Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02466234 2004-05-04
HERBAL PHARMACEUTICAL COMPOSITIONS FOR
PROPHYLAXIS AND/OR TREATMENT OF CARDIOVASCULAR
DISEASES AND THE METHOD OF PREPARING THE SAME
FIELD OF THE INVENTION
The present invention relates to herbal pharmaceutical compositions which
contain the root of scutellaria (Radix Scutellariae), the rhizome of coptis
(Rhizoma
Coptidis), the root and rhizome of rhubarb (Radix et Rhizoma Rhei), and the
root of
ginseng (Radix Ginseng) or American ginseng (Radix Panacis Quinguefolii) for
prophylaxis or treatment of cardiovascular diseases. Optionally, the root of
ginseng or
American ginseng can be replaced with the rhizome of ginger (Rhizoma
Zingiberis). The
herbs can be prepared as dry powders or extracts. The present invention also
relates to
the methods of preparing and using the herbal pharmaceutical compositions.
BACKGROUND OF THE INVENTION
Based on data from the World Health Organization (WHO), cardiovascular
diseases contribute to a third of global deaths in 1999 and are estimated to
be the leading
cause of death in developing countries by 2010. Cardiovascular diseases are
the name for
a group of disorders in the heart and blood vessels, including, but not
limited to,
hypertension (high blood pressure), coronary heart disease (heart attack),
cerebrovascular
disease (stroke), peripheral vascular disease, heart failure, rheumatic heart
disease,
congenital heart disease, and cardiomyopathies.
Hypertension is by far the most prevalent cardiovascular disease. It is
estimated
that more than a third of Americans aged 45 or older have high blood pressure
and,
among them, more than 50% are aged 60 or older. Untreated hypertension can
lead to
la
CA 02466234 2004-05-04
serious and life-threatening complications, ~, stroke, coronary heart disease,
arteriosclerosis, atherosclerosis, heart failure, kidney failure and
blindness.
As indicated in the United States Sixth Report of the Joint National Committee
(JNC VI) on High Blood Pressure, current treatment for hypertension includes
diuretics,
S a-blockers, (3-Mockers, calcium channel blockers, ACE inhibitors, and
angiotensin
antagonists. These agents can be used as monotherapy or in combination.
However,
most of these agents ameliorate the symptoms but not curing the diseases.
These agents
are also frequently accompanied with side effects.
One of the major mechanisms for causing human hypertension is the dysfunction
of endothelium. Endothelium is the layer of epithelial cells that lines the
cavities of the
heart and of the blood and lymph vessels. Its main role is to modulate both
vascular tone
and structure by producing vasodilator and vasoconstrictor mediators.
When activated by specific agonists such as acetylcholine, endothelial cells
produce nitric oxide ("NO"), a labile substance derived by L-arginine
degradation
1 S through the activity of the endothelial NO synthase ("eNOS"). NO is a
powerful relaxing
agent which also inhibits platelet aggregation and smooth muscle cell
proliferation.
Under pathological conditions, such as hypertension or aging, agonist-induced
stimulation of endothelium leads to activation of a cyclooxygenase pathway and
consequent production of cyclooxygenase-dependent factors, including
thromboxane AZ
or prostaglandin H2, or free radicals (such as superoxide anions).
Dysfunctional
endothelium can also cause vascular damage, in particular, atherosclerosis.
There are two isoforms of cyclooxygenase, cyclooxygenase 1 and 2 (COX-1 and
COX-2), also referred to as prostaglandin endoperoxide synthase 1 and 2, which
are key
CA 02466234 2004-05-04
enzymes in the conversion of arachidonic acid to prostaglandins, thromboxanes
and other
eicosanoids. It is believed that COX-1 and COX-2 have different physiologic
functions
due to striking differences in their tissue expression and regulation. COX-1
is a
constitutive enzyme that is present at all times in the body and is
responsible for the
production of cytoprotective prostaglandins important for homeostatic
functions, such as
maintaining the integrity of the gastric mucosa, mediating normal platelet
function, and
regulating renal blood flow. In contrast, COX-2 is a rapidly inducible form of
cyclooxygenase that leads to the production of proinflammatory prostaglandins.
While
COX-2 expression is highly restricted under basal conditions, it is
dramatically up-
regulated during inflammation. The involvement of COX-2 and the elevated
production
of prostaglandins are associated with a variety of diseases and disorders,
such as brain
ischemia and cancers, as well as diseases and disorders in which elevated
levels of NO is
present.
NO modulates the activity of COX-2 and participates in inflammatory and
autoimmune-mediated tissue destruction. The effect of NO on COX-2 is dose-
dependent.
Low levels of NO activate COX-2. In contrast, large amounts of NO produced by
inducible nitric oxide synthase ("iNOS") can inhibit the induction of COX-2
and suppress
the formation of COX-2 metabolites.
iNOS is expressed in the myocardium after myocardial infarction (MI) and in
heart failure. Myocardium is the middle and thickest layer of the heart wall
composes of
cardiac muscle. Upregulation or overexpression of iNOS is associated with mild
inflammatory cell infiltrate, cardiac fibrosis, hypertrophy, and dilatation.
Cardiac
hypertrophy is a significant risk factor for the development of congestive
heart failure
CA 02466234 2004-05-04
(CHF). Overexpression of iNOS results in overproduction of NO, causing
myocardial
dysfunction and CHF.
CHF is a form of heart disease in which weakened heart function exists with
concommitant edema. CHF has many different causes, including narrowing of the
arteries supplying blood to the heart muscle (coronary heart disease), prior
heart attack
(myocardial infarction) resulting in scar tissue large enough or located so to
interfere with
normal electrocardiac function, high blood pressure, etc. CHF is one of the
most serious
cardiovascular diseases affecting adults. Over 4 million people have CHF and
the
incidence is on the rise. The incidence of this disease or condition is
increasing with the
aging of the population and is currently the most common cause for hospital
admission in
the elderly. The total U.S. healthcare expenditure on CHF is over five billion
dollars per
year.
Atrial fibrillation (AF) is atrial arrhythmia characterized by rapid
randomized
contractions of the atrial myocardium, causing a totally irregular, often
rapid ventricular
rate. AF may persist due to structural changes in the atria that are promoted
by
inflammation. C-reactive protein (CRP) is a marker of systemic inflammation
which
predicts cardiovascular events and stroke, a common sequela of AF. CRP also
induces
adhesion molecule expression by endothelial cells.
While a panacea has been hunted for in western medicine for years, researchers
turn to traditional Chinese herbal medicine for medications of various
diseases. Chinese
herbal medicine has existed and been use for treating various diseases for
thousands of
years.
