterol level in the subject relative to the subject before
administration of the composition.
40. A composition comprising substantially similar amounts of
tetramethylscutellarein and nobiletin by weight, wherein the
combined amount of tetramethylscutellarein and nobiletin is less
than about 60% of the composition by weight.
41. The composition of claim 40, comprising about 14-21% of each of
tetramethylscutellarein and nobiletin by weight.
42. A composition of claim 40, further comprising sinensetin,
tangeretin, heptamethoxyflavone, and
hexamethyl-o-quercetagetin.
43. The composition of claim 42, comprising about 15-21% nobiletin,
about 14.5-20.5% tetramethylscutellarein, about 4-10% sinensetin,
about 13-19% tangeretin, about 0.3-2.3% hexamethyl-o-quercetagetin,
and about 0.5-3.5% heptamethoxyflavone by weight.
44. The composition of claim 43, comprising about 18% nobiletin,
about 17.5% tetramethylscutellarein, about 7% sinensetin, about 16%
tangeretin, about 1.3% hexamethyl-o-quercetagetin, and about 2%
heptamethoxyflavone by weight.
45. A composition of claim 40, comprising substantially similar
amounts of tetramethylscutellarein, nobiletin and tangeretin by
weight, wherein the combined amount of tetramethylscutellarein,
nobiletin and tangeretin is less than about 60% of the composition
by weight.
46. A pharmaceutical formulation comprising: (a) a pharmaceutically
acceptable carrier or vehicle; and (b) an effective amount of the
composition of any one of claims 41-45.
47. A pharmaceutical formulation comprising: (a) a pharmaceutically
acceptable carrier or vehicle; and (b) an effective amount of a
polar solvent soluble extract from Ortanique peel.
48. The pharmaceutical formulation of claim 47, wherein the extract
is methanol soluble.
Description
1. REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application No. 61/081,916, filed Jul. 18, 2008,
the entire disclosure of which is incorporated by reference herein
in its entirety.
2. FIELD OF THE INVENTION
[0002] The present invention relates generally to treatment and
prevention of hypercholesterolemia.
3. BACKGROUND OF THE INVENTION
[0003] Familial hypercholesterolemia affects approximately 1 in 500
people worldwide, and the elevated serum cholesterol concentrations
associated with it contributes more than 50% to the risk of fatal
or non-fatal coronary heart disease by age 50 in men, and at least
30% in women aged 60 and above. The prevalence of
hypercholesterolemia in Jamaica is estimated at 31 %. It is
estimated that about 37%, or 101 million people in the United
States suffer from hypercholesterolemia.
[0004] Hypercholesterolemia is implicated in many cardiovascular
diseases affecting people worldwide and pose tremendous burden on
the global healthcare system. In the last decade cardiovascular
disease (CVD) has become the leading cause of death across the
Caribbean and accounted for 19% of deaths overall in 1995. The
World Health Organization report also states that coronary heart
disease burden is projected to rise from around 47 million DALYs
(disability-adjusted life years) globally in 1990 to 82 million
DALYs in 2020 (WHO, 2004).
[0005] The major conventional drugs used to treat
hypercholesterolemia include the statins (lovastatin, pravastatin,
simvastatin, etc.) and niacin (nicotinic acid). Although these
drugs are effective in lowering serum cholesterol levels, they
cause several adverse effects. Statins, for example, cause
gastrointestinal upset, muscle aches, and hepatitis. Rarer problems
associated with statins include myopathy (defined as muscle pain
with serum creatine kinase concentrations of more than 1000 U per
liter), rash, peripheral neuropathy, and insomnia. Niacin can cause
adverse effects such as flushing, abdominal pain, vomiting,
headache, or elevated serum aminotransferase levels indicating
liver damage.
[0006] Flavonoids are a ubiquitous family of phytochemicals that
exhibit a broad spectrum of pharmacological properties.
Polymethoxylated flavones are flavonoids that are almost exclusive
to citrus peels and display potent anti-hypercholesterolemic,
anti-cancer, anti-atherosclerotic and anti-diabetic properties.
[0007] Jamaica is the leading producer of citrus in the Caribbean.
A vast majority of the citrus fruits that are harvested are used
for citrus juice production, which results in large quantities of
citrus peel and other citrus byproducts which end up in river beds
and constitute environmental problems.
4. SUMMARY OF THE INVENTION
[0008] The invention is based, in part, on the discovery that the
Ortanique peel extract is effective in treating
hypercholesterolemia without the side effects of existing drugs,
such as statins and niacin, and in part, on the discovery that the
Ortanique peel extract is more effective in treating
hypercholesterolemia compared to peel extracts from other citrus
fruits.
[0009] In one aspect, the invention provides a method for reducing
LDL cholesterol level in a subject comprising administering to a
subject in need thereof an effective amount of a composition
comprising a polar solvent soluble extract from Ortanique peel,
wherein the reduction of LDL cholesterol level is observed relative
to the subject before administration of the composition.
[0010] In another aspect, the invention provides a method for
increasing HDL cholesterol level in a subject comprising
administering to a subject in need thereof an effective amount of a
composition comprising a polar solvent soluble extract from
Ortanique peel, wherein the increase of HDL cholesterol level is
observed relative to the subject before administration of the
composition.
