Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NUTRITIONAL COMPOSITION AND METHOD OF INHIBITING SMOOTH
MUSCLE CELL CONTRACTION THEREOF
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of the U.S. Utility Application
Serial
No. 10/449,828 filed May 30, 2003, the disclosure of which is incorporated by
reference
in its entirety herein.
FIELD OF THE INVENTION
This invention relates to nutritional composition and method of inhibiting the
contraction of smooth muscle cells, and hence may lower blood pressure in
hypertensive
patients.
BACKGROUND OF THE INVENTION
There are many documented pathophysiological and clinical effects of
hypertension. These effects include the short-term effects resulting in poor
health and .
bad work performance and the longer-term effects resulting in myocardial
infarction,
stroke, cardiac arrest, kidney disease, kidney failure and others. Moreover,
the effects of
hypertension may be exacerbated in conjunction with other diseases such as
diabetes. In
recent years it is estimated that more than 50% of deaths relating to
cardiovascular
disease in the United States alone was related to or resulted from
hypertension.
Hypertension remains the most common cause of cardiac failure or other disease
states
requiring some amount of hospitalization.
There has been significant and extensive research for treatment for
hypertension.
However, present treatments for such disorders are treatments such as
administration of
angiotensin converting enzyme inhibitors (ACE inhibitors). These treatments
have
serious shortcomings in long-term effectiveness, most notable the cost
associated with
these treatments and significant adverse effects.
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There are also a vast number of publications with regard to the mechanisms of
pathogenesis of hypertension. Extensive production and activity of angiotensin
II is well
accepted as one of the major sources in the development of hypertension, since
its excess
causes abnormally strong contraction of arteries, compromises process of
arteries
relaxation and lead therefore to elevated blood pressure. Thus, a massive
effort is being
undertaken to develop pharmaceutical compounds capable either to reduce
formation of
angiotensin II (i.e. ACE inhibitors which block a conversion of angiotensin I
to
angiotensin II by arterial wall cells) or to block a biological activity of
angiotensin II (i.e.
agonists of angiotensin receptors). Both classes of compounds are being tested
in
1o experimental conditions for their capacity to block angiotensin-dependent
contraction of
arterial wall either using arteries isolated from laboratory animals or a
model of cultured
smooth muscle cells embedded in collagen gel. A capacity of a tested compound
to
block a contractile activity of angitensin II in such experimental models
unequivocally
means that this compound will block angiotensin II activity in in vivo
conditions and will
~ reduce angiotensin-driven hypertension.
Carini et al. describe procyanidins from grape seeds that enhance relaxation
of
human artery (Life Sci. 2003 Oct. 17; 73(22):2883-98). Shen et al. describe
green tea
catecins that evoke a phasic contraction in rat aorta, and Chen et al.
describe purified
2o green tea epicatechins on contraction. Sanae et al. describe the effects of
catechins on
vascular tone in rat thoracic aorta with endothelium. Huang et al. describe
role of
endotheliurnlnitric oxide in vascular response to flavonoids and epicatechin
(Acta
Pharmacol. Sin. 2000 Dec; 21(12): 1119-24). While these references suggest a
possible
role of green tea extracts in regulating vascular tone, its direct effect to
smooth muscle
cells is less clear. Little is know if other ingredients may enhance the
effect of green tea
extract on smooth muscle cell contraction.
In view of the foregoing, there is a need for a nutritional composition and
method
to directly inhibit smooth muscle cell contraction and hence treat the
underlying
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hypertension disease. There is a need for a method of using such a nutritional
composition to preserve and restore the sensitivity of the arteries to stimuli
that would
allow for proper contraction and relaxation of smooth muscle cells in the
arteries.
SUMMARY OF THE~INVENTION
The present invention provides a method of inhibiting smooth muscle cell
contraction comprising the step of treating smooth muscle cells with a
nutritional
composition comprising a green tea extract, ascorbic acid, lysine, proline,
arginine,
magnesium, N-acetyl cystein, selenium, copper, and manganese.
