Note: Descriptions are shown in the official language in which they were submitted.
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ANTICHOLESTEROLEMIC EDIBLE OIL
This application is based on U.S. Provisional Patent Application Serial No.
60/104,227, filed October 14, 1998, the disclosure of which is incorporated in
its entirety herein by reference.
Field of the Invention
The present invention relates to an edible oil that is useful in improving
blood lipid levels in a human patient, and to methods for making and using the
oil.
Background of the Invention
More than 750,000 people in the United States die from coronary heart
disease and strokes every year. About 1 .25 million people have heart attacks
every year, half of which occur without warning. Coronary heart disease is the
most frequent killer of men and women in the United States. Despite a century
of drug development, ten times as many Americans die of heart attacks as at
the
turn of the century.
According to the American Heart Association, cholesterol levels are the
major predictors of cardiovascular disease. Cholesterol, a soft, waxy
substance
found among the lipids in the blood stream, is an important part of a healthy
body because it is used to form cell membranes, some hormones and other
needed tissues. However, a high level of cholesterol in the blood (hyper-
cholesterolemia) is a major risk factor for coronary heart disease, which
leads to
heart attack.
Cholesterol is insoluble in the blood, and must be transported to and from
the cells by a,_special carrier of lipids and proteins called lipoproteins.
There are
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several kinds of lipoproteins, the most important of which are low-density
lipoprotein (LDL) and high-density lipoprotein (HDL1.
Low-density lipoprotein is the major cholesterol carrier in the blood. Excess
LDL cholesterol circulating in the blood can slowly build up within the walls
of
the arteries feeding the heart and brain. Together with other substances it
can
form plaque, a thick, hard deposit that can clog those arteries. This
condition is
known as atherosclerosis. The formation of a clot (or thrombus) in the region
of
this plaque can block the flow of blood to part of the heart muscle and cause
a
heart attack. If a clot blocks the flow of blood to part of the brain, the
result is
a stroke. A high level of LDL cholesterol reflects an increased risk of heart
disease. Thus, LDL cholesterol is often called "bad cholesterol."
High density lipoprotein ("HDL") carries about one-third to one-fourth of
blood cholesterol. It is believed that HDL carries cholesterol away from the
arteries and back to the liver, from which it is ultimately passed from the
body.
Some experts believe HDL removes excess cholesterol from atherosclerotic
plaques and thus slows their growth. HDL is known as "good cholesterol"
because a high level of HDL seems to protect against heart attack. The
opposite
is also true: a low HDL level indicates a greater risk.
Cholesterol comes from .two sources. It is produced in the body, mostly
in the liver (about 1,000 milligrams a day), and is also found in foods that
come
from animals, such as meat, poultry, fish, seafood and dairy products. Foods
from plants (fruits, vegetables, grains, nuts and seeds) do not contain
cholesterol.
Saturated fatty acids are the chief culprit in raising blood cholesterol,
which
increases the risk of heart disease. But dietary cholesterol also plays a
part. The
average American man consumes about 360 milligrams of cholesterol a day; the
average American woman, between 220 and 260 milligrams.
One hundred (100) million adults have blood cholesterol levels of 200
milligrams per deciliter (mg/dl) or higher, and nearly 40 million Americans
have
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levels of 240 mg/dl or above. It is estimated that there are 53 million
Americans
with LDL levels that require treatment, but that less than one-third of those
in
need are receiving the necessary treatment. Furthermore, most patients who are
treated fail to attain treatment goals. The yearly cost of treatment is
estimated
at more than S 100 billion, yet coronary heart disease still remains the No. 1
killer
of Americans.
Thus, the risk of having a heart attack or stroke is strongly predicted by the
amounts of low-density lipoprotein (LDL1, high-density lipoprotein (HDL), and
triglycerides in the blood.
Cholesterol and triglyceride levels can be reduced through medical
intervention and/or dietary modification, such as reduction of the dietary
intake
of cholesterol and saturated fats. However, some dietary modifications have
given rise to new problems. For example, in recent years the substitution of
margarine for butter has been promoted. Butter is high in cholesterol and
saturated fats. Stick margarine, on the other hand, has a semi-solid
consistency
based on their content of hydrogenated oils. The hydrogenation process,
however, forms trans fats. Clinical studies have demonstrated that trans fats
are
atherogenic, causing two to three times the cardiovascular risk of the
naturally
saturated fats which give butter its stability. The health advantage of
margarine
over butter is now suspect in that margarine, particularly stick margarine,
can
contain 20% to 30% of trans fats. The American Heart Association now
recommends soft margarine. Such margarine, so called trans-free margarine,
which is formulated from either completely hydrogenated palm oil or palm oil
fractions, has been introduced recently. This margarine, while free of trans
fats,
contain increased levels of saturated fats, the second most dangerous
component of margarine.