[0011] In one aspect, the invention provides a method for reducing
total cholesterol level in a subject comprising administering to a
subject in need thereof an effective amount of a composition
comprising a polar solvent soluble extract from Ortanique peel,
wherein the reduction of total cholesterol level is observed
relative to the subject before administration of the
composition.
[0012] In another aspect, the invention provides a method for
reducing triglyceride levels in a subject comprising administering
to a subject in need thereof an effective amount of a composition
comprising a polar solvent soluble extract from Ortanique peel,
wherein the reduction of triglyceride levels is observed relative
to the subject before administration of the composition.
[0013] In one or more embodiments, the polar solvent is
methanol.
[0014] In one or more embodiments, administration of the
composition to the subject also results in an increase in HDL
cholesterol level in the subject relative to the subject before
administration of the composition.
[0015] In some embodiments, the subject is a human.
[0016] In one or more embodiments, the composition is suitable for
oral administration.
[0017] In one or more embodiments, the extract comprises at least
about 60% polymethoxylated flavones by weight.
[0018] In one or more embodiments, the extract comprises six
different polymethoxylated flavones.
[0019] In one or more embodiments, the six polymethoxlylated
flavones are nobiletin, sinensetin, tangeretin,
heptamethoxyflavone, hexamethyl-o-quercetagetin, and
tetramethylscutellarein.
[0020] In one or more embodiments, the extract comprises about
15-21 % nobiletin, about 14.5-20.5% tetramethylscutellarein, about
4-10% sinensetin, about 13-19% tangeretin, about 0.3-2.3%
hexamethyl-o-quercetagetin, and about 0.5-3.5% heptamethoxyflavone
by weight.
[0021] In one or more embodiments, the extract comprises about 18%
nobiletin, about 17.5% tetramethylscutellarein, about 7%
sinensetin, about 16% tangeretin, about 1.3%
hexamethyl-o-quercetagetin, and about 2% heptamethoxyflavone by
weight.
[0022] In one aspect, the invention provides a method for reducing
LDL cholesterol level in a subject comprising administering to a
subject in need thereof an effective amount of a composition
comprising substantially similar amounts of tetramethylscutellarein
and nobiletin by weight, wherein the reduction of total cholesterol
level is observed relative to the subject before administration of
the composition.
[0023] In one or more embodiments, the composition comprises about
16-19% tetramethylscutellarein and about 16-19% nobiletin by
weight.
[0024] In one or more embodiments, the composition comprises about
17.5% tetramethylscutellarein and about 18% nobiletin by
weight.
[0025] In one or more embodiments, administration of the
composition to the subject also results in an increase in HDL
cholesterol level in the subject relative to the subject before
administration of the composition.
[0026] In one or more embodiments, administration of the
composition to the subject also results in a decrease in total
cholesterol level in the subject relative to the subject before
administration of the composition.
[0027] In one aspect, the invention provides a composition
comprising substantially similar amounts of tetramethylscutellarein
and nobiletin by weight, wherein the combined amount of
tetramethylscutellarein and nobiletin is less than about 60% of the
composition by weight.
[0028] In one or more embodiments, the composition comprises about
14-21% of each of tetramethylscutellarein and nobiletin by
weight.
[0029] In some embodiments, the composition further comprises
sinensetin, tangeretin, heptamethoxyflavone, and
hexamethyl-o-quercetagetin.
[0030] In one or more embodiments, the composition comprises about
15-21% nobiletin, about 14.5-20.5% tetramethylscutellarein, about
4-10% sinensetin, about 13-19% tangeretin, about 0.3-2.3%
hexamethyl-o-quercetagetin, and about 0.5-3.5% heptamethoxyflavone
by weight.
[0031] In one or more embodiments, the composition comprises about
18% nobiletin, about 17.5% tetramethylscutellarein, about 7%
sinensetin, about 16% tangeretin, about 1.3%
hexamethyl-o-quercetagetin, and about 2% heptamethoxyflavone by
weight.
[0032] In one or more embodiments, the composition comprises
substantially similar amounts of tetramethylscutellarein, nobiletin
and tangeretin by weight, wherein the combined amount of
tetramethylscutellarein, nobiletin and tangeretin is less than
about 60% of the composition by weight.
[0033] In one aspect, the invention provides a pharmaceutical
formulation comprising: (a) a pharmaceutically acceptable carrier
or vehicle; and (b) an effective amount of a composition of the
invention.
[0034] In another aspect, the invention provides a pharmaceutical
formulation comprising: (a) a pharmaceutically acceptable carrier
or vehicle; and (b) an effective amount of a polar solvent soluble
extract from Ortanique peel.
[0035] In one or more embodiments, the extract is methanol
soluble.
5. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The invention is described with reference to the following
figure, which is presented for purposes of illustration only and
which is not intending to be limiting of the invention.
[0037] FIG. 1 is a representation of the effects of Ortanique peel
extract on HMG-CoA Reductase activity in (a) control rats fed a
normal diet; (b) rats fed a diet with Ortanique peel extract; (c)
diet-induced hypercholesterolemic rats fed a normal diet; (d)
diet-induced hypercholesterolemic rats fed a diet with Ortanique
peel extract; and (e) diet-induced hypercholesterolemic rats fed a
diet with niacin.