Preferably, the green tea extract is at least one compound selected from the
group
consisting of epicatechin, epicatechin-3-gallate, epigallocatechin and
epigallocatechin-3-
gallate. More preferably, the green tea extract is epigallocatechin-3-gallate.
Preferably,, the ascorbic acid is calciurr~ ascorbate, magnesium ascorbate or
ascorbyl palmitate.
Preferably, the step of treating is the step of administering to a human
subject.
Preferably, the administered nutritional composition comprises 1,000 mg green
tea
extract, 710 mg ascorbic acid, 1,000 mg lysine, 750 mg proline, 500 mg
arginine, 1 mg
magnesium, 30 mg N-acetyl cystein, 30 pg selenium, 2 mg copper, and 1 mg
manganese.
Preferably, the nutritional composition further comprises at least one
ingredient
selected from the group consisting of resveratrol and genistein.
It is an object that the present invention provides a method of administering
a
nutritional composition that is useful in lowering blood pressure.
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It is another object of the present invention to use nutritional compounds
from a
natural source that is safe.
It is another object of the present invention to provide a method of retarding
adverse effects of stimuli, which lead to contraction of smooth muscle cells,
which in turn
increase blood pressure and results in hypertension.
It is yet another object of the present invention to provide method of
administering
a nutritional composition wherein the nutritional composition is administered
in daily
to amounts indicated in Table 1.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the effects of 0.1 IU/ml thrombin on smooth muscle cells in
SMC gels
containing composition 1 ("composition EF") and a control without composition
EF.
15 Control SMC gel is without thrombin.
Figure 2 depicts the effects of 1.0 ~.M angiotensin II on smooth muscle cells
in SMC gels
containing composition 1 ("composition EF") and a control without composition
EF.
Control SMC gel is without angiotensin II.
Figure 3 depicts SMC gel contraction by 1 ~.M angiotensin II and in the
presence of
2o increasing concentrations of composition EF.
Figure 4 depicts SMC gel contraction by increasing concentrations of 110 nM,
330 nM,
and 1,000 nM angiotensin II and in the presence of 100 ~.g/ml of composition
EF.
Figure 5 depicts SMC gel contraction by angiotensin II and in the presence of
composition EF, ascorbic Acid, EGCG, and ascorbic Acid-EGCG combination.
25 Figure 6 depicts SMC gel contraction by angiotensin II and in the presence
of arginine at
various concentrations.
Figure 7 depicts SMC gel contraction by angiotensin II and in the presence of
calcium
chloride, magnesium chloride, and calcium chloride-magnesium chloride
combination.
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Figure 8 depicts SMC gel contraction by angiotensin II and the effects of
resveratrol and
genistein, and in the presence of 100 ~g/ml of composition EF.
Figure 9 depicts SMC gel contraction by angiotensin II in presence of various
concentrations of N-acetyl cystein.
Figure 10 depicts SMC gel contraction by angiotensin II at 1 ~M in the
presence of
various concentrations of lysine and proline.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "EF" refers to a nutritional composition comprising
1,000 mg green tea extract, 710 mg ascorbic acid, 1,000 mg lysine, 750 mg
proline, 500
mg arginine, 1 mg magnesium, 30 mg N-acetyl cystein, 30 pg selenium, 2 mg
copper, and
1 mg manganese; lysine includes L-lysine and its derivative, proline includes
L-proline nd
its derivatives, arginine includes L-arginine and its derivatives; SMC refers
to smooth
muscle cells, EGCG refers to (-)-epigallocatechin-3-gallate; EC refers to
epicatechin
which refers to (-)-epicatechin, ECG refers to eipcatechin-3-gallate which
refers to (-)-
epicatechin-3-gallate, EGC refers to epigallocatechin which refers to (-)-
epigallocatechin.
Plant-derived bioflavonoids include catechins (which include EGCG, EG, ECG and
EC)
and is implicated to support arterial wall structural integrity and interfere
with a variety of
pro-atherosclerotic stimuli.