Other compounds have been reported to reduce cholesterol levels in
humans. For example, plant sterols, particularly beta-sitosterol, have been
reported to have anticholesterolemic effects, and are believed to inhibit
cholesterol absorption in the small intestine. Plant sterols are thought to
displace
cholesterol in bile salt micelles. Approximately half of the dietary
cholesterol
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ingested is absorbed whereas less than 5% of beta-sitosterol is absorbed. When
the plant sterols displace cholesterol of the bile salt micelles, the
cholesterol is
fecally excreted.
Plant sterols exist naturally in saturated and unsaturated forms, as free
alcohols and as esters. The unsaturated forms dominate. It is known that
natural sitosterols may be converted to sitostanols by hydrogenation, and it
has
been reported that stanols are more effective per unit weight than sterols in
blocking cholesterol absorption and that stanols are not absorbed. Further,
the
amount of beta-sitosterol absorbed appears to be relatively constant even when
doses administered vary by an order of magnitude. Both sterols and stanols
have
been used as relative markers of cholesterol absorption because of their
unabsorbability. However, it seems clear that while sitostanol is completely
unabsorbed, some sitosterol is.
Further, the addition of sitostanol to the diet reduces not only cholesterol
absorption but also sitosterol and vitamin absorption. Some have characterized
this as an advantage, but the fact that sitostanols block the normal
absorption
of micronutrients may be problematic.
The Lancet 1995; 345: 1529-1532, reported on the use of beta-sitosterol
(20 milligrams per day) for the treatment of benign prostatic hyperplasia
(BPH).
This condition is a slow enlargement of the fibromuscular and epithelial
structures within the prostate gland, eventually leading to obstructive
urinary
symptoms which are experienced to some extent by most men over the age of
50 years. Using sitostanols alone as an anti-cholesterolemic thus may increase
the risk of BPH.
Other compounds that have been studied in connection with the treatment
and prevention of diseases including arteriosclerosis and high cholesterol
levels
include tocotrienols, which are natural forms of vitamin E found in wheat
germ,
rice bran, oats and palm.
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In vitro, the concentration-dependent impact of tocotrienols on cholesterol
can be demonstrated to involve post-transcriptional down regulation of 3-
hydoxy-
3-methyl-glutaryl coenzyme A reductase (HMGCoA reductase) activity. This is
the enzyme targeted by statins, the anti-cholesterolemic drug with annual
sales
of eight (8) billion dollars in the U. S. alone. Statins act directly,
blocking
HMGCoA reductase. However, statins also sometimes cause liver dysfunction.
Unfortunately, many patients taking statins or tocotrienols respond to the
decreased rate of cholesterol synthesis by a compensatory increase in the rate
at which dietary cholesterol is absorbed from food. A recent study reports
that
80% of patients taking statins as a monotherapy failed to reach treatment
goals.
With respect to statins, increasing the dosage to the levels frequently
required
to overcome compensatory increase in cholesterol absorption, produces an 1 1-
fold increase in the incidence of liver complications as noted above. Because
of
the risk of liver complications, statins must be taken under a doctor's
supervision. Similarly, while tocotrienols have shown promise in vitro, the
results
of clinical trials have been equivocal. Qureshi, Am. J Clin. Nutr. 53: Suppl.
4:
1021 S-1026S, ( 1991 Apr.) reported significant improvements in lipid
parameters
amongst "responders" in a short study, but three subsequent studies of free
living patients supplemented with the same material (palm-derived tocotrienol-
rich fraction, i. e., palm-derived TRF) failed to confirm his results. See
Antila, et
a/, Helsinki Antioxidant Symposium, 1991 Wahlquist, M., et al, Nutrition
Research 12: Suppl. 1 : S181-S201 (19921; Tomeo, A., etal., Lipids 30: 1179-
1 183, 1995] In response, Qureshi has suggested Qureshi, A., et al, Lipids 30
(121: 1 171-1 177, (1995) that d-tocopherol inhibits the anticholesterolemic
effect
of tocotrienols. The results of clinical trials with oils according to the
present
invention do not support this conclusion, but rather show that synergy with
other
non-saponifiable components is required to effect blood lipid modulation.
A margarine recently introduced in Finland, Benecol, that contains
hydrogenated plant sterols extracted from pulp and paper waste, has been found
to achieve a 10-15% reduction in cholesterol levels in patients substituting
Benecol margarine for standard margarine in their diets. This reduction
corresponds to a twenty to thirty percent decrease in cardiovascular risk.