6. DETAILED DESCRIPTION OF THE INVENTION
6.1 Definitions and Abbreviations
[0038] A "polymethoxylated flavone" or "PMF" is a flavone
substituted with methoxy groups, for example, with 2, 3, 4, 5, 6, 7
or 8 methoxy groups and optionally substituted with one or more
hydroxy groups, for example, 1, 2 or 3 hydroxy groups.
Polymethoxylated flavones are almost exclusively found in citrus
fruits with a specific characteristic distribution for each
variety. Illustrative polymethoxylated flavones include, but are
not limited to: (a) 5,6,7,8,3',4'-hexamethoxyflavone (nobiletin);
(b) 5,6,7,3',4'-pentamethoxyflavone (sinensetin); (c)
5,6,7,8,4'-pentamethoxyflavone (tangeretin); (d)
3,5,6,7,8,3',4'-heptamethoxyflavone (heptamethoxyflavone); (e)
3,5,7,8,3',4'-hexamethoxyflavone (hexamethyl-o-quercetagetin); (f)
5,6,7,4'-tetramethoxyflavone (tetramethylscutellarein); (g)
5-hydroxy-6,7,8,3',4'-pentamethoxyflavone; (h)
5,7,8,3',4'-pentamethoxyflavone; (i)
7-hydroxy-3,5,6,7,3',4'-hexamethoxyflavone; (j)
5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone; (k)
5,6,7,3',4',5'-hexamethoxyflavone; and (l)
5,7,3',4'-tetramethoxyflavone.
[0039] Two parameter values are "substantially similar" if they
have values within .+-.10% of each other. The parameter values can
be expressed as absolute values (e.g., weight) or as relative
values (e.g., percentage).
[0040] As used herein, "about" means .+-.10% of the value that
follows it.
[0041] A "polar solvent" is a protic or aprotic polar solvent.
Aprotic polar solvents include, but are not limited to,
1,4-dioxane, tetrahydrofuran, dichloromethane, acetonitrile,
dimethylformamide, dimethyl sulfoxide. Protic polar solvents
include, but are not limited to, acetic acid, n-butanol,
isopropanol, n-propanol, ethanol, methanol, and formic acid.
[0042] As used herein, "PMF-ORT" is the Orthanique peel extract
described herein.
[0043] As used herein, a solid is "soluble" in a given solvent if
the solid has solubility of at least 0. 1 g/L in the solvent under
desired conditions, such as temperature, pressure, and pH.
[0044] The following abbreviations are used herein and have the
indicated definitions: LDL is low-density lipoprotein, HDL is
high-density lipoprotein, VLDL is very-low-density lipoprotein,
DMSO is dimethyl sulfoxide, PMF is polymethoxylated flavone, MDA is
malondialdehyde, HPLC is High Performance Liquid Chromatography,
HMG-CoA reductase is 3-hydroxy-3-methyl-glutaryl-CoA reductase, and
U is unit.
6.2 Ortanique Citrus Fruit
[0045] The Ortanique fruit, a type of tangor, is a Jamaican hybrid
between tangerine (Citrus reticulata) and sweet orange (Citrus
sinensis). The name "Ortanique" is derived from a combination of
the words "orange," "tangerine," and "unique" and is indigenous to
Jamaica. Ortanique fruit is grown commercially in Jamaica. The
Ortanique grows well in hot, dry weather on shallow bauxite soil at
an elevation of about 2,500 feet. In Jamaica, Ortanique is
cultivated on approximately 1550 acres and over 270,000 boxes of
fresh fruits are produced annually, the majority of which (over
90%) is consumed locally.
[0046] The Ortanique peel contains a high percentage by weight of
polymethoxylated flavones, and further contains a unique
combination of six known polymethoxylated flavones, namely,
nobiletin, sinensetin, tangeretin, heptamethoxyflavone,
hexamethyl-o-quercetagetin, and tetramethylscutellarein. The
Ortanique peel contains a higher overall percentage by weight of
polymethoxylated flavones than either the tangerine peel or the
sweet orange peel. Tangerine peel and sweet orange peel were
previously reported to contain the highest percentage by weight of
polymethoxylated flavones.
6.3 Compositions of Ortanique Peel Extract (PMF-ORT)
[0047] In some embodiments, the Ortanique peel extract contains
over 40%, over 50%, over 60% or over 70% of polymethoxylated
flavones by weight. By weight, the Ortanique peel extract can
contain about 15-21% nobiletin, about 14.5-20.5%
tetramethylscutellarein, about 4-10% sinensetin, about 13-19%
tangeretin, about 0.3-2.3% hexamethyl-o-quercetagetin, and about
0.5-3.5% heptamethoxyflavone.
[0048] In a specific embodiment, the Ortanique peel extract
contains the following approximate weight percentages (w/w) of
polymethoxylated flavones: 18% nobiletin, 17.5%
tetramethylscutellarein, 7% sinensetin, 16% tangeretin, 1.3%
hexamethyl-o-quercetagetin, and 2% heptamethoxyflavone. This
distribution of polymethoxylated flavones is unique to the
Ortanique peel, and is believed to have an important role in the
potency of the Ortanique peel extract (PMF-ORT). Of course, the
percentages of polymethoxylated flavones listed above can vary by
as much as a few percent from the values listed above
[0049] Compositions described herein and extracts from the
Ortanique peel can comprise substantially similar amounts of
tetramethylscutellarein and nobiletin by weight. This amount can
be, for example, 14-21%, 15-20%, 16-19%, or 17-18% for each of
tetramethylscutellarein and nobiletin. In a specific embodiment,
the composition comprises about 18% nobiletin and about 17.5%
tetramethylscutellarein by weight.