Hypertension as used in this application includes and is defined using the
guidelines of the American Heart Associate (AHA) for both hypertensive and pre-
hypertensive states. The AHA defines pre-hypertensive state as a systolic
blood pressure
of between 120 and 139 mmHg and a diastolic blood pressure between 80 and 89
mmHg.
The AHA defines hypertensive state as systolic blood pressure of greater 140
mmHg and
a diastolic blood pressure greater than 90 mmHg.
The nutritional composition of the present invention includes at least one
flavonoid component. The flavonoid component includes green tea extract. The
green
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tea extract is commercially available from U.S. Pharma Lab. (Somerset, NJ)
(product
name: GreenHerb --- green tea powder extract). The green tea extract contains
total
polyphenols of about 80% wt. Within the polyphenols, catechins are present in
an
amount of about 60% wt. Within the catechins, EGCG is present in an amount of
about
35% wt. Caffeine is present in the green tea extract (about 1.0% wt).
The nutritional composition of the present invention comprises a green tea
extract,
ascorbic acid, lysine, proline, arginine, magnesium, N-acetyl cystein,
selenium, copper,
and manganese.
Preferably, the nutritional composition of the present invention comprises 500
mg
- 2,000 mg green tea extract, 400 mg -1,500 mg ascorbic acid, 400 mg - 1,500
mg
lysine, 500 mg -1,500 mg proline, 200 mg - 1,000 mg arginine, 0.5 mg - 2 mg
magnesium, 10 mg - 60 mg N-acetyl cystein, 10 ~.g - 60 p,g selenium, 0.5 mg -
5 mg
copper, and 0.5 mg - 2 mg manganese.
More preferably, the nutritional composition of the present invention
comprises
1,000 mg green tea extract, 710 mg ascorbic acid, 1,000 mg lysine, 750 mg
proline, 500
mg arginine, 1 mg magnesium, 30 mg N-acetyl cystein, 30 ~.g selenium, 2 mg
copper, and
1 mg manganese.
Preferably, the nutritional composition further comprises resveratrol or
genistein.
The preferred doses for resveratrol and genistein are 10-50 ~M; and more
preferred doses
of 10 p.M - 30 ~,M.
The nutritional composition of the present invention is intended for
administered
to a mammal, in particular a human being, in a suitable dosage form as is
known in the
art. Suitable dosage forms known in the art include parenteral, enteral, and
especially
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oral. Oral solid and liquid dosage forms are particularly preferred. Oral
solid dosage
forms are well known in the art and include tablets, capsules, and edible food
items. Oral
solid dosage forms can be made with one or more pharmaceutically acceptable
excipients.
Pharmaceutical acceptable excipients assist or make possible the formation of
a dosage
form for a bioactive material and include diluents, binding agents,
lubricants, glidants,
disintegrants, coloring agents, and flavorants. An excipient is
pharmaceutically
acceptable if, in addition to performing its desired function, it is non-
toxic, well tolerated
upon ingestion, and does not interfere with absorption of bioactive
ingredients. In
another embodiment, these ingredients are prepared in a tablet form. Tablets
can be made
1o by well-known compression techniques using wet, dry, or fluidized bed
granulation
methods. The effective proportions of each specified ingredients (i.e., within
the EF
composition) are combined with desired amount of a pharmaceutically acceptable
excipient (e.g., lactose, starch, dextrin, ethyl cellulose and the like. The
ingredients are
mixed in a blender. Useful blenders include the twin-shell type, the planetary
mixer type,
~ and the high-speed high shear type, all of which are known in the art.
Tablets can be
either coated or uncoated as is knbwn in the art. Capsules, also known as dry
filled
capsules, are oral solid dosage forms in which the composition is contained in
a
swallowable container of suitable size, typical made of gelatin. Hard empty
capsules
suitable for containing the nutritional composition of the present invention
are
2o commercially available. The art of capsule filing is well known in the art
(Edward
Rudnic and Joseph B. Schwartz, Oral Solid Dosage Forms, in Volume II,
Remington:
The Science and Practice of Pharmacy, Chapter 92, 1615, 1642-1647 (Alfonso R.