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However, Benecol suffers from the disadvantage that the plant sterol extracts
require regulatory approval in the United States and other countries as a new
food additive.
Toxic forms of oxygen have been associated with many chronic,
debilitating diseases. These include cardiovascular, neoplastic, arthritic,
age
related macular degenerative and progeria, among others. As tissue levels of
these toxic forms of oxygen rise, tissue levels of protective antioxidants,
such
as antioxidants of the vitamin E family, decline. These risk factors have been
confirmed in the case of cardiovascular disease by Gey, who showed that as
blood vitamin E values decrease in a population, the incidence of ischemic
heart
disease rises. To assess the blood levels of peroxides, many researchers have
measured adducts of thiobarbituric acid (a.k.a TEARS, thiobarbituric acid
reactive
substances, also called malonaldehyde modified material), or peroxides.
Holvoet,
Collen and van de Werf recently documented the relation of malonaldehyde-
modified LDL as a marker of acute coronary syndromes. These scientists
showed that malonaldehyde (TBARS) type pollution in the blood indicates
endothelial injury and plaque instability, and more accurately indicates acute
coronary syndromes than other commonly used indices, such as troponin I. In
an intervention study of patients who had had at least one stroke who were
supplemented daily with Redeem, their serum levels of TBARS material
decreased significantly from pre-study values. See: Tomeo, A. C., e1 al.
Antioxidant effects of tocotrienols in patients with hyperlipidemia and
carotid
stenosis. Lipids 30: 1179-1183, 1995; Watkins, T. R. et al.
Hypocholesterolemic and antioxidant effects of rice bran oil non-saponifiables
in
hypercholesterolemic subjects. Environ. Nutr. Interactions, 3: (2) 1-8, 19991.
Further, their serum vitamin E levels nearly doubled over pre-study values.
This
same group of researchers of the Jordan Heart Research Foundation had
previously documented the same relation in the laboratory rat model. See:
Watkins, T. R., et al y-tocotrienol as a hypocholesterolemic and antioxidant
agent
in pats fed atherogenic diets. Lipids, 28: 1 1 13-1 1 18, 19931.
Accordingly, there is a need for an edible oil that is traps-free, low in
saturated fats and suitable for use in the manufacture of margarine.
Preferably,
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the edible oil does not require additives that must be chemically processed
(e. g.,
hydrogenatedl.
There is also a need for an edible oil product that is a safe, effective
alternative to known oil products and that can be made available over-the-
counter (OTC) or incorporated into staple foods.
Furthermore, there is a need for a new intervention strategy against
cardiovascular disease, one which recognizes the difficulty patients have in
changing life-long bad eating habits and which, unlike cardiovascular drugs,
is
safe enough to be taken without direct medical supervision.
Summary of the Preferred Embodiments
In accordance with one aspect of the present invention, there is provided
an edible oil that reduces the synthesis and absorption of cholesterol by the
human patient and promotes the excretion of cholesterol from the human
patient.
Preferably, the edible oil is substantially free of trans fatty acids.
In preferred embodiments, the inventive oils are vegetable oil or mixtures
of vegetable oils. Very preferably, the inventive oils are refined rice bran
oils or
mixtures of rice bran and palm oils.
In accordance with still other aspects of the present invention, there are
provided food products that include any of the foregoing oils.
According to additional aspects of the present invention, there are provided
methods of reducing total cholesterol and LDL and raising HDL in a human
patient comprising the step of administering to said patient an effective
amount
of any of the foregoing oils.
According to still another aspect of the present invention, there is provided
a method of making an anticholesterolemic edible oil. The method includes the
steps of providing an edible oil, and adjusting the content of tocopherols,
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tocotrienols, free sterols,. steryl esters and cycloartenols of the edible oil
such
that the oil, when consumed at a pre-selected dosage and in a pre-selected
dosage form, provides on a daily basis about 25 to 750 mg of tocopherols,
tocotrienols or combinations thereof, about 5 to about 500 mg of steryl
esters,
and about 5 to about 500 mg of cycloartenols.
In a preferred embodiment, a crude vegetable oil ("Oil A"1, in particular a
crude rice bran oil, is dewaxed and degummed, and held under vacuum at
elevated temperature. Free fatty acids are then removed from Oil A at mild pH
using an alkaline hydrous sodium silicate and small quantities of potassium
hydroxide so that the free fatty acids are converted to soap (saponified) at
conditions which minimize the loss of esters of sterols and cycloartenols to
the
soap stock. Next, a tocotrienol-rich distillate, preferably a rice bran or
palm oil
deodorizer distillate, is substantially saponified, preferably in isopropanol,
and the
non-saponifiable fraction is extracted, preferably with hexane and water to
yield
an extract 1"Oil B"). Finally, appropriate portions of Oil A and Oil B are
mixed to
produce a product having the desired concentration of tocopherols,
tocotrienols,
free sterols, steryl esters and cycloartenols.