4
CA 02466234 2004-05-04
For example, San-Huang-Hsie-Hsin-Tang is an ancient herbal decoction which
was first described in Chin-Kuei-Yao-Lueh (translated into English as "the
Prescriptions
From the Golden Chamber") for "purging fire and clearing the three torsos" and
wherefore it is indicated for insufficient cardiac "Chi," hematemesis, and
epistaxis. The
decoction is made of equal amounts of the root of scutellaria (Radix
Scutellariae), the
rhizome of coptis (Rhizoma Coptidis), and the root and rhizome of rhubarb
(Radix et
Rhizoma Rhei). The decoction has a bitter taste and with a cold nature. The
decoction is
intended for patients with congestion, flush up, fidgets, shoulder stiffness,
gastric
obstructive depression, constipation, and forceful pulse. However, the
decoction is
contraindicated for not suitable for patients with symptoms of prolonged
bleeding,
marked anemia, and minute-weak pulses.
U.S. Patent No. 5,443,839 discloses a composition having anti-inflammatory,
anti-allergic or anti-aging activity comprising, inter alia, an extract of
Scutellaria. There
is no indication that the composition is effective in treating cardiovascular
disease and
hypertension.
U.S. Patent No. 6,274,177 discloses a method of preparing an extract from
Zingiber officinale, which is potent in anti-inflammation and anti-platelet
aggregation.
There is no indication that the herbal composition is effective in treating
cardiovascular
disease and hypertension.
U.S. Patent No. 6,340,480 discloses a composition and method for treating
circulatory conditions including hypertension by promoting systemic vascular
relaxation
and dilation. The composition is a natural combination of L-arginine, ginseng,
and
Zizyphi fructus in an orally or topically administered form. The combination
works
CA 02466234 2004-05-04
synergistically to synthesize NO and thereby promote systemic vascular
relaxation and
dilation. The combined constituents may work to maintain a critical threshold
level of
NO in areas that cannot themselves produce it, thereby promoting systemic
vascular
relaxation and dilation in order to reduce hypertension. However, it is not
clear whether
the herbal composition is effective in treating cardiovascular disease.
In the invention to be presented in the following section, an herbal
pharmaceutical
composition is described. This herbal pharmaceutical composition is effective
in both
prophylaxis and treatment of cardiovascular diseases. The composition is also
non-toxic
and thus can be used by patients in all ages and physical conditions,
including the week,
the early and the debilitated.
In addition, the herbal pharmaceutical composition of the present invention
provides multiple mechanisms of pharmacologically effects, including lowering
and
stabilization of blood pressure; inhibition of expression of iNOS; inhibition
of COX-2
activity; reduction of blood CRP; and reduction of blood cholesterol.
SUMMARY OF THE INVENTION
The present invention provides herbal pharmaceutical compositions which
contain Radix scutellariae (root of scutellaria); Rhizoma Coptidis (rhizome of
coptis);
Radix et Rhizoma Rhei (root and rhizome of rhubarb); and Radix Ginseng (root
of
ginseng) or Radix Panacis Quin9uefolii (American ginseng). The preferred
weight ratio
of the root of scutellaria, the rhizome of coptis, the root and rhizome of
rhubarb, and the
root of ginseng or American ginseng is about 1-2 : 1-2 : 1-2 : 1-2; and most
favorably 1
1 : 1 : 1. Optionally, the root of ginseng or American ginseng can be replaced
with
6
CA 02466234 2004-05-04
Rhizoma Zingiberis (rhizome of ginger). The preferred weight ratio of the root
of
scutellaria, the rhizome of coptis, the root and rhizome of rhubarb, and the
rhizome of
ginger is about 1-2 : 1-2 : 1-2 : 1-2; and most favorably 1 : 1 : 1 : 1.
The herbs of the present invention can be prepared in the form of dry powders
or
extracts. The herbal pharmaceutical composition containing dry powders of the
root of
scutellaria, the rhizome of coptis, the root and rhizome of rhubarb, and the
root of
ginseng or American ginseng (as shown in Example 1, infra) is pharmaceutically
active
and possesses the properties of lowering and maintaining blood pressure as
well as
treating other cardiovascular diseases. However, the preferred pharmaceutical
compositions of the present invention are the ones containing a mixture of
both herbal
extracts and dry powders. Preferably, the root of scutellaria, the rhizome of
coptis, and
the root and rhizome of rhubarb are prepared as extracts, where the active
ingredients of
the herbs are extracted by a solvent, which can be water, alcohol, or a
mixture thereof.
The preferred solvent for the root of scutellaria and the rhizome of coptis is
water. The
preferred solvent for the root and rhizome of rhubarb is alcohol, most
favorably 95%
alcohol (in water).
The preferred form of ginseng/American ginseng or ginger used in the herbal
pharmaceutical compositions is dry powders, which are prepared by cutting and
grinding
the herbs, followed by drying.
In addition to the herbs, the herbal pharmaceutical compositions of the
present
invention can contain a pharmaceutically acceptable excipient and/or carrier
and be
formulated in various dosage form such as granule, capsule, tablet, powder,
and bolus, for
orally administration. The preferred formulation is tablet.
7
CA 02466234 2004-05-04
The present invention provides a methods for preparing the herbal
pharmaceutical
compositions which contain a mixture of herbal extracts and dry powders as
follows: (1)
individually extracting the root of scutellaria, the rhizoma of coptis, and
the root and
rhizome of rhubarb with an appropriate solvent to form respective extracts of
the herbs;
(2) individually filtering the respective extracts of the herbs; (3) mixing
the respectively
filtered extracts of the herbs to form an herbal mixture; (4) condensing the
herbal
mixture to form an herbal paste; (5) grinding the root of ginseng or American
ginseng to
make dry powders of ginseng or American ginseng; (6) adding the dry powders of
ginseng or American ginseng to the herbal paste; and (7) drying the paste to
form the
herbal pharmaceutical compositions.
Optionally, the root of ginseng or American ginseng can be replaced with
Rhizoma Zingiberis (rhizome of ginger).
The root of scutellaria and the rhizome of coptis are preferably extracted by
water.
The root and rhizome of rhubarb can either be extracted by alcohol or by a
mixture of
alcohol and water, preferably 95% alcohol in water.
The herbal pharmaceutical composition of the present invention has
therapeutical
effect on cardiovascular diseases and can be used for prevention and/or
treatment of
cardiovascular diseases, including, but not limited to, hypertension, coronary
heart
disease, cerebrovascular disease, peripheral vascular disease, heart failure,
rheumatic
heart disease, congenital heart disease, and cardiomyopathies.
Specifically, the herbal pharmaceutical composition of the present invention
can
stabilize and lower blood pressure, prevent damage to endothelial cell (e.~.,
by inhibiting
iNOS activity, inhibiting COX-2 activity, reducing blood CRP concentration,
inhibiting
CA 02466234 2004-05-04
smooth muscular cell proliferation, and reduce blood cholesterol level). The
herbal
pharmaceutical composition can be safely used by patients at any ages and
physical
conditions, including the weak, the elderly, and the debilitated.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows the anti-hypertensive effects of: (1) the root of scutellaria
alone
(H1); (2) the root and rhizome of rhubarb alone (H3); (3) a combination of the
root of
scutellaria and the rhizome of coptis (H1+2); (4) the modified San-Huang-Hsie-
Hsin-
Tang decoction (M) as shown in COMPARATIVE EXAMPLE 1, infra; (S) STC
(Example 3), infra; and (6) a combination of captopril and nifedipine (A) in
rats.