[0050] In some embodiments, the composition comprises substantially
similar amounts of tetramethylscutellarein, nobiletin, and
tangeretin by weight. This amount can be 14-21%, 15-20%, 16-19% or
17-18% for each of tetramethylscutellarein, nobiletin, and
tangeretin. In a specific embodiment, the composition comprises
about 18% nobiletin, about 17.5% tetramethylscutellarein, and about
16% tangeretin by weight.
6.4 Method of Making Ortanique Peel Extract (PMF-ORT)
[0051] Because only the peel of the Ortanique is used, PMF-ORT can
be made from Ortanique fruit that has been used for juice
production. The juice production results in large quantities of the
peel and other byproducts which end up being discarded and can even
cause environmental problems.
[0052] Ortanique peel extract (PMF-ORT) can be made by the
following process. First, Ortanique fruits are peeled, and the peel
is dried. The dried peel is then milled to a fine powder.
Extraction of the polymethoxylated flavones from the peel is
performed by soaking the peel in a polar solvent such as methanol.
The extract is then filtered and the solvent is evaporated. The
resultant solids are dried to provide the Ortanique peel extract
(PMF-ORT).
[0053] In some embodiments, the Ortanique peel extract contains
over 60% polymethoxylated flavones by weight. This concentration is
greater than the concentration of polymethoxylated flavones in
tangerine peel extract or sweet orange peel extract. By weight, the
Ortanique peel extract can contain the following approximate
percentages (w/w) of polymethoxylated flavones: 18% nobiletin,
17.5% tetramethylscutellarein, 7% sinensetin, 16% tangeretin, 1.3%
hexamethyl-o-quercetagetin, and 2% heptamethoxyflavone. This
distribution of polymethoxylated flavones is unique to the
Ortanique peel, and is believed to have an important role in the
potency of the Ortanique peel extract (PMF-ORT). Of course, the
percentages of polymethoxylated flavones listed above can vary by
as much as a few percent from the values listed above. Thus, it is
contemplated that the Ortanique peel extract can contain about
15-21% nobiletin, about 14.5-20.5% tetramethylscutellarein, about
4-10% sinensetin, about 13-19% tangeretin, about 0.3-2.3%
hexamethyl-o-quercetagetin, and about 0.5-3.5%
heptamethoxyflavone.
6.5 Methods of using Ortanique Peel Extract
6.5.1 Methods for Treating Hypercholesterolemia
[0054] The Ortanique peel extract is useful for treatment or
prevention of hypercholesterolemia. Accordingly, the invention
provides methods for treating or preventing hypercholesterolemia in
a subject, comprising administering to a subject in need of such
treatment or such prevention an effective amount of Ortanique peel
extract.
[0055] Experiments carried out on hypercholesterolemic rats
indicated that the supplementation of the rats' diets with
Ortanique peel extract resulted in statistically significant
reductions in serum levels of total cholesterol and LDL
cholesterol, as well as triglycerides, when compared to
hypercholesterolemic rats fed normal food without supplementation
and when compared to hypercholesterolemic rats fed food
supplemented with niacin, a standard cholesterol reducing drug.
Unexpectedly, the hypercholesterolemic rats fed Ortanique peel
extract also experienced an elevation in serum HDL cholesterol
levels compared to hypercholesterolemic rats fed normal food
without supplementation and hypercholesterolemic rats fed food
supplemented with niacin. This experimental data indicates that
Ortanique peel extract is useful for reducing serum levels of total
cholesterol in a subject, reducing serum levels of LDL cholesterol
in a subject, reducing serum levels of triglycerides in a subject,
and increasing serum levels of HDL cholesterol in a subject.
[0056] Unexpectedly, the Ortanique peel extract is more effective
in treating hypercholesterolemia compared to peel extracts from
other citrus fruits. For instance, a study performed using
tangerine peel extract in hypercholesterolemic hamster diets
resulted in significantly reduced serum total cholesterol and serum
LDL cholesterol levels, and tended to reduce serum
triacylglycerols, but resulted in no increase in serum HDL levels.
By contrast, supplementation of diet with the Ortanique peel
extract resulted in even greater reduction in total serum
cholesterol, serum LDL cholesterol, and serum triglyceride levels,
and further resulted in an increase in serum HDL levels. (See
Kurowska and Manthey, Hypolipidemic Effects and Absorption of
Citrus Polymethoxylated Flavones in Hamsters with Diet-Induced
Hypercholesterolemia, J. Agric. Food Chem. 52: 2879-2886
(2004)).
6.5.2 Methods for Reducing LDL Cholesterol Levels
[0057] The Ortanique peel extract is useful for reducing LDL
cholesterol levels in a subject in need thereof. Accordingly, the
invention provides methods for reducing LDL cholesterol levels in a
subject, comprising administering to a subject in need of such
reduction an effective amount of Ortanique peel extract.
Experiments carried out on hypercholesterolemic rats indicated that
the supplementation of the rats' diets with Ortanique peel extract
resulted in significant reductions in serum levels of LDL
cholesterol compared to hypercholesterolemic rats fed normal food
without supplementation and hypercholesterolemic rats fed food
supplemented with niacin, a standard cholesterol reducing drug.