Other objects, features and advantages of the present invention will
become apparent to those skilled in the art from the following detailed
description. It is to be understood, however, that the detailed description
and
specific examples, while indicating preferred embodiments of the present
invention, are given by way of illustration and not limitation. Many changes
and
modifications within the scope of the present invention may be made without
departing from the spirit thereof, and the invention includes all such
modifications.
Detailed Description of the Preferred Embodiments
Cholesterol levels in the human body are regulated by three concurrent
mechanisms, namely synthesis, absorption and excretion. Most known anti-
cholesterolemic compounds and compositions target only one of these
mechanisms, and thus must have a relatively large impact on the targeted
mechanism in order to function.
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Edible oils according to the present invention reduce the synthesis and
absorption of cholesterol while increasing the excretion of cholesterol. All
three
mechanisms are affected simultaneously, resulting in a gentle, balanced
improvement in LDL, HDL and triglyceride levels.
Reductions and increases in cholesterol synthesis, absorption and excretion
in human patients are determined by comparison with the same quantities
measured in human patients before and after administration of edible oils
according to the invention.
The edible oils, according to the invention, can be characterized as
"functional foods," as opposed to drugs or nutraceuticals. Functional foods
have
been defined by the European Union as "ordinary foods processed or modified in
such a way that they have scientifically documented health promoting effects
and can be marketed with a health claim." In Japan, "functional foods" are
defined as ordinary foods that are derived only from naturally occurring
ingredients and that are consumed as part of the diet and not in supplement
form
(i.e., not as tablets or capsulesl.
Preferably, the edible oil according to the invention includes at least one
compound that reduces cholesterol synthesis in a human patient, such as at
least
one tocotrienol. Specific examples of such compounds include a-tocotrienol, p-
tocotrienol, y-tocotrienol and b-tocotrienol.
The edible oil also preferably includes at least one compound that reduces
cholesterol absorption in a human patient, for example, at least one free
sterol
or steryl ester. Specific compounds useful according to the invention include
ferulic and fatty acid esters of campesterol, (3-sitosterol and other sterols
and
stanols.
Preferably included in the inventive oil is at least one compound that
promotes cholesterol excretion in a human patient. Such compounds include
cycloartenol esters of ferulic acid (C~oH,o04), variously referred to as 3-(4-
hydroxy-3-methoxyphenyl)-2-propanoic acid, 4hydroxy-3-methoxy-cinnarnic acid
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or 3-methoxy-4-hydroxy-cinnamic acid. Specific examples of these compounds
include ferulic esters of 24-methylene-cylcoartenol and cycloartenol.
The edible oil, according to the invention, preferably attenuates the
accumulation in and blood level of peroxidized lipid--and other substrates,
such
as protein, carbohydrate and nucleic acid--called peroxides, but also called
on
analysis TBARS (thiobarbituric acid reactive substances) and malonaldehyde-
like
compounds (i.e., TBA), known cardiovascular risk factors.
Very preferably, the edible oil provides at least one compound which limits
the formation and accumulation of TEARS, and similar peroxidation adducts, in
a human patient.
According to the invention, the edible oil also provides vitamin-E like
activity, whether derived from the tocotrienol or tocopherol family, which
confers
antioxidant activity to tissues, such as the blood, which can be measured as
tocotrienol or tocopherol.
And, very preferably, at least one compound in the oil is derived from the
tocotrienol or tocopherol family (as described in detail herein), which
results in
elevated serum tocopherol or tocotrienol levels in the blood of a human
patient
at risk of cardiovascular disease.
The tocopherols, tocotrienols, sterols, steryl esters and cycloartenols
employed according to the invention are preferably derived from natural
sources,
but can also be synthetically produced, if desired. In particular, one or more
of
the ingredients can be synthetic or can be derived from a source other than
the
vegetable oil base.
Very preferably, the inventive oil is substantially free of traps fatty acids.
"Substantially free" as used herein means less than about 2% (weight/weight).
Optimally, the inventive oil includes no traps fatty acids.
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It has been discovered in clinical trials that the optimal ratio of (i1
tocotrienols and/or tocopherols to (iil free sterols and/or steryl esters to
(iii)
cycloartenols ranges from about 1 :0.5:0.05 to 1 :5:0.5, and very preferably
is
about 1:1:0.05. Thus, in a preferred embodiment, the ratio of the foregoing
ingredients in the inventive oil falls within this preferred range, and
optimally is
about 1:1:0.05.