Captopril is an ACE inhibitor. Nifedipine is a calcium channel blocker. *
indicates
statististical significance.
Figures 2a and 2b show the effects of : (1) placebo control (C); (2) the
modified
San-Huang-Hsie-Hsin-Tang decoction as shown in COMPARATIVE EXAMPLE 2 (M),
infra; (3) STC (Example 3, infra); and (4) the combination of captopril and
nifedipine
(A); on mean arterial blood pressure (maBP) and heart rate (HR) of rats,
respectively, on
the day after a completion of 2-week dose administration.
Figures 3a and 3b show the effects of STC (Example 3, infra) on heart rate
(HR)
and blood pressures, respectively, of rats, after the animals were given a
daily dose of
14.4 mg/kg for one week. SHR represents the baseline control of the
spontaneous
hypertensive rats. WKY represents the baseline control of the normal rats. STC
were
given to the spontaneous hypertensive rats. SYS, DIA, and maBP represent the
systolic,
diastolic and mean arterial blood pressure, respectively.
9
CA 02466234 2004-05-04
Figures 4a and 4b compare the heart rates (HR), and the systolic (SYS),
diastolic
(DIA) and mean arterial blood pressures (maBP), respectively, in 8-week-old
WKY rats
(Young), 10-month-old WKY rats (Old) and 10-month-old WKY rats treated with 48
mg/kg of the herbal pharmaceutical composition of the present invention (STC).
Figure 5 shows inhibition of nitrite formation in the mouse macrophage cell
line
RAW 264.7 by STC. The symbol, C, represents the untreated control mouse
macrophage
cells. LPS and STC represent the mouse macrophage cells treated with lipopoly-
saccharide and the herbal pharmaceutical composition of the present invention,
respectively.
Figure 6 shows the inhibitory effects of the herbal pharmaceutical composition
of
the present invention on PEG-2 formation by mouse macrophage cell line RAW
264.7.
LPS and STC represent the mouse macrophage cells treated with
lipopolysaccharide and
10 mg/mL of the herbal pharmaceutical composition of the present invention.
Figures 7a and 7b show inhibition on the protein syntheses of COX-2 and iNOS,
respectively, by the herbal pharmaceutical composition of the present
invention (STC).
Column 1 presents the enzymes isolated from the heart of spontaneous
hypertensive rats
treated with 20 mg/kg of lipopolysaccharide. Column 2 presents the enzymes
isolated
from the lungs of spontaneous hypertensive rats treated with the herbal
pharmaceutical
composition of the present invention. Column 3 presents the enzymes isolated
from the
lungs of the control, untreated spontaneous hypertensive rats.
Figures 8a and 8b show RNA expressions of (3-actin and iNOS, respectively, in
the lung of spontaneous hypertensive rats that were treated with (1) the
modified San-
CA 02466234 2004-05-04
Huang-Hsie-Hsin-Tang decoction (M) (cDNA marker), (2) STC (Example 3, infra)
(S),
(3) control (C), and (4) a combination of captopril and nifedipine (A).
Figure 9 shows the ELISA analysis of CRP in the blood of spontaneous
hypertensive rats of the control group (C) and the group treated with 48 mg/kg
of STC
(Example 3, infra) for two weeks.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel herbal pharmaceutical compositions for
preventing and treating cardiovascular diseases, which are suitable for
patients of all age
groups and physical conditions, including elderly and debilitated.
Recent progress in the scientific and medical understanding of the
cardiovascular
diseases provide more knowledge on the involvement of endothelial damages,
nitric
oxide (NO), inflammation reactions and C-reactive protein (CRP) in these
diseases. NO
produced in the endothelium by the endothelial nitric oxide synthase (eNOS) is
not only a
potent vasodilator but also inhibits platelet aggregation, smooth muscle cell
proliferation,
monocyte adhesion and adhesion molecule expression, thus, maintain the
integrity of the
endothelial tissues. Production of cyclooxygenase (COX)-dependent factors,
including
prostanoids and oxygen free radicals, may be the main cause for endothelial
dysfunction.
Dysfunctional endothelium can then be one of the main mechanisms causing
vascular
damage which can further lead to more severe cardiovascular diseases. As
inhibition of
cyclooxygenase may restore NO-mediated vasodilation in essential hypertension,
anti-
inflammatory interventions may have therapeutic utility.
11
CA 02466234 2004-05-04
During diseased states, ~, cardiac hypertrophy, myocardial infarction (MI),
ischemia, myocarditis and septic shock, overexpression of the inducible nitric
oxide
synthase (iNOS) leads to increased production of NO. The elevated NO levels
can result
in more severe complications, ~, myocardial dysfunction, congestive heart
failure and
sudden cardiac death.
During the inflammation process, an acute-phase reactant, C-reactive protein
(CRP), is formed. CRP, frequently used as a systemic inflammation marker,
promotes
the expression of the adhesion molecules and may plays a direct role in the
pathogenesis
of vascular inflammation, particularly atherosclerosis. CRP has been
associated with
vascular risk factors and with prevalent and incident atherothrombotic
cardiovascular
diseases, i.e., coronary heart disease, stroke, and peripheral arterial
disease.
A new approach for treatment of cardiovascular diseases should consider all
aspects of the diseases. For example, in addition to the anti-hypertensive
activity, the
new medicine should not only be able to protect healthy endothelium but also
improve
the functions of dysfunctional endothelium.
The present invention provides herbal pharmaceutical compositions that have
the
ttJnctions of reducing higj blood pressure, maintaining normal blood pressure,
improving
and protecting cardiovascular endothelial cell functions, so as to treat and
prevent
cardiovascular diseases.
The herbal pharmaceutical compositions of the present invention contain four
herbs: the root of scutellaria, the rhizome of coptis, the root and rhizome of
rhubarb, and
the root of ginseng or American ginseng. Optionally, the root of ginseng or
American
ginseng can be replaced with the rhizome of ginger.
12
CA 02466234 2004-05-04
The herbs used in the compositions can be any variants of the herbs mentioned
above. For example, the root of scutellaria has three closely related
variants, which are,
Scutellaria baicalensis Georgi, Scutellaria viscidula Bge., and Scutellaria
amoena C. H.
Wright. The rhizome of coptis has four closely related variants, which are,
Coptis
chinensis Franch., Coptis deltoidea C. Y. Cheng et Hsiao, Coptis teetoides
C.Y. Cheng,
and Coptis omeiensis (Chen) C.Y.Cheng. The root and rhizome of rhubarb has
three
closely related variants, which are Rheum palmatum L., Rheum tanguticum
Maxim., and
Rheum officinale Baill.
The pharmaceutical effect of scutellaria is in the dried root, which has the
pharmaceutical name of Radix Scutellariae. Scutellaria belongs to the family
of
Labiatae. The herb is mainly produced in the provinces of Hebei, Shanxi, and
inner
Mongolia of China. The best harvest seasons for the herb are in spring or
autumn. The
root of scutellaria is dried under the sunlight, sliced, and used unprepared
or stir-baked
with wine or stir-baked to charcoal. The herb is bitter in flavor and cold in
property.