Gennaro, Ed., 19th Ed., 1995).
Experimental Protocol
The following starting material and equipment were used.
1. Cultured vascular smooth muscle cells (SMC) isolated from human aorta.
Cells
are used from 4th to gih passages.
2. Human collagen type I.
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3. Angiotensin II.
4. Thrombin.
5. Composition EF (lysine, proline, arginine, vitamin C (as ascorbic acid,
calcium
ascorbate, magnesium ascorbate, or ascorbyl palmitate), magnesium, N-acetyl
cystein, selenium, copper, and manganese. 6 capsules of composition EF contain
1,000 mg of lysine, 750 mg proline, 500 mg L-Arginine, 710 mg of vitamin C, 50
mg magnesium, 1000 mg standardized green tea extract (80% polyphenols - 800
mg (decaffeinated)) 30 mg N-acetyl cystein, 30 ~g selenium, 2 mg copper, 1 mg
manganese. (all ingredients commercially available)
l0 6. Epigallocatechin gallate (EGCG)
7. Resveratrol
8. Cell culture medium (DMEM)
9. 24 well plastic cell culture plate pre-incubated with 2 mg/ml bovine serum
albumin.
10. Digital camera.
11. Digital image analyzing software (Scion Corporation).
Table 1. Composition 1 ("Composition EF")
Compound Dosage per day
L-Lysine 1,000 mg
L-Proline 750 mg
L-Arginine 500 mg
Vitamin C, as ascorbic 710 mg
acid,
Calcium Ascorbate, magnesium
Ascorbate or Ascorbyl
Palmitate
Magnesium 50 mg
Standardized Green Tea 1,000 mg
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Extract, 80% polyphenols
- 800
mg (decaffeinated)
N-Acetyl -Cystein 200 mg
Selenium 30 mcg
Copper 2 mg
Manganese 1 mg
METHODS:
We tested the ability of green tea extracts (i.e., bioflavonoids) and various
ingredients on inhibiting the contractile activity of smooth muscle cells.
Cultured human
aortic smooth muscle cells (SMC) (commercially available from Clonetics) were
used
and embedded in a three-dimensional type I collagen (1 mg/mL) matrix. Collagen
was
obtained from Sigma and the matrix preparation is described below. Gel
contraction was
stimulated by adding 1 .molar angiotensin II (Ang II) in serum-free media and
the gel
area was assessed by digital image analysis after 24 hours.
l0
Culture of Smooth Muscle Cells
Confluent cultures of SMC were removed from culture flask by trypsinization
and
washed with phosphate-buffered saline (PBS) from serum-containing medium. Cell
concentration in suspension was brought to 500,000 cell/mL in serum-free DMEM.
Cell
suspension was then mixed 1:1 with ice-cold 2 mg/ml collagen type I solution
in
phosphate buffered solution (PBS). Final concentration of collagen type I was
1 mg/mL,
final cell concentration was 250,000/mL.
Collagen-SMC suspensions were distributed by 300 ~l to 24 well plates in such
a
2o manner to cover the entire bottom surface of the wells. The plates were
then incubated
for one hour at 37°C to allow gel to polymerize. 0.5 mL of experimental
serum-free
medium containing no additions (control), or I micromol/L angiotensin II with
or without
tested compound was added to polymerized gel. Plates were then gently tapped
on the
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side to detach gel from the bottom of plastic well, and plates were then
placed to
incubator with the controlled atmosphere containing 5%COz at 37°C for
incubation.
After 24-hour incubation plates were taken from the incubator and plate image
with
floating gels were taken using digital camera. Gel flat surface area is
measured with
digital image analyzing software from Scion Corporation. Experiments were
performed
in triplicates and results are presented as mean +/- SD.
Studies were carried out to observe the effects of various components in
composition EF and to determine the synergistic effect of the ingredients in
composition
EF, if any, in inhibiting smooth muscle cell contraction. These studies may
shed light on
the treatment and/or prevention of hypertension.