The amount of tocopherols and tocotrienols administered to a human
patient preferably ranges from about 50 to 500 mg per day, or alternatively,
about 10 to 200 mg/dosage unit. The amount of sterols and steryl esters
likewise preferably ranges from about 50 to 500 mg/day. The amount of
cycloartenols preferably ranges from about 2.5 to 25 mg per day. In a
preferred
embodiment, a human patient is administered about 400 milligrams of
tocopherols and tocotrienols, 400 milligrams of sterols and steryl esters and
20
milligrams of cycloartenols. This corresponds to the preferred ratio of
ingredients
of about 1 :1:0.05. The percentages of the various ingredients in the
inventive
oil can vary within a wide range, so long as the proportions of the
ingredients are
within the stated ranges and the patient consumes a total amount of each
ingredient within the stated ranges each day.
In a preferred embodiment, the edible oil according to the invention is a
vegetable oil, in particular a refined rice bran oil or a mixture of refined
rice bran
oil and palm oil.
Very preferably, the edible oil is a refined rice bran oil. Crude rice bran
oil
contains the highest percentage of non-saponifiables of any commercial
vegetable oil. Total non-saponifiables often exceed 4% (by weight),
approximately four times more than the oils currently used in margarine
manufacture.
Crude rice bran oil is preferably refined for use according to the invention.
When the oilbearing bran is separated from rice, a particularly active lipase
enzyme is activated which causes a very rapid increase in free fatty acids.
Even
when the rice is stabilized by heat or chemicals shortly after milling, free
fatty
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acid (FFA) levels of 5 to 10% are common; industrial rice bran oils may have
FFA
levels as high as 30%. There is furthermore, a seasonal variation in FFA
levels.
Wax levels are also high and generally related to the temperature at which the
bran is extracted with solvent. Rice bran oil thus is among the most
challenging
of oils to refine. Industrially, rice bran oil is processed by chemical
refining.
Palm oil, another useful source of tocotrienols, also has high FFA levels
because palm fruit releases a lipase enzyme when bruised. FFA levels of palm
oil range from 2% to 5%. Most commercial production of palm oil uses physical
refining processes. However, such refining methods produce low-grade
distillates of low tocopherol/tocotrienol concentration, typically 3,000 -
5,000
ppm. Deodorizer distillates obtained from chemical refineries are of higher
concentration, typically 1 to 3% tocopherol/tocotrienol. However, during the
chemical refining process, the ferulic and fatty acid esters of sterols and
cycloartenols are ionized and lost to the soap stock. Free sterols, and
triterpene
alcohols (cycloartenols) are soluble to various extents in both polar and non-
polar
solvents, whereas non-polar solvents are selective with respect to their
esters
of fatty acids and ferulic acids. These molecules are structurally similar to
cholesterol. In the ester form they more readily displace cholesterol from the
micelles in the digestive tract, but are themselves not absorbed, or if
absorbed,
quickly excreted.
Thus, while a tocotrienol-rich fraction can be recovered from chemically
refined rice deodorizer distillate, it is substantially depleted of these
useful esters.
Known processes for production of tocopherol and tocotrienol-rich fractions
from deodorizer distillates include processes such as ion exchange,
saponification
and extraction from hard soap, methyl esterification, esterification of free
fatty
acids and molecular distillation, and desterolization. In each of these
processes,
the method of separation of tocotrienol-rich concentrates purposely or
incidentally removes the natural steryl esters of ferulates and cycloartenols.
As crude rice bran oil ages, it has been observed that the free sterols
become esterified with free fatty acids. It has been discovered that this
process
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can be accelerated by aging the oil at elevated temperatures under vacuum,
under mild conditions such that the free sterols and free cycloartenols are
esterified and the tocopherols are not. Rogers, et al, J. Am. Oil Chem.
Assoc., 70: No. 3, 1993, analyzed five commercially available rice bran oils
from
different manufacturers. The content of ferulic esters of cycloartenols and
plant
sterols (quantitated as oryzanols) ranged between 1 15 ppm and 787 ppm, with
an average of 400 ppm. In the same oils, tocotrienol content ranged between
72 ppm and 1 157 ppm, with an average of 500 ppm. More than 95% of the
oryzanols and 60% of the tocotrienols are lost in conventional refining
processes.
To obtain effective amounts of the anticholesterolemic active principles, a
patient could be required to consume more than a kilogram of oil per day.