According to traditional Chinese medicine, the herb can be used to cure
diseases in lung,
gallbladder, stomach, and large intestine channels. Specifically, the herb can
be used to
remove damp-heat, counteract toxicity, arrest bleeding, and prevent abortion
in patients.
The root of scutellaria contains active ingredients which include, but are not
limited to, baicalin, oroxylin A-glucuronide, wogonin-7-O-glucuronide,
baicalein,
wogonin, and oroxylin A. Baicalin can be used as a denominator for qualitative
or
quantitative control of the herb.
The pharmaceutical effect of coptis is in the dried rhizome of coptis, which
has
the pharmaceutical name of Rhizoma Coptidis. Coptis belongs to the family of
13
CA 02466234 2004-05-04
Ranunculaceae. It is mainly produced in the provinces of Sichuan, Hubei, and
Yunnan of
China. The preferred harvest season is in autumn. The rhizome of coptis is
dried under
the sunlight after the rootlets and earth have been removed and used
unprepared or stir-
baked with ginger juice. The herb is bitter in flavor and cold in property.
According to
traditional Chinese medicine, the herb can be used to cure diseases in the
heart, stomach,
liver, and large intestine channels.
The rhizome of coptis contains active ingredients, which include, but are not
limited to, berberastine, columbamine, jatrorrhizine, epiberberine, coptisine,
palmatine,
and berberine. Berberine can be used as a denominator for the qualitative or
quantitative
control of the herb.
The pharmaceutical effect of rhubarb is in the dried root and rhizome of
rhubarb,
which has the pharmaceutical name of Radix et Rhizoma Rhei. Rhubarb belongs to
the
family of Polygonaceae. It is mainly produced in the provinces of Qinghai and
Sichuan
of China. The root and rhizome of rhubarb is dug in the late autumn when its
stem and
leaves begin to wither, or in the early spring before the plant begins to
sprout. The
harvested herb is dried and sliced. The root and rhizome of rhubarb can be
used
unprepared, stir-baked with wine, or carbonized. It is bitter in flavor and
cold in
property. According to traditional Chinese medicine, the root and rhizome of
rhubarb
can cure diseases in spleen, large intestine, liver, and heart channels.
The root and rhizome of rhubarb contains active ingredients, which include,
but
are not limited to, sennoside B, sennoside A, aloe-emodin, rhein, emodin, and
chrysophanol. Sennoside A and/or emodin can be used as denominators for
qualitative or
quantitative control of the herb.
14
CA 02466234 2004-05-04
There are two kinds of ginseng, Radix Ginseng (root of ginseng) and Radix
Panacis Quinquefolii (root of American ginseng), which can be used in the
herbal
pharmaceutical composition of the present invention. Radix Ginseng is Panax
ginseng C.
A. Mey. Radix Panacis Quinquefolii is Panax quinquefollum L. Radix Ginseng
belongs
to the family of Araliaceae. Ginseng is mainly produced in the provinces of
Jilin,
Liaoning, and Heilongjiang of China. Ginseng produced in Fusong of Jilin is
particularly
of good quality. The herb can also be cultivated, which is called "garden
ginseng," as
opposed to "mountain ginseng" which refers to the ginseng found in the wild.
Cultivated
ginseng is harvested in autumn. The harvested ginseng is dried in the sun or
roasted,
which is called "sun-dried ginseng," or dried after being steamed, which is
called "red
ginseng," or soaked in syrup, which is known as "sugar-processed ginseng." The
fibrous
rootlets are known as ginseng rootlets. Wild ginseng dried in the sun is known
as sun-
cured wild ginseng. The herb is sliced for use. The herb has a sweet and
slightly bitter
flavor and is neutral in property. According to traditional Chinese medicine,
ginseng is
particularly good for curing diseases in spleen, lung, and heart channels.
The pharmaceutical effects of ginseng are in its dried root. Ginseng also has
effects on central nervous system. It enhances both stimulatory and inhibitory
processes
in the central nervous system, thereby improving the adaptability of nervous
responses.
Ginseng can also lower serum glucose and cholesterol. It also shows
therapeutic and
preventive effect on peptic ulcer.
Radix Panacis Quinquefolii also known as American ginseng, belongs to the
family of Araliaceae. The medicinal effects of American ginseng is in the
root.
American ginseng can be found in northern United States and Canada. It has
also been
CA 02466234 2004-05-04
widely cultivated in France and northern China. The best harvest season for
American
ginseng is in autumn.
The active ingredients in the root of ginseng or American ginseng include, but
are
not limited to, ginsenoside Rgl, ginsenoside Re, and ginsenoside Rbl, among
which
ginsenoside Rbl can be used as a denominator for qualitative or quantitative
control of
the herb.
The pharmaceutical effect of ginger is in the dried rhizome of ginger, which
has
the pharmaceutical name of Rhizoma Zingiberis. Ginger belongs to the family of
Zingiberaceae. Fresh rhizome or dried rhizome of ginger can be prepared by
baking,
roasting, and simmering. Ginger can be found throughout China, especially
Sichuan,
Guizhou, Guangdong. It is primarily cultivated. The best season for harvest is
before the
winter solstice. Ginger is acrid and has warm property. According to
traditional Chinese
medicine, ginger is particularly good for curing diseases in spleen, lung, and
stomach.
The active ingredients in the rhizome of ginger include, but are not limited
to,
gingerol, shogaol, and zingerone, among which gingerol can be used as a
denominator for
qualitative and quantitative control of the herb.
The pharmaceutical names, botanical or zoological names, family names,
common descriptions, and major ingredients of the herbs used in the present
invention are
shown in Table 1.
16
CA 02466234 2004-05-04
Table 1. Herbs of the Present Pharmaceutical Composition
PharmaceuticalBotanical Family Common Major Ingredients
Name Name Descri
tion
Radix ScutellariaeScutellariaLabiatae scutellariabaicalein, baicalin,
or wogonin,
baicalensis scute wogonin-7-0-glucuronide,
Georgi neobaicalein, oroxylin
A
glucuronide, camphesterol,
(3-
sitosterol, benzoic
acid
Rhizoma CoptidisCoptis chinensisRanunculaceaecoptis berberine, coptisine,
rhizome worenine,
Franch., palmatine, columbamine,
C.
deltoidea obacunone, obaculactone,
C.Y.
Cheng, C. palmatine, jatrorrhizine,
omeiensis magnoflorine, ferulic
acid
(Chen) C.Y.
Cheng, or
C.
teetoides
C. Y.
Chen
Radix et Rheum polygonaceaerhubarb derivatives of anthraquinone
Rhizoma root and
Rhei palmatum rhizome glycosides including
L. or
R. tanguticum chrysophanol, emodin,
aloe-
Maxim. et emodin, rhein, and
Reg. physcion,
(used in rheum tannic acids,
north gallic acid,
China) or catechin, tetrarin,
R. glucogallin,
officinale cinnamic acid, rheosmin,
Baill. fatty
(used in acids, calcium oxalate,
south glucose,
China fructose, sennoside
A, B, and C
Rhizoma Zingiber ingiberaceaeginger, gingerol, shogaol,
ginger and zingerone
Zingiberis officinale rhizome
Roscoe
Radix PanacisPanax AraliaceaeAmerican saponins, panaquilon
uin ue olii uin ue olium insen
L.