Various ingredients including epigallocatechin gallate (EGCG) was studied.
Epigallocatechin gallate and other ingredients were first studied by
evaluating the single
effect of epigallocatechin gallate and respective ingredients. Synergistic
effects between
epigallocatechin gallate with other ingredients were then studied.
RESULTS
Both angiotensin II and thrombin (used as agonists) caused contraction of the
2o smooth muscle cells in the SMC gel. These agonists further caused
contraction of the
entire gel. Addition of angiotensin II or thrombin caused a reduced gel
surface area. The
differential between the gel surface area at 24 hours after pouring of the SMC
gel that
does not contain a contracting agent, and the gel surface area at 24 hours
after pouring of
an SMC gel that does contain a contracting agent is attributed to the effect
of the
contracting agent.
Using this SMC gel contraction assay, we evaluated various compounds for their
ability to inhibit the smooth muscle cell contraction. Among the ingredients
in the green
tea extracts, epigallocatechin gallate is noted to be the most active
inhibitor of gel
to
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contraction tested. When added at the concentration of 30 ~.mole/L. Inhibition
of gel
contraction by bioflavonoids (including EGCG) did not depend on antioxidant
activity,
since ascorbic acid did not have any activity in this assay.
Example 1
Fig. 1 shows the ability of composition EF in inhibiting smooth muscle cell
contraction as induced by thrombin. In this study, a SMC gel without the
contracting
agent (control) and a gel with the contracting agent (thrombin at 0.1 ICJ/ml)
were
compared to a gel with thrombin at 0.1 IU/mI treated with 100 ~g/ml of
composition EF.
to Control SMC gels without a contracting agent and treating agent showed some
contraction. Thus, smooth muscle cells have a tendency to contract, even
without the
presence of a contracting agent.
SMC gel with contracting agent thrombin showed greater contraction of the SMC
gel. however, when an SMC gel was treated with thrombin at 0.I IU/ml and
composition
EF, the SMC gel did not contract as much as an SM.C, gel with or without the
cr~ntracting:
agent by themselves. Thus, composition EF showed significant effect in
inhibiting the
contraction of the SMC gel and in acting as an anti-hypertensive.
2o Example 2
Fig. 2 shows the ability of composition EF in inhibiting smooth muscle cell
contraction as induced by angiotensin II (as an contracting agent). SMC gel
without the
contracting agent angiotensin II and a gel with the contracting agent
angiotensin II at 1.0
~M were compared to a gel with angiotensin II at 1.0 ~M treated with 100
~.g/ml of
composition EF.
SMC gel with contracting agent angiotensin II showed greater contraction of
the
SMC gel. When a SMC gel was treated with angiotensin II at 1.0 ~M and
composition
EF, the SMC gel did not contract as much as an SMC gel with or without the
contracting
agent by themselves. Both of these experiments at least tested the premise
that
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composition EF was effective as an anti-hypertensive agent. Accordingly, these
data
together (Fig. 1 and 2) clearly show that composition EF is effective in
inhibiting the
contraction of smooth muscle cells, and thereby may be useful in anti-
hypertensive
purposes.
Example 3
Fig. 3 shows a dose-dependent effect of composition EF on inhibiting smooth
muscle cell contraction as induced by angiotensin II. SMC gel containing
angiotensin II
at I .0 yM was treated with increasing concentrations of composition EF at 11,
33, and
l0 100 ~tg/ml, and compared to a control of angiotensin II without composition
EF. This
produced a dose response curve, showing less contraction (greater reduction in
SMC gel
surface area loss) with increased concentrations of composition EF.
Example 4
We next tested respective constituent of the composition EF in inhibiting
smooth
cell contraction. We also tested ifvarious constituents of composition EF
might act in a
synergistic manner. To test this, various constituents of composition EF were
tested
either alone or in combination with other ingredients in their ability to
inhibit smooth
muscle cell contraction.
Fig. 4 shows the effects of ascorbic acid, EGCG, and ascorbic acid + EGCG on
their ability to inhibit smooth muscle cell contraction. SMC gels were induced
to
contract by angiotensin II (1.0 ~M). Control SMC gel contained only
angiotensin II.