The invention thus meets the need for a new process in which the optimal
proportions of tocotrienols, steryl esters and cycloartenols are retained in
the
product and the sterols present are substantially in the form of steryl esters
whose increased solubility in lipids underlies the efficacy of the compound in
decreasing the absorption of dietary cholesterol.
In general, the inventive method begins with crude rice bran oil ("Oil A"),
which is dewaxed and degummed, and free sterols and triterpene alcohols
esterified with free fatty acids. The remaining free fatty acids are then
removed
under conditions that preserve the esterified state of the sterols and
cycloartenols, by distillation or at mild pH using an alkaline hydrous sodium
silicate and small quantities of potassium hydroxide so that the free fatty
acids
are converted to soap (saponified) at conditions which minimize the loss of
esters
of sterols and cycloartenols to the soap stock. Next, a tocotrienol-rich
deodorizer
distillate, preferably of rice bran or palm oil, is substantially saponified
in
isopropanol and the non-saponifiable fraction is extracted with hexane and
water
to produce and extract ("Oil B"). Finally, appropriate portions of Oil A and
Oil B
are mixed to form a product having the required concentration of tocopherols,
tocotrienols, free sterols, steryl esters and cycloartenols.
In contrast to products such as Benecol, which include synthetic
ingredients requiring regulatory approval, preferred embodiments of the edible
oils
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according to the present invention, such as refined rice bran oil, raise no
regulatory issues since they naturally contain suitable steryl and stanyl
esters,
which, unlike Benecol, do not require hydrogenation and chemical processing
with attendant risks of trans fatty acid formation.
The edible oils according to the invention can be incorporated into a variety
of food products, including, without limitation, butter, margarine, ice cream
and
mayonnaise- chocolate products; liquid such as soybean milk and rice milk- and
water-based drinks such as wines and mineral waters. The inventive oils are
also suitable for encapsulation in gelatin shells to form soft gels.
Regardless of
the particular form in which the inventive oil is prepared, the daily dosage
of the
various ingredients to a human patient should fall within the ranges set forth
above. Depending on the concentration of the inventive oil in the given form,
the
total amount of the food product per serving, or encapsulated oil, etc., will
also
vary. Highly concentrated forms, such as soft gels, will be administered in
lower
total amounts than diluted forms, such as drinks.
The invention is further illustrated by the following non-limiting examples.
Example 1 discloses a method for the preparation of an embodiment of the
inventive oil. Examples 2, 3, 4 and 5 compare the results of human studies in
which patients received the inventive oil or other preparations containing the
elements of the inventive oils individually or in proportions which differ
substantially from those of the invention. Example 2 compares the
administration of palm-derived tocotrienols with the inventive oil. Example 3
compares rice tocotrienols processed by another method with the inventive oil.
Example 4 compares a conventionally processed rice oil with the inventive oil.
Example 5 compares the performance of the inventive oil against two
commercial margarines incorporating high levels of sterols and steryl esters.
In each case, it is demonstrated that the desired changes in blood lipid
values are achieved only when the proportions of the active components are
according to the invention. It is shown that the inventive oil is markedly
superior, decreasing LDL levels and uniquely superior in elevating HDL and
diminishing triglyceride values.
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Example 1
One hundred ( 100) grams of rice oil ("Oil A") are analyzed, dewaxed and
degummed, and the acid value of the oil is determined by AOAC methods. The
oil contains 5% sterols, stanols, and cycloartenols as:
Campesterol 15
Sitosterol 10
Campestanol 1 .4
Stigmasterol 1 .5
Sitostanol 1 .5
Cycloartenol 30
24-methylene-
cycloartenol 40
and, further, contains 1120 ppm of tocopherols and tocotrienols, 58% as
gamma-tocotrienol. Oil A is held overnight at moderate vacuum at 125 °
C, so
that water generated during esterfication is removed and the reaction driven
to
the right, accelerating the natural aging process in which fatty acid esters
of
sterols and cycloartenols are formed. Oil A is then cooled to 50° C.
A mild, caustic agent is prepared by combining potassium hydroxide and
alkaline hydrous sodium silicate (Britesorb~ NC, commercially available from
PQ
Corporation, Valley Forge, PA) in a slurry, in the ratio of 1 part potassium
hydroxide to 4 parts Britesorb° to 6 parts of water. An amount of
slurry equal
to 5% stoichiometric excess of the acid value previously measured is added to
the cooled Oil A above, and the mixture is stirred at 60° C for one
hour, after
which the temperature is increased to 80° C and the mixture filtered.
The refined oil is washed and dried, leaving a neutral oil rich in sterol
esters
and cycloartenol esters of ferulic acids, but substantially free of free fatty
acids
and free sterols.