Radix GinsengPanax ginsengAraliaceaeginseng, panaxatriol, panaxadiol,
red other
(Rubra) C.A. Mey ginseng panoxisides, panoquilon,
panaxin, ginsenin,
a-panaxin,
protopanaxadiol,
protopanaxtriol, panacene,
panaxynol, panaenic
acid,
panose, dammarane,
glucose,
fructose, maltose,
sucrose,
nicrotinic acid, riboflavin,
thiamine
In the pharmaceutical compositions of the present invention, the root of
scutellaria, the rhizome of coptis, and the root and rhizome of rhubarb are
best prepared
by solvent extraction. The solvent can be water or any pharmaceutically
acceptable
17
CA 02466234 2004-05-04
organic solvent or a mixture of water and the organic solvent. The organic
solvent is
preferred to be alcohol.
In the pharmaceutical composition of the present invention, the weight ratio
of the
root of scutellaria, the rhizome of coptis, the root and rhizome of rhubarb,
and the root of
S ginseng or American ginseng is about 1-2 : 1-2 : 1-2 : 1-2, most favorably 1
: 1 : 1 : 1.
Optionally, the rhizome of ginger can replace the root of ginseng or American
ginseng.
The pharmaceutical compositions of the present invention can be formulated
into
tablet, bolus, powder, capsule, and granule by means suitable and known in the
art and
the pharmaceutical industry. The preferred formulation is tablet.
QUALITY CONTROL OF THE HERBS
The present invention uses High Performance Liquid Chromatography (HPLC) to
fingerprint each herb for the purpose of ensuring quality of individual herbal
ingredients.
The HPLC method and the test results of the herbal components of the present
invention
are described as follows:
A. Preparation of Herbal Extracts for HPLC
( 1 ) 0.5 gram of the herbal component was precisely weighed and placed in a
50-mL sample bottle.
(2) 20 mL of 70% methanol was added to the sample bottle of (1).
(3) The mixture of (2) was sonicated at room temperature for 15 minutes and
further shaked in a 40°C water bath at 160 rpm for 20 minutes; the
sample
18
CA 02466234 2004-05-04
was then sat for 30 minutes or more until two layers of the solution was
formed.
(4) The clear, upper layer of the solution was taken out and passed through a
0.45 ~m PVDF filter made by Whatman, England.
(5) About 20 ~L of the filtered solution was injected into the HPLC for
quantitative analysis.
B. Instruments for HPLC Anal,
The instruments used include Waters 600E Pump, Waters 717P1us Autosampler,
and Waters 996 Photodiode Array Detector.
C. HPLC Conditions and Results of Individual Herbs
1. Rhubarb
(a) HPLC conditions:
Guard column: Lichrospher RP-18 endcapped
(5 Vim, 4.0 ID x 10 mm, Merck, German)
Column: Symmetry Shield RP18
(5 pm, 4.6 ID x 250 mm, Waters, USA)
Column temperature: 40°C
Mobile phase: A: 0.5% acetic acid (CH3COOH) in water
B: acetonitrile (CH3CN)
Elution Gradients:
Time (minutes) A (%) B(%) Linearity
0 86 14
40 75 25 linear
60 55 45 linear
19
CA 02466234 2004-05-04
70 55 45 linear
90 0 100 linear
100 86 14 linear
Flow rate: 0.85 mL/min
Detection wavelength: 270 nm
(b) Results:
The HPLC chromatogram of the root and rhizome of rhubarb contains the
indicative ingredients of sennoside B, sennoside A, aloe-emodin, rhein,
emodin,
and chrysophanol. The retention times and maximum absorption wavelengths of
these ingredients are shown in Table 2.
Table 2: Retention Times and Wavelengths of the Ingredients in Rhubarb
Retention Time Maximum absorption
Compound (minutes) wavelength (7~max)
Sennoside B (SB) ~ 38 268 nm
Sennoside A (SA) ~ 46 269 nm
Aloe-emodin (AL) ~ 72 277 nm
Rhein (RH) ~ 87 257 nm
Emodin (EM) ~ 92.5 287 nm
Chrysophenol (CH) ~ 94 256 nm
2. Scutellaria
(a) HPLC conditions:
Guard column: Lichrospher RP-18 endcapped
(5 Vim, 4.0 ID x 10 mm, Merck, German)
Column: Cosmosil SC18-MS
(5 Vim, 4.6 ID x 250 mm, Nacalai tesque, Japan)
Column temperature: 35°C
CA 02466234 2004-05-04
Mobile phase: A: 20 mM KHZP04 and 0.01 % H3P04 in water
B: acetonitrile (CH3CN)
C: water (HZO)
Elution Gradients:
Time (minutes) A (%) B (%) C (%) Linearity
0 87 13 0
25 75 25 0 Linear
40 65 35 0 Linear
55 0 75 25 Linear
60 87 13 0 Linear
Flow rate: 1.0 mL/min
Detection wavelength: 280 nm
(b) Results:
The HPLC chromatogram of the root of scutellaria contains the indicative
ingredients of baicalin, oroxylin A-glucuronide, wogonin-7-O-glucuronide,
baicalein, wogonin, and oroxylin A. The retention times and maximum
absorption wavelengths of these ingredients are shown in Table 3.
Table 3: Retention Times and Wavelengths of the Ingredients in Scutellaria
21
CA 02466234 2004-05-04
Compound Retention Time Maximum absorption
(minutes) wavelength (a,max)
Baicalin (BG) ~ 30 276 nm
Oroxylin A - glucuronide ~ 36 269 nm
(OG)
Wogonin-7-O-glucuronide ~ 39 272 nm
(WG)
Baicalein (B) ~ 51 275 nm
Wogonin (W) ~ 56 274 nm
Oroxylin A (O) ~ 57 269 nm
3. Coptis
(a) HPLC conditions
Guard column: Lichrospher RP-18 endcapped
(5 Vim, 4.0 ID x 10 mm, Merck, German)
Column: Cosmosil 5C18-MS
(5 Vim, 4.6 ID x250 mm, Nacalai tesque, Japan)
Column temperature: 35°C
Mobile phase: A: buffered acetonitrile (The buffer contains 50
mM of CH3COONa, 2% CH3COOH, and 5 mM
CizHzsOS03Na)
B: H20 : CH3CN : CH30H = 10 : 45 : 45 (v/v)
Elution Gradients:
Time (minutes) A (%) B(%) Linearity
0 100 0
65 35 linear
30 65 35 linear
40 100 0 linear
Flow rate: 0.85 mL/min
Detection wavelength: 270 nm
(b) Results:
22
CA 02466234 2004-05-04
The HPLC chromatogram of the rhizome of coptis contains the indicative
ingredients of berberastine, columbamine, jatrorrhizine, epiberberine,
coptisine,
palmatine, and berberine. The retention times and maximum absorption
wavelengths of these ingredients are shown in Table 4.