This experimental data indicates that Ortanique peel extract is
useful for reducing serum levels of LDL cholesterol in a
subject.
6.5.3 Methods for Reducing Triglyceride Levels
[0058] The Ortanique peel extract is useful for reducing
triglyceride levels in a subject in need thereof. Accordingly, the
invention provides methods for reducing triglyceride levels in a
subject, comprising administering to a subject in need of such
reduction an effective amount of Ortanique peel extract.
Experiments carried out on hypercholesterolemic rats indicated that
the supplementation of the rats' diets with Ortanique peel extract
resulted in significant reductions in serum levels of triglycerides
compared to hypercholesterolemic rats fed normal food without
supplementation and hypercholesterolemic rats fed food supplemented
with niacin, a standard cholesterol reducing drug. This
experimental data indicates that Ortanique peel extract is useful
for reducing serum levels of triglycerides in a subject.
6.5.4 Methods for Increasing HDL Cholesterol Levels
[0059] The Ortanique peel extract is useful for increasing HDL
cholesterol levels in a subject in need thereof. Accordingly, the
invention provides methods for increasing HDL cholesterol levels in
a subject, comprising administering to a subject in need of such
reduction an effective amount of Ortanique peel extract.
Experiments carried out on hypercholesterolemic rats indicated that
the supplementation of the rats' diets with Ortanique peel extract
resulted in significant increases in serum levels of HDL
cholesterol compared to hypercholesterolemic rats fed normal food
without supplementation and hypercholesterolemic rats fed food
supplemented with niacin, a standard cholesterol reducing drug.
This experimental data indicates that Ortanique peel extract is
useful for reducing serum levels of triglycerides in a subject.
[0060] Furthermore, with respect to HDL cholesterol levels, the
Ortanique peel extract yields different, unexpected results when
compared to peel extracts from other citrus fruits. For instance, a
study performed using tangerine peel extract in
hypercholesterolemic hamster diets resulted in no increase in serum
HDL cholesterol levels. By contrast, the Ortanique peel extract
study resulted in a significant increase in serum HDL cholesterol
levels. (See Kurowska and Manthey, Hypolipidemic Effects and
Absorption of Citrus Polymethoxylated Flavones in Hamsters with
Diet-Induced Hypercholesterolemia, J. Agric. Food Chem. 52:
2879-2886 (2004)).
6.5.5 Methods for Administering Ortanique Peel Extract
[0061] The Ortanique peel extract can be administered to subjects
in a variety of ways. In one embodiment, the Ortanique peel extract
is administered orally to a subject, e.g., in the form of a tablet.
The tablet can comprise Ortanique peel extract combined, in
appropriate quantities, with a suitable medium to form a tablet. In
another embodiment, the Ortanique peel extract is administered
orally to a subject in the form of a capsule. The capsule can
comprise Ortanique peel extract encapsulated in a standard
ingestible capsule.
[0062] The Ortanique peel extract can also be incorporated into
various foods and beverages, thus forming a nutraceutical. Examples
of suitable beverages include, but are not limited to, fruit juices
and sodas (e.g., colas). Examples of suitable foods include, but
are not limited to, chocolates, snacks, confectionery, pizza, foods
made from cereal flour (e.g., breads, cakes, crackers, cookies,
biscuits, and noodles), and seasonings and spices used to prepare
meat.
[0063] The Ortanique peel extract can be formulated into
pharmaceutical compositions together with a pharmaceutically
acceptable carrier or vehicle for oral administration in solid or
liquid form, or for intravenous, intramuscular, or subcutaneous
administration.
[0064] Pharmaceutically acceptable carriers for oral administration
include capsules, tablets, pills, powders, troches, and granules.
In the case of solid dosage forms, the pharmaceutically acceptable
carrier can comprise at least one inert diluent such as sucrose,
lactose or starch. Such pharmaceutically acceptable carriers can
also comprise additional substances other than diluents, e.g.,
lubricating agents such as magnesium stearate. In the case of
capsules, tablets, troches and pills, the pharmaceutically
acceptable carrier can also comprise buffering agents. Carriers,
such as tablets, pills and granules, can be prepared with enteric
coatings on the surfaces of the tablets, pills or granules.
Alternatively, the enteric coated compounds can be pressed into
tablets, pills, or granules.
[0065] Pharmaceutically acceptable carriers include liquid dosage
forms for oral administration, e.g., emulsions, solutions,
suspensions, syrups and elixirs containing inert diluents commonly
used in the art, such as water. Besides such inert diluents,
compositions can also include adjuvants, such as wetting agents,
emulsifying and suspending agents, and sweetening, flavoring
agents.
[0066] Pharmaceutical compositions of the invention for parenteral
administration comprise product according to the invention in
combination with one or more pharmaceutically acceptable sterile
isotonic aqueous or non-aqueous solutions, dispersions, suspensions
or emulsions, or sterile powders which may be reconstituted into
sterile injectable solutions or dispersions just prior to use,
which may contain antioxidants, buffers, bacteriostats, solutes
which render the formulation isotonic with the blood of the
intended recipient or suspending or thickening agents.