Composition EF at 100 ~g/ml greatly inhibit smooth muscle cell contraction.
Ascorbic
acid at 100 ~,M alone did not affect angiotensin II induced smooth muscle cell
contraction. EGCG at 15 pM alone did not have an appreciable inhibitory
effect. The
combination of ascorbic acid and EGCG also did not have any appreciable
inhibitory
effect. These data show that there is a synergistic effect among the various
components
of composition EF that inhibiting smooth muscle cell contraction. Note that
ascorbic acid
3o and EGCG were used at equivalent concentrations found in composition EF.
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Example 6
Fig. 5 shows the single effect of arginine on inhibiting smooth muscle cell
contraction. Arginine (0.50 mM and 1.0 mM) was applied to SMC gels containing
1.0
~M of angiotensin II and 0.5 mM of ascorbic acid. Equivalent concentrations of
arginine
were applied to SMC gels containing 1.0 pM of angiotensin II but with no
ascorbic acid.
The concentration of arginine in 100 pg/ml of composition EF is 50 p.M.
Therefore the concentrations of arginine applied singly to the SMC gels were
respectively
times and 20 times greater than the concentration of arginine in the
composition EF.
1o The concentration of ascorbic acid in SMC gels containing ascorbic acid was
0.5 mM,
which is 5 times greater than the concentration of ascorbic acid in EF.
Despite these
higher concentrations, ascorbic Acid and arginine, either alone or in
combination did not
produce a detectable effect in inhibiting smooth muscle cell contraction.
Example 7
Fig: 7 shows the single and' combined effect of calcium and magnesiurn (in the
form of calcium chloride and magnesium chloride) on inhibiting smooth muscle
cell
contraction. The concentration of calcium in 100 p.g/ml of composition EF is
12 ~M.
The concentration of magnesium in composition EF is 50 p.M. The concentration
of
2o calcium and magnesium used in this study for SMC gel contraction was 2.0
mM.
Therefore, the concentrations of calcium and magnesium applied to the SMC gels
were
respectively approximately 160 times and 40 times greater than the
concentration of
calcium and magnesium in composition EF. Angiotensin II was added at 1 ~.M as
contracting agent to all SMC gels. Despite these higher concentrations,
calcium chloride
and magnesium chloride, either alone or in combination, did not produce a
detectable
inhibition on smooth muscle cell contraction induced by angiotension II.
Although composition EF did not contain any resveratrol or genistein, we
tested
their combined effect with composition EF. Fig. ~ shows the effects of
genistein and
3o resveratrol to either individually or in combination with each other, in
inhibiting smooth
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muscle cell contraction. Resveratrol was applied to SMC gels and compared with
SMC
gels that did not contain resveratrol. The concentration of resveratrol
applied to the SMC
gel was 15 ~M and 30 ~.M. In one SMC gel genistein was added at a
concentration of 30
~M to test the effect, if any, of genistein by itself. The concentration of
resveratrol
applied to the SMC gels was 15 ~M and 30 ~M. Genistein and resveratrol in
combination
were applied to the SMC gel both at 15 pM. Angiotensin II was added at 1 pM as
contracting agent to all SMC gels. Two groups of experiments were carried out,
one set
of SMC gels without composition EF, and the other set SMC gels containing
composition
EF at 100 ~M.
to
While resveratrol, genistein, and their combination tended to show some
inhibiting effect, this effect was more pronounced when composition EF was
present.
There was a clear detectable additive anti-hypertensive effect in all SMC
gels, whether
containing only resveratrol, only ginestein, or both. A dose response curve
was evident in
the groups containing 15 ~M and 30 ~M of resveratrol in groups with or without
composition EF, however the dose response curve in the resveratrol groups
containing
Composition EF was more pronounced.