Table 4: Retention Times and Wavelengths of the Ingredients in Coatis
Compound Retention Time Maximum absorption
-(minutes) wavelength (7~max)
Berberastine (Berber)~ 17 264 nm; 357 nm
Columnbamine (Col) ~ 21 264 nm; 345 nm
Jatrorrhizine (Jat) ~ 21.5 264 nm; 345 nm
Epiberberine (Epi) ~ 22.5 267 nm; 357 nm
Coptisine (Cop) ~ 23.5 264 nm; 358 nm
Palmatine (Pal) ~ 26 272 nm; 345 nm
Berberine (Ber) ~ 27 263 nm; 347 nm
4. Ginseng
(a) HPLC conditions
Guard column: Lichrospher RP-18 endcapped
(5 Vim, 4.0 ID x 10 mm, Merck, German)
Column: Cosmosil 5C18-MS
(5 ~.m, 4.6 ID x 250 mm, Nacalai tesque, Japan)
Column temperature: 35°C
Mobile phase: A: 20 mM KHZP04
B: CH3CN
C: H20
Elution Gradients:
Time (minutes) A (%) B (%) C (%) Linearity
0 80 20 0
23
CA 02466234 2004-05-04
20 75 25 0 Linear
40 65 35 0 Linear
55 0 80 20 Linear
60 0 20 80 Linear
65 80 20 0 Linear
Flow rate: 1.0 mL/min
Detection wavelength: 203 nm
(c) Results:
The HPLC chromatogram of the root of ginseng contains the indicative
ingredients of ginsenoside Rgl, ginsenoside Re, and ginsenoside Rb,. The
retention
times and maximum absorption wavelengths of these ingredients are shown in
Table 5.
Table 5: Retention Times and Wavelengths of the Ingredients in Ginseng
Compound Retention Time (minutes) Maximum absorption
wavelength (~,max)
Ginsenoside Rg~ (Rg,) ~ 23.5 204 mn
Ginsenoside Re (Re) ~ 23.8 203 nm
Ginsenoside Rbl (Rb~) ~ 38.5 203 nm
5. Giyer
(a) HPLC conditions
Guard column: Lichrospher RP-18 endcapped
(5 Vim, 4.0 ID x 10 mm, Merck, German)
Column: Cosmosil 5C 18-MS
(5 qm, 4.6 ID x 250 mm, Nacalai tesque, Japan)
Column temperature: 35°C
24
CA 02466234 2004-05-04
Mobile phase: A: 20 mM KHZP04
B: CH3CN
C: HZO
Elution Gradients:
Time (minutes) A (%) B (%) C (%) Linearity
0 70 30 0
20 40 60 0 Linear
30 0 80 20 Linear
35 0 30 70 Linear
Flow rate: 1.0 mL/min
Detection wavelength: 280 nm
(d) Results:
The HPLC chromatogram of the rhizome of ginger contains the indicative
ingredients of 6-gingerol, and 6-shogaol. The retention times and maximum
absorption wavelengths of these ingredients are shown in Table 6.
Table 6: Retention Times and Wavelengths of the Ingredients in Ginger
Compound Retention Time Maximum absorption
(minutes) wavelength (~,max)
6-Gingerol (G) 17 230 nm; 285 nm
6-Shogaol (S) 26 230 nm; 285 nm
PHARMACEUTICAL COMPOSITIONS
The pharmaceutical compositions of the present invention is prepared by the
following procedures:
(1) Preparation of Herbal Extracts
CA 02466234 2004-05-04
Some of the herbs (e.g., scutellaria, coptis, and rhubarb) are preferred to be
individually extracted by a solvent. The solvent can be water or organic
solvent that is
pharmaceutically acceptable for extraction purpose, or a mixture of water and
the organic
solvent. The preferred organic solvent is alcohol. It is preferred that the
root of
scutellaria and the rhizome of coptis are extracted by water, and the root and
rhizome of
rhubarb is by alcohol, especially 95% of alcohol in water (v/v). The extracts
are further
filtered.
(2) Condensation of the Herbal Extracts
For the herbs that have been prepared by extraction, the individual herbal
extracts,
after filtration, are pooled together and condensed under reduced pressure in
a water bath
(maintained at 50°C) until an herbal paste is formed.
(3) Preparation of Dry Powders
All of the herbs used in the herbal pharmaceutical compositions can be used in
the
form of dry powders. However, for better therapeutic effects, all herbs,
except
ginseng/American ginseng or ginger, are preferred to be prepared by solvent
extraction.
As to ginseng/American ginseng or ginger, the dry powders form of the herbs
are
prepared by cutting the herbs into small pieces, followed by grinding and
drying them
into powders. The dry powders of the herbs are then passed through a sieve to
ensure
that the size of the dry powders are within certain ranges.
(4) Preparation of A Herbal Pharmaceutical Composition
The dry powders of the herbs are mixed in with the herbal paste as shown in
(2),
supra, to form an herbal mixture, which is further dried to form the
pharmaceutical
composition. Additionally, the pharmaceutical composition can be processed
into tablet,
26
CA 02466234 2004-05-04
bolus, powder, capsule, and granule by means of formulation which is well-
known to
those ordinary skill in the art, particularly in the pharmaceutical industry.
Excipients,
binders, carriers, fillers can be added to the herbal mixture.
The following examples are for illustrative purpose and are not intended to
limit
the scope of the invention. Reasonable variations, such as those understood by
reasonable artisans, can be made herein without departing from the scope of
the present
invention.
~~r a r~rnr ~ ~
Preparation of Herbal Pharmaceutical Composition 1
Herbal pharmaceutical composition 1 of the present invention was prepared as
follows:
(1) About 20 grams of each of the root of scutellaria, the rhizome of coptis,
the root and rhizome of rhubarb, and ginseng, in the form of "Yin Pian"
(meaning
"drinking pieces"), which contained small thin slices of the herb that were
ready for
decoction use, were individaully meansured.
(2) The herbs of (1) were individually ground in a grinder into individual
powder forms.
(3) The individaul powders of (2) were passed though a 120-mesh sieve
respectively and the resultant powders were collected.
(4) The individal powders of (3) were mixed together until a homogeneous
mixture was obtained.
27
CA 02466234 2004-05-04
Preparation of Herbal Pharmaceutical Composition 2
The herbal pharmaceutical composition 2 of the present invention was prepared
as
follows:
(1) About 20 grams of each of the root of scutellaria, the rhizome of coptis,
and the root and rhizome of rhubarb, in the form of "Yin Pian" (meaning
"drinking
pieces"), which contained small thin slices of the herb that were ready for
decoction use,
were individaully meansured.
(2) The individually measured herbs of (1) were separately simmered and/or
boiled in about 20 volumes of water for about 60 minutes to produce individual
herbal
extracts.
(3) The individually boiled herbal extracts were passed through a 100-mesh
sieve when the extracts were still hot; and the filtered extracts were
individually
collected.
(4) The individually filtered herbs of (3) were mixed together and condensed
under condensed pressure in a SO°C water bath until a paste was formed.