Next, a deodorizer distillate from chemical refining of rice bran oil is
obtained and analyzed. The distillate is found to contain the following
ingredients:
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2.0% tocopherols and tocotrienols, including:
a-tocopherol 0.5
y-tocopherol 0.4%
a-tocotrienol 0.1
y-tocotrienol 1.0%
8.0 % sterols, total
2.0% steryl esters
75.2% glycerides, total, ncluding:
i
free fatty acids 43.5%
monoglycerides 6.7%
diglycerides 8.6%
triglycerides 16.4%
The distribution of fatty acids is as follows:
C 12:0 0.1 % (by weight)
C 14:0 1 .0
C 16:0 27.5
C16:1 0.3
C 18:0 2.0
C 18:1 39.0
C 18:2 27.0
C 18.3 0.8
C20:0 0.8
C20:1 0.6
C22:0 0.2
C24:0 0.4
Next, 50 grams of the distillate is mixed with 5 volumes of isopropanol and
the saponification calculated from the analysis above using the AOAC method.
Then an amount of 80% potassium hydroxide at 150% of the calculated
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stoichiometric weight required to saponify all of the glyceride components is
determined, and is added slowly to the reaction mixture. The reaction mixture
is held in a water bath for 30 minutes at 60° C and allowed to, cool,
then
neutralized and extracted with 10 volumes of hexane and 20 volumes of water
over night. The hexane phase is separated, washed and dried, and the semi-
solid
resultant phase ("Oil B") is combined with the neutral oil obtained above. The
resultant refined oil is enriched in tocopherols and tocotrienols, and esters
of
cycloartenols and sterols, but depleted of free fatty acids, mono-, di- and
triglycerides and free sterols.
Optionally, the semi-solid resultant phase above can be de-sterolized by
precipitating free sterols from methanol in 4° C, and further
concentrated by
distillation, prior to blending.
The yield of tocopherols and tocotrienols ranges between 45% and 75%,
depending upon the degree of saponification of the reaction mixture. The ratio
of tocotrienols and tocopherols to cycloartenols and ferulic and sterol esters
can
also be adjusted by the degree of saponification.
The ratio of Oil A and Oil B combined can be varied to produce a re-
proportioned oil ranging in concentration of tocopherols and tocotrienols
between
about 0.5% and 25% (weight/weight, based on the total weight of the oil). At
higher concentrations, the oil is suitable for encapsulation into soft gels as
a
nutraceutical or therapeutic. At lower concentrations, the oil can be
incorporated
directly into food products, such as margarine or mayonnaise. In each case,
the
desired concentration is that sufficient to provide between 50 - 500 mg/day
(or,
alternatively, about 20 - 200 mg/serving) of tocotrienols/tocopherols to a
patient
consuming the product.
Example 2
A test group was administered a palm oil-derived tocotrienol-rich fraction
(TRF), processed in a manner which depletes sterols, steryl esters and
cycloartenols, in an amount of 160 - 240 mg three times per day for one year.
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Tomeo, A., et al Lipids 30: 1179-1183, 1995. The dosage was increased to
240 mg of tocotrienols three times per day for two additional years. Kooyenga,
D., et al. Asia Pacific J Clin. Nutr. 6: 72-75, 1996. No change in total
cholesterol, LDL or HDL cholesterol, or triglyceride levels was observed for
two
years. The test group was then administered 2.4 grams per day of an oil
according to the invention containing 200 mg of tocotrienols three times per
day
for one year. The blood lipids improved: a 20% decrease in LDL cholesterol, a
20% increase in HDL cholesterol and a 23% decrease in triglycerides was
observed. See Table 1.
Table 1. Changes in blood lipids during supplementation with palm-derived
tocotrienol or the invention, rice bran tocotrienols with non-saponifiables.
Data
in mg/dl. N = 50 subjects.
Lipid Baseline (start)Year 1 Year 3 Year 4
----------Palm The invention
tocotrienol
period----------
Cholesterol, 239 239 239 206*
total
LDL cholesterol165 165 165 132"
HDL cholesterol40 40 40 48~
Triglycerides 21 1 21 1 21 1 162 ~'
Data of years 3 and 4 differ significantly, p < 0.01 .
Example 3
Ten members of the study of Example 2 were switched to NuTriene°,
a
tocotrienol-rich concentrate from rice oil manufactured by Eastman Chemical
Company, processed in a manner that depletes the oil of steryl esters and
cycloartenols. The lowering of LDL cholesterol observed in Example 2 was
maintained, but 25% of the increase in HDL cholesterol levels and 50% of the
decrease in triglycerides was lost. See Table 2.