Pharmaceutically acceptable carriers for intravenous administration
include solutions containing pharmaceutically acceptable salts or
sugars. Pharmaceutically acceptable carriers for intramuscular or
subcutaneous injection include salts, oils, or sugars.
[0067] Furthermore, carriers such as solvents, water, buffers,
alkanols, cyclodextrins and aralkanols can be used. Other
auxiliary, non-toxic agents may be included, for example,
polyethylene glycols or wetting agents.
[0068] The pharmaceutically acceptable carriers and compositions of
the invention are formulated into unit dosage forms for
administration to the patients. The dosage levels of active
ingredient (i.e., Ortanique peel extract) in the unit dosage may be
varied so as to obtain an amount of active ingredient that is
effective to achieve a therapeutic effect in accordance with the
desired method of administration. The selected dosage level
therefore mainly depends upon the nature of the active ingredient,
the route of administration, and the desired duration of treatment.
If desired, the unit dosage can be such that the daily requirement
for the Ortanique peel extract product is in one dose, or divided
among multiple doses for administration, e.g., two, three, or four
times per day.
[0069] This invention is further illustrated by the following
examples, which should not be construed as limiting. Those skilled
in the art will recognize, or be able to ascertain, using no more
than routine experimentation, numerous equivalents to the specific
substances and procedures described herein. Such equivalents are
intended to be encompassed in the scope of the claims that follow
the examples below. A number of references have been cited, the
entire disclosures of which have been incorporated herein in their
entirety.
7. EXAMPLES
7.1 Example 1
Preparation of Ortanique Peel Extract
[0070] Jamaican indigenous Ortanique fruit was harvested. The
fruits were thoroughly washed and peeled. The peel was dried in the
sun for 4-8 days and milled to fine powder using a commercial
blender. The powdered peel was then extracted with reagent grade
methanol (3.times.500 mL) at room temperature for 24 hours with
constant stirring. The mixture was filtered, and the filtrate was
evaporated via rotary evaporation. The resulting solids were first
air-dried, then further dried in an oven at 70.degree. C. to
provide the Ortanique peel extract (PMF-ORT) as a solid.
7.2 Example 2
Determination of the Composition of Ortanique Peel Extract
[0071] The Ortanique peel extract was characterized using
reverse-phase high performance liquid chromatography (HPLC) with UV
detection. Elution was carried out in the gradient mode, using a
ternary mobile phase. The polymethoxylated flavone components of
Ortanique citrus peel extract and their respective quantities were
determined using this method.
[0072] The Ortanique PMF extract was found to contain the following
combination of six different PMFs: nobiletin
(5,6,7,8,3',4'-hexamethoxyflavone), sinensetin
(5,6,7,3',4'-pentamethoxyflavone), tangeretin
(5,6,7,8,4'-pentamethoxyflavone), heptamethoxyflavone
(3,5,6,7,8,3',4'-heptamethoxyflavone), hexamethyl-o-quercetagetin
(3,5,7,8,3',4'-hexamethoxyflavone) and tetramethylscutellarein
(5,6,7,4'-tetramethoxyflavone), in the following proportions by
weight: 18% nobiletin, 17.5% tetramethylscutellarein, 7%
sinensetin, 16% tangeretin, 1.3% hexamethyl-o-quercetagetin, and 2%
heptamethoxyflavone.
7.3 Example 3
Determination of Efficacy of Ortanique Peel Extract
[0073] To determine the effects of Ortanique peel extract on
various cholesterol levels, the following animal model experiments
were carried out.
[0074] Thirty Sprague-Dawley rates were divided into five groups.
Group 1 consisted of healthy rats fed a normal diet. Group 2
consisted of diet-induced hypercholesterolemic rats. Group 3
consisted of healthy rats fed Ortanique peel extract. Group 4
consisted of diet-induced hypercholesterolemic rats fed Ortanique
peel extract. Group 5 consisted of diet-induced
hypercholesterolemic rats fed niacin, a common cholesterol reducing
drug. The normal diet consisted of normal rat chow. Rats were made
hypercholesterolemic by being fed a diet consisting of 1% cholic
acid and 4% cholesterol mixed with the normal rat chow. Rats that
were fed Ortanique peel extract were fed normal rat chow mixed with
Ortanique peel extract that consisted of 1.5% Ortanique peel
extract by weight. Rats were fed their respective diets for
forty-nine (49) days, after which they were sacrificed. Blood serum
and organs were obtained for analyses and several biochemical
parameters were tested.
[0075] Hypercholesterolemic rats fed diets supplemented with
Ortanique peel extract (Group 4) exhibited significantly lower
total serum cholesterol concentration (3.11.+-.0.10 mmol/l;
p<0.05) compared to Group 2 hypercholesterolemic control rats
(5.49.+-.0.49 mmol/l) and Group 5 hypercholesterolemic rats fed
with niacin (4.57.+-.0.44 mmol/l).
[0076] Hypercholesterolemic rats fed diets supplemented with
Ortanique peel extract (Group 4) also exhibited reduced serum LDL
cholesterol concentration (0.97.+-.0.24 mmol/l; p<0.05) compared
to Group 2 hypercholesterolemic control rats (3.12.+-.0.53 mmol/l)
and Group 5 hypercholesterolemic rats fed with niacin (2.36.+-.0.27
mmol/l).