(5) About 20 grams of the dry powders of ginseng or American ginseng were
prepared according to (1) and (2) of Example 1, supra.
(6) The herbal paste of (4) and the dry powders of (5) were mixed together
until a homogeneous paste was obtained.
28
CA 02466234 2004-05-04
EXAMPLE 3
Preparation of Herbal Pharmaceutical Composition 3 (STC)
The herbal pharmaceutical composition 3 of the present invention was prepared
as
follows (NOTE: The herbal pharmaceutical composition 3 is also known as STC,
which
is named after "SunTen Cardiovascular Drug"):
(1) About 20 grams of each of the root of scutellaria, the rhizome of coptis,
and the root and rhizome of rhubarb, in the form of "Yin Pian" (meaning
"drinking
pieces"), which contained small thin slices of the herb that were ready for
decoction use,
were individaully meansured.
(2) The small pieces of scutellaria and coptis were individually simmered
and/or boiled in about 20 volumes of water for about 60 minutes to produce the
extracts
of scutellaria and coptis, respectively.
(3) The extracts of scutallaria and coptis were separately passed through a
100-mesh sieve when the extracts were still hot; and the filtered herbal
extracts were
individually collected.
(4) The filtered herbal extracts of (3) were mixed together and condensed
under reduced pressure in a 50°C water bath until a paste was formed.
(5) About 20 grams of root and rhizome of rhubarb, in the form of "Yin Pian"
were meansured, and extracted under refluxing in about 20 volumes of alcohol :
water
(95:5, v/v) for 60 minutes to produce a rhubarb extract.
(6) The rhubarb extract of (6) was passed through a 100-mesh sieve; and the
filtered rhubarb extract was colleced.
29
CA 02466234 2004-05-04
(7) The filtered rhubarb extract was condensed under reduced pressure in a
50°C water bath until a paste was formed.
(8) About 20 grams of dry powders of ginseng or American ginseng was
prepared according to (1) and (2) of Example 1, supra.
(9) The dry powders of (8) were mixed with the pastes of (4) and (7) until a
homogeneous paste was obtained.
DVATiTDTD A
Preparation of Herbal Composition 4
The herbal pharmaceutical composition 4 of the present invention was prepared
using the same procedures as Example 2, except that the root of scutellaria,
the rhizome
of coptis, and the root and rhizome of rhubarb were extracted in alcohol :
water (50:50,
v/v), rather than water.
DVATifDTD G
Preparation of Herbal Pharmaceutical Composition 5
The herbal pharmaceutical composition 5 of the present invention was prepared
using the same procedures as in Example 2, except that the root of
scutellaria, the
rhizome of coptis, and the root and rhizome of rhubarb were extracted in
alcohol : water
(95:5, v/v).
EXAMPLE 6
Preparation of Herbal Pharmaceutical Composition 6
The herbal pharmaceutical composition 6 of the present invention was prepared
CA 02466234 2004-05-04
using the same procedures as in Example 1, except that ginger was used to
replace
ginseng or American ginseng.
EXAMPLE 7
Preparation of Herbal Pharmaceutical Composition 7
The herbal pharmaceutical composition 7 of the present invention was prepared
using the same procedures as in Example 2, except that ginger was used to
replace
ginseng or American ginseng.
EXAMPLE 8
Preparation of Herbal Pharmaceutical Composition 8
The herbal pharmaceutical composition 8 of the present invention was prepared
using the same procedures as in Example 4, except that ginger was used to
replace
ginseng or American ginseng.
EXAMPLE 9
Preparation of the Herbal Pharmaceutical Composition 9
The herbal pharmaceutical composition 9 of the present invention was prepared
using the same procedures as in Example 4, except that ginger was used to
replace
ginseng or American ginseng.
EXAMPLE 10
Preparation of Herbal Pharmaceutical Composition 10
31
CA 02466234 2004-05-04
The herbal pharmaceutical composition 10 of the present invention was prepared
using the same procedures as in Example 5, except that ginger was used to
replace
ginseng or American ginseng.
COMPARATNE EXAMPLE 1
Preparation of Modified San-Huang-Hsie-Hsin-Tang
San-Huang-Hsie-Hsin-Tang contains the root of scutellaria, the rhizome of
coptis,
and the root and rhizome of rhubarb. The commercially available San-Huang-Hsie-
Hsin-
Tang is a decoction, which contains the "Yin-Pians" (small thin slices) of the
root of
scutellaria, the rhizome of coptis, and the root and rhizome of rhubarb. When
in use, the
"Yin-Pians" were placed in a bowl and boiled water was added to the "Yin-
Pians"-
containing bowl for drink as a soup or a beverage. Alternatively, the "Yin-
Pians" of San-
Huang-Hsie-Hsin-Tang can be placed in a boiler to be cooked with water.
To properly compare the herbal pharmaceutical compositions of the present
invention (i.e., Examples 1-10) with San-Huang-Hsie-Hsin-Tang, the three
ingredients of
the San-Huang-Hsie-Hsin-Tang were prepared the same way as that in Example 2,
i.e.,
by boiling the "Yin-Pians" of the root of scutellaria, the rhizome of coptis,
and the root
and rhizome of rhubarb individually in water. The extracts were filtered,
combined and
then condensed into a paste.
COMPARATNE EXAMPLE 2
Preparation of Modified San-Huang-Hsie-Hsin-Tank
To properly compare the herbal pharmaceutical compositions of the present
32
CA 02466234 2004-05-04
invention (i.e., Examples 1-10) with San-Huang-Hsie-Hsin-Tang, the three
ingredients of
the San-Huang-Hsie-Hsin-Tang were prepared the same way as that in Example 3,
i.e.,
by boiling the "Yin-Pians" of the root of scutellaria and the rhizome of
coptis
individually in water and extracting the root and rhizome of rhubarb in
alcohol : water
(95 : 1, v/v) under refluxing. The extracts of scutellaria and coptis were
separately
filtered, combined, and condensed into a paste. The extract of rhubarb was
filtered and
condensed into a paste. The paste of scutellaria and coptis and the paste of
rhubarb was
then combined and mixed thoroughly.
PHARMACOLOGICAL STUDIES
The following pharmacological studies confirm that the herbal pharmaceutical
compositions of Examples 1-10, supra, were capable of lowering high blood
pressure,
maintaining stable blood pressure (in the normal range), improvimg
cardiovascular
diseases in the elderly, inhibiting the formation of nitrite, inhibiting the
proliferation of
smooth muscle, reducing CRP formation, inhibiting iNOS expression, and
inhibiting
COX-2 activity. Such herbal compositions were not only useful for treating
patients with
normal physique, but also safe and effective for treating patients who were
elderly or
debilitated.
Although herbal pharmaceutical compositions 1-10, supra, all demonstrated
therapeutic effectiveness in treating patients with cardiovascular diseases,
Example 3,
also known as STC, appeared to be superior to the rest of the Examples.
Therefore, for
the purpose of simplifying the studies to be presented below, only STC was
chosen as
representing the pharmaceutical composition of the present invention.