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Table 2. Changes in blood lipids in subjects supplemented with the invention,
rice bran tocotrienols and non-saponifiables, or NuTriene°, between
years 4 and
5. Data as mg/dl. N = 10.
Lipid Year 4 (endl Vear 5 (endl
Cholesterol, total 206 205
LDL cholesterol 132 127
HDL cholesterol 48 46
Triglycerides 162 187
Example 4
Patients were administered a margarine prepared from a physically refined
rice oil rich in sterols, steryl esters and cycloartenois (1 .5 grams total
per day)
but poor in tocotrienols (2.5 mg per day). No change in blood lipid values was
observed. Weststrate, J. A., et al European J. Clin. Nutr. 52: 334-343, 1998.
See Table 3.
Table 3. Percent change in blood lipid components in patients administered
sterol and stanol esters and tocotrienols from a physically refined rice bran
oil
compared with those obtained with the inventive oil.
Lipid Rice bran oii'* The invention * ~
Cholesterol, total -1 .1 -14
LDL cholesterol -1 .5 -20
HDL cholesterol -1 .3 + 20
LDL/HDL cholesterol -0.3 -28
~' 1 .5 g/d in 30 g oil providing 2.5 mg/d tocotrienols. ~'* As described
above.
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Example 5
The patients of Example 4 were switched to margarine preparations
containing either stanol esters (Benecol) or steryl esters. Both groups
received
margarine containing approximately 3 grams per day of the stated esters.
Decreases of LDL cholesterol concentrations between 8% and 13% were
observed, but'no significant changes in HDL cholesterol or triglycerides were
found. This study confirms the findings of other studies of sitostanol esters
but
reported significant depletion of plasma antioxidants. Miettinen, T. A. et al
New
Engl. J Med 333: 1308-1312, 1995; Weststrate, J. A., et al. Eur. J Clin. Nuir.
52: 334-343, 1998. See Table 4.
Table 4. Percentage change in blood lipids in patients administered sterol and
stanol esters compared with those obtained with the inventive oil.
Lipid Steryi esters'* Stanoi esters The inventive
~"* oil's *'
Cholesterol, -8.3 -7.3 -14
total
LDL cholesterol -13 -13 -20
HDL cholesterol n.s. n.s. + 20
Triglycerides n.s. n.s. -23
LDL/HDL chol. -14 -12 -28
*~Steryl esters: 3 g/d in 30 g oil; 0 mg/d tocotrienols. *~~" Stanol esters: 3
g/d in
g oil; 0 mg/d tocotrienols. ~ ~ ~* As described above.
25 Conclusion
The foregoing data demonstrate that the control of the amounts of
tocotrienols/tocopherols, steryl esters, free sterols, and cycloartenols
according
to the invention modulate blood lipid values in an optimally therapeutic
manner.
30 The results in Example 2 show that the administration of tocotrienols alone
does
not affect blood lipid values. Example 2 further demonstrates that
administration
of an edible oil according to the invention lowers total cholesterol, LDL
cholesterol and triglycerides, while increasing HDL cholesterol. Example 3
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demonstrates that a rice-derived tocotrienol concentrate depleted in steryl
esters
and cycloartenols is ineffective in increasing HDL levels or decreasing
triglycerides. Example 4 shows that a physically refined rice oil poor in
tocotrienols is ineffective, and Example 5 shows that administration of
sterols
and steryl esters without tocotrienols does not result in changes in HDL or
triglyceride values.
The decreases in serum total cholesterol and LDL cholesterol achieved by
patients administered the oil according to the invention are comparable to the
best results achieve by drug therapy. The increase in HDL cholesterol levels
of
20% and the decrease in triglycerides of 23% are particularly significant.
Watkins, T. et al. Environmental Nutr. Interactions 3:8 - 18, 1999. The total
cholesterol (TC1/HDL cholesterol ratio has been found to be a superior measure
of risk of coronary heart disease compared with either total cholesterol or
LDL
cholesterol levels alone. In the Framingham study, the eight-year likelihood
ratios
for coronary heart disease increased 10 times in men with the lowest TC/HDL
ratios compared with men with the highest ratios. Kinosian, B. et al L J
Invest
Med 43.- 443-450, 1995. On average, the TC/HDL ratio of patients
administered the oil according to the invention decreased by 28% from 5.97 to
4.29. The potential impact of the inventive oil to improve the public health
is
self-evident. A fifty-year-old man who achieves the 28% reduction in TC/HDL
ratios reported in the clinical trial with the inventive oil is twice as
likely to live
beyond the age of 75 years.
The foregoing examples are for illustrative purposes only, and do not in any
way limit the scope of applicants' invention which is identified by the claims
appended below.