[0077] Hypercholesterolemic rats fed diets supplemented with
Ortanique peel extract (Group 4) further exhibited reduced serum
triglyceride concentration (3.45.+-.0.29 mmol/l, p<0.05)
compared to Group 2 hypercholesterolemic control rats (4.51.+-.0.64
mmol/l) and Group 5 hypercholesterolemic rats fed with niacin
(3.97.+-.0.53 mmol/l).
[0078] Hypercholesterolemic rats fed diets supplemented with
Ortanique peel extract (Group 4) also exhibited increased serum HDL
cholesterol concentration (0.58.+-.0.05 mmol/l; p<0.05) compared
to Group 2 hypercholesterolemic control rats (0.32.+-.0.05 mmol/l)
and Group 5 hypercholesterolemic rats fed with niacin (0.39.+-.0.07
mmol/l).
[0079] In addition, hypercholesterolemic rats fed diets
supplemented with Ortanique peel extract (Group 4) exhibited
reduced hepatic activity of HMG-CoA Reductase, the enzyme that
catalyses the rate limiting step in cholesterol synthesis, compared
to Group 2 hypercholesterolemic control rats and Group 5
hypercholesterolemic rats fed with niacin. Thus, supplementation of
the diets with Ortanique peel extract inhibited the hepatic
activity of the rate limiting enzyme involved in cholesterol
synthesis--HMG-CoA Reductase, as seen in FIG. 1. Different letter
superscripts (above each column) denote significant differences
between values (p<0.05).
7.4 Example 4
Effect of Jamaican Ortanique Peel Polymethoxylated Flavones on
Liver and Kidney Function in Diet-Induced Hypercholesterolemic
Rats
[0080] Malonedialdehyde (MDA) level was used as a measure of lipid
peroxidation. Hypercholesterolemic rats fed diets supplemented with
PMF-ORT were found to have significantly reduced lipid peroxide
levels (650.86.+-.128.28 nmol MDA/g wet weight; p<0.05) in their
liver compared to hypercholesterolemic control (3,381.56.+-.954
nmol MDA/g wet weight) and hypercholesterolemic rats fed niacin
(1,316.07.+-.201.72 nmol MDA/g wet wt). No significant difference
was however observed in the lipid peroxide levels in the kidneys of
hypercholesterolemic rats fed PMF-ORT (1,243.03.+-.421.16 nmol
MDA/g wet weight) when compared to the hypercholesterolemic control
(1,363.70.+-.440.9 nmol MDA/g wet weight) as well as
hypercholesterolemic rats fed niacin (1,127.28.+-.354.65 nmol MDA/g
wet weight), respectively. Liver function analyses supported
results obtained from lipid peroxidation analysis. Reduced
activities in serum aspartate aminotransferase and alkaline
phosphatase were found in hypercholesterolemic rats whose diets
were supplemented with PMF-ORT (69.+-.3.34 and 100.+-.9.20 U/L)
when compared to hypercholesterolemic control (118.+-.9.18 and
132.60.+-.15.71 U/L) and hypercholesterolemic rats fed niacin
(136.40.+-.27.34 and 80.6.+-.4.45 U/L), respectively. There were
however, no significant differences between the levels of serum
creatinine or urea among the groups.
[0081] As indicated in Example 4, supplementation of the diets of
hypercholesterolemic rats with Jamaican Ortanique peel extract
(PMF-ORT) resulted in statistically significant reductions in lipid
peroxide levels in organs as well as reductions in biochemical
indicators of kidney and liver function compared with untreated
hypercholesterolemic rats.
7.5 Example 5
Effect of Jamaican Ortanique Peel Polymethoxylated Flavones on
Activities of Endogenous Antioxidant Enzymes in Diet-Induced
Hypercholesterolemic Rats
[0082] Supplementation of the diets with PMF-ORT resulted in
reductions in the activities of endogenous antioxidant enzymes
(glutathione reductase, glutathione peroxidase, glutathione
transferase and catalase) in the liver, kidney, brain, spleen, and
heart of rats.
[0083] Table 1 shows activity of glutathione reductase in the
liver, kidney, brain, spleen, and heart of rats.
TABLE-US-00001 TABLE 1 Glutathione Reductase Activity (nmol/min/mg
protein)* Group Heart Liver Kidney Spleen Brain Normal control
14.14 .+-. 0.60.sup.a 29.28 .+-. 2.0.sup.a 10.44 .+-. 0.51.sup.a
23.94 .+-. 4.86.sup.b 25.52 .+-. 0.27.sup.a Hyp. Control 20.08 .+-.
0.18.sup.a 41.05 .+-. 3.45.sup.ab 16.71 .+-. 1.52.sup.a 43.62 .+-.
3.67.sup.a 24.05 .+-. 1.69.sup.a Normal Fed PMFs 21.44 .+-.
0.55.sup.a 30.62 .+-. 5.06.sup.a 4.83 .+-. 0.12.sup.a 31.00 .+-.
2.01.sup.ab 16.27 .+-. 3.92.sup.a Hyp. Fed PMFs 20.23 .+-.
0.95.sup.a 18.20 .+-. 1.63.sup.a 7.99 .+-. 0.11.sup.a 16.56 .+-.
3.12.sup.b 11.45 .+-. 2.25.sup.a Hyp. Fed Niacin 12.53 .+-.