Example 8
2o Fig. 9 shows the effectiveness of N-acetyl cystein for inhibiting smooth
muscle
cell contraction. The concentration of N-acetyl cystein in 100 pg/ml of
composition EF is
pM. The concentration of N-acetyl cystein applied to the SMC gels was 2.2,
6.7, 20
and 60 p,M respectively. Angiotensin II was added at 1 p.M as contracting
agent to all
SMC gels. Despite these higher concentrations, N-acetyl cystein did not
produce a
detectable anti-contracting effect.
Example 9
Fig. 10 shows the effects of lysine and proline, either individually or in
combination with each other, to inhibit smooth muscle cell contraction. The
3o concentration of lysine in 100 p.g/ml of composition EF is 110 ~.M. The
concentration of
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lysine applied to the SMC gels was 0.25, 0.50, and 1 mM. Therefore, the
concentrations
applied to the SMC gel were respectively approximately 2 times, 4.5 times, and
9 times
greater than the concentration of lysine in composition EF. The concentration
of proline
in 100 ~.g/ml of composition EF is 100 ~.M. The concentration of proline
applied to the
SMC gels was 0.25, 0.50, and 1 mM. Therefore, the concentrations applied to
the SMC
gel were respectively 2.5 times, 5 times and 10 times greater than the
concentration of
proline in composition EF. Lysine and proline were added as,a combination to
an SMC
gel at a concentration of 0.54 mM. Angiotensin II was added at 1 p,M as
contracting
agent to all SMC gels. Despite these higher concentrations, proline and
lysine, either
1o alone or in combination did not produce a detectable anti-contracting
effect.
Together, the results show that a nutritional composition comprising a green
tea
extract (including ECGC as a bioflavonoid), ascorbic acid, lysine, proline,
arginine,
magnesium, N-acetyl cystein, selenium, copper, and manganese, has a
synergistic effect
~ 5 '.in regulation of SMC-mediated. contraction. .Th.e nutritional
composition has a strong
potential in counteracting pathophysiological effects of agonists such as
thrombin and
angiotensin II. While not being bound by a particular mechanism, the
synergistic effect
seen in composition EF may relate tQ extracellular matrix integrity.
2o Example 10
We studied the effects of individual catechins on angiotensin II-stimulated
contraction of human aortic smooth muscle cells. Catechin (30 pM), epicatechin
(30
p.M), epicatechin gallate (30 p.M), and epigallocatechin gallate (30 pM) were
used and
angiotensin II was used as stimulant for smooth muscle cell contraction. Gel
contraction
25 is represented as percentage of reduction in gel surface area over 24 hour
incubation
times. Angiotensin II (1 p.M) caused 85.26 ~ 1.18 % (mean ~ SD) reduction.
Angiotensin II (1 p.M) plus catechin (30 pM) caused 76.83 X1.63 % reduction.
Angiotensin II (1 p,M) plus epicatechin (30 pM) caused 78.59 ~ 7.03 %
reduction.
Angiotensin II (1 p.M) plus epicatechin gallate (30 pM) caused 65.70 ~ 6.56 %
reduction.
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Angiotensin II (1 pM) plus epigallocatechin gallate (30 ~.M) caused 61.23 ~
9.14
reduction.
Accordingly, the present invention provides a possible therapy for a
nutritional
composition. The components present in the nutritional composition act
synergistic in
inhibiting smooth muscle cell contraction and hence, reverse and minimize the
lack of
sensitivity of arteries that lead to hypertension. Furthermore, the present
invention
provides a potential therapy for a nutritional composition that may retard
adverse effects
of stimuli, which lead to contraction of smooth muscle cells, which increase
blood
pressure and results in chronic hypertension. The present invention relates to
the
selection of compounds and extracts from nature, which are more effective
without undue
side-effects of pharmaceutical compounds, not to mention its further
advantages of
economic cost.
~t will be understood that there is no intent to limit the present invention
to the
prefen-ed embodiment disclosed, but ratr~er it is intended to cover all
modifications and
alternate constructions falling within the spirit and scope of the invention.
All
publications and other references mentioned herein are incorporated by
reference in their
entirety.
16
SUBSTITUTE SHEET (RULE 26)