0.85.sup.a 61.23 .+-. 5.45.sup.ab 12.36 .+-. 0.10.sup.a 49.48 .+-.
3.32.sup.a 14.95 .+-. 1.67.sup.a *Different letter superscripts (in
each column) denote significant differences between values (p <
0.05).
[0084] Table 2 shows activity of glutathione peroxidase in the
liver, kidney, brain, spleen. and heart of rats.
TABLE-US-00002 TABLE 2 Glutathione Peroxidase Activity (nmol/min/mg
protein)* Group Heart Liver Kidney Spleen Brain Normal control 4.57
.+-. 0.94.sup.a 1.30 .+-. 0.27.sup.a 5.92 .+-. 0.71.sup.a 6.14 .+-.
0.42.sup.a 0.49 .+-. 0.086.sup.a Hyp. Control 9.54 .+-. 1.81.sup.a
2.54 .+-. 0.76.sup.a 5.88 .+-. 1.33.sup.a 25.95 .+-. 5.32.sup.b
1.63 .+-. 0.11.sup.b Normal Fed PMFs 7.36 .+-. 0.81.sup.a 0.81 .+-.
0.027.sup.a 5.31 .+-. 0.75.sup.a 10.85 .+-. 1.22.sup.a 0.72 .+-.
0.02.sup.a Hyp. Fed PMFs 5.61 .+-. 0.22.sup.a 1.18 .+-. 0.33.sup.a
4.86 .+-. 1.05.sup.a 1.18 .+-. 0.33.sup.a 0.54 .+-. 0.018.sup.a
Hyp. Fed Niacin 5.14 .+-. 0.63.sup.a 0.95 .+-. 0.03.sup.a 3.35 .+-.
1.30.sup.a 0.21 .+-. 0.034.sup.a 0.59 .+-. 0.18.sup.a *Different
letter superscripts (in each column) denote significant differences
between values (p < 0.05).
[0085] Table 3 shows activity of glutathione transferase in the
liver, kidney, brain, spleen, and heart of rats.
TABLE-US-00003 TABLE 3 Glutathione Transferase Activity
(nmol/min/mg protein)* Group Heart Liver Kidney Spleen Brain Normal
control 0.21 .+-. 0.012.sup.a 1.46 .+-. 0.55.sup.a 0.089 .+-.
0.006.sup.a 0.077 .+-. 0.016.sup.a 0.25 .+-. 0.03.sup.a Hyp.
Control 0.22 .+-. 0.038.sup.a 3.04 .+-. 0.68.sup.a 0.53 .+-.
0.18.sup.b 0.12 .+-. 0.033.sup.b 0.44 .+-. 0.05.sup.b Normal Fed
PMFs 0.19 .+-. 0.013.sup.a 1.50 .+-. 0.41.sup.a 0.16 .+-.
0.03.sup.a 0.019 .+-. 0.0021.sup.a 0.28 .+-. 0.046.sup.a Hyp. Fed
PMFs 0.18 .+-. 0.011.sup.a 2.49 .+-. 0.17.sup.a 0.14 .+-.
0.029.sup.a 0.067 .+-. 0.018.sup.a 0.26 .+-. 0.011.sup.a Hyp. Fed
Niacin 0.21 .+-. 0.011.sup.a 2.37 .+-. 0.42.sup.a 0.20 .+-.
0.05.sup.b 0.16 .+-. 0.07.sup.b 0.28 .+-. 0.04.sup.a *Different
letter superscripts (in each column) denote significant differences
between values (p < 0.05).
[0086] Table 4 shows activity of catalase in the liver, kidney,
brain, spleen and heart of rats.
TABLE-US-00004 TABLE 4 Catalase Activity (nmol/min/mg protein)*
Group Heart Liver Kidney Spleen Brain Normal control 10.50 .+-.
5.54.sup.a 5.22 .+-. 0.22.sup.a 5.82 .+-. 0.047.sup.a 172.00 .+-.
10.45.sup.a 2.95 .+-. .015.sup.a Hyp. Control 216.98 .+-.
0.14.sup.b 114.21 .+-. 9.21.sup.b 15.45 .+-. 0.84.sup.b 223.87 .+-.
28.56.sup.a 21.49 .+-. 1.40.sup.b Normal Fed PMFs 19.40 .+-.
10.50.sup.a 2.37 .+-. 0.37.sup.a 4.38 .+-. 0.21.sup.a 158.21 .+-.
19.68.sup.a 3.18 .+-. 0.09.sup.a Hyp. Fed PMFs 3.09 .+-. 0.09.sup.a
1.00 .+-. 0.04.sup.a 8.48 .+-. 0.32.sup.a 288.74 .+-. 9.67.sup.a
1.13 .+-. 0.04.sup.a Hyp. Fed Niacin 18.07 .+-. 1.33.sup.a 19.94
.+-. 1.25.sup.a 5.51 .+-. 0.69.sup.a 329.74 .+-. 35.02.sup.a 2.23
.+-. 0.04.sup.a *Different letter superscripts (in each column)
denote significant differences between values (p < 0.05).
[0087] As indicated in Tables 1-4, supplementation of the diets of
hypercholesterolemic with PMF-ORT resulted in statistically
significant reductions activities of antioxidant enzymes in liver,
kidney, brain, spleen, and heart.
* * * * *