33
CA 02466234 2004-05-04
STUDY 1
Effects of the herbal compositions on lowerin bghpressure
Spontaneous hypertensive rats (SHR) were obtained from the National
Experimental Animal Center, Taiwan. The SHR rats were first anaesthetized by
intraperitoneal injection of urethane at 9g/kg. The SHR rats were then
inserted with a
trachea tube, maintained on a respiration apparatus for small animals, and
inserted with
catheters into the femoral vein and femoral artery.
The catheter from the femoral artery was connected to a pressure transducer,
which transmitted the change in pressure to a multi-function recorder for
monitoring of
the artery blood pressure, mean arterial blood pressure, and heart rate. The
femoral vein
catheter was in place for intravenous injection. The body temperature of the
rats was
maintained at 37-38°C by an electric blanket. The baseline values were
recorded after
the blood pressure of the SHR rats were stabilized.
The SHR rats were separated into six groups and received a single dose of one
of
the following medications through gavage: (1) 144 mg/kg of the root of
scutellaria alone
(H1); (2) 144 mg/kg of the root and rhizome of rhubarb alone (H3); (3) 144
mg/kg of the
combination of the root of scutellaria and the rhizome of coptis (H1+2); (4)
144 mg/kg of
the compositions San-Huang-Hsie-Hsin-Tang as in COMPARATNE EXAMPLE 1 (M);
(5) 144 mg/kg of STC (Example 3); and (6) the combination of captopril (ACE
inhibitor)
1 mg/kg and nifedipine (calcium channel Mocker) 0.4 mg/kg (A). The SHR rats in
the
Western medicine control group (A) had been pre-treated with one week of
captopril and
nifedipine at the same doses.
34
CA 02466234 2004-05-04
Blood pressure and heart rate were monitored continuously throughout the study
period. Percent change in the mean arterial blood pressure (maBP) was
expressed as the
ratio of maBP after treatment to the baseline maBP before treatment. The
within-group
baseline and post-treatment results were analyzed using the paired Student's T
test.
As presented in Figure l, all medications showed significant reduction of the
blood pressure (indicated with an asterisk, *). However, the root and rhizome
of rhubarb
alone (H3) was less effective in lowering the blood pressure. Also, the
combination of
the root of scutellaria and the rhizome of coptis (H1+2) show a large
variation in the
blood pressure reduction.
STUDY 2
Effects of the herbal compositions on
maintaining and controllin bg lood rep ssure
Spontaneous hypertensive rats (SHR) were obtained from the National
Experimental Animal Center, Taiwan. The SHR rats were anaesthetized by
intraperitoneal injection of urethane at 9g/kg. The SHR rats were then
inserted with a
trachea tube, maintained on a respiration apparatus for small animals, and
inserted with
catheters into the femoral vein and femoral artery.
The catheter from the femoral artery was connected to a pressure transducer,
which transmitted the change in pressure to a mufti-function recorder for
monitoring of
the artery blood pressure, mean arterial blood pressure, and heart rate. The
femoral vein
catheter was in place for intravenous injection. The body temperature of the
rats was
maintained at 37-38°C by an electric blanket. The baseline values were
recorded for 60
minutes after the blood pressure of the SHR rats were stabilized.
CA 02466234 2004-05-04
The SHR rats were separated into groups (seven rats per group) and received
through gavage daily for two weeks of (1) no treatment (control); (2) 48 mg/kg
of
composition of COMPARATIVE EXAMPLE 2 (M); (3) 48 mg/kg of STC(Example 3);
or (4) the combination of captopril 1 mg/kg and nifedipine 0.4 mg/kg (A). The
dosages
of M and STC were one-third of those of STUDY 1.
Blood pressure and heart rate were monitored continuously throughout the study
period. Values of the mean arterial blood pressure (maBP) and heart rate (HR)
were
compared on the day after the completion of the 2-week dosing. The between-
group
results were analyzed using the unpaired Student's T test. The within-group,
baseline
and post-treatment results were analyzed using paired Student's T test.
As shown in Figure 2a, only the group giving STC demonstrated lower and
maintained mean arterial blood pressure on the day after the completion of the
doses. In
contrast, the traditional San-Huang-Hsie-Hsin-Tang decoction (M) and the
combination
of captopril and nifedipine (A) did not maintain their anti-hypertensive
effects on the day
after dose completion, despite the multiple dose administration for two weeks.
As shown in Figure 2b, all three treatments had no effects on the heart rate.
STUDY 3
Effect of the Herbal Compositions on Preventing H~,rpertension
The spontaneous hypertension rats (SHR) and normal rats (WKY) younger than 8
weeks old were obtained from the National Experimental Animal Center, Taiwan.
Baseline measures of heart rate and blood pressures at the tail are first
recorded. The
SHR rats were then treated with 14.4 mg/kg/day of the herbal compositions of
the present
36
CA 02466234 2004-05-04
invention as described in the below Example 4 through gavage for one week. The
between-group comparison of the baseline measures of the WKY and SHR rats was
carried out using the unpaired Student's T test with the statistical
significance indicated
by an asterisk in the figures. The within-group comparison of the baseline and
post-
treatment measures of the SHR rats was carned out using paired Student's T
test with the
statistical significance indicated by an "a" in the figures.
As shown in Figures 3a and 3b, the untreated spontaneous hypertensive rats had
statistically significantly higher heart rate (HR), systolic pressure (SYS),
diastolic
pressure (DIA) and mean arterial blood pressure (MED) than the normal rats
(WKY).
Treatment with the herbal compositions of the present invention (STC)
significantly
reduced the systolic pressure and mean arterial blood pressure in the
spontaneous
hypertensive rats (p < 0.05, n = 5) to levels that were comparable to the
normal WKY
rats.
STUDY 4
Effect of the Herbal Compositions on Improving
Cardiovascular Conditions in the Elderly
Part 1. In vivo test
Normal WKY rats that were 8 weeks and 10 months of age were used in this
study. Baseline blood pressures and heart rate measured at the tails were
first recorded
for both groups. The 10-month old rats were then treated with one single dose
of 48
mg/kg of the herbal compositions of the present invention. Heart rate and
blood
pressures were measured at 1 hour after the dosing of STC. The between-group
comparison of the baseline measures of the 8-week old (Young) and the 10-month
old
37
CA 02466234 2004-05-04
WKY rats (Old) was carried out using the unpaired Student's T test with the
statistical
significance indicated by an asterisk in the figures. The within-group
comparison of the
baseline (Old) and post-treatment measures of the 10-week old WKY rats (STC)
was
carned out using paired Student's T test with the statistical significance
indicated by an
"a" in the figures.
As shown in Figures 4a and 4b, the heart rate (HR), diastolic pressure (DIA)
and
mean arterial blood pressure (maBP) were higher, but not significantly, in the
old WKY
rats than the young rats. Treatment with the herbal compositions of the
present invention
(STC) significantly reduce the diastolic pressure and mean arterial blood
pressure in the
old WKY rats (p < 0.05, n = 4, indicated with "a" in Figure 4b) at one hour
after the dose
administration.
Part 2. Non-circulation perfusion test
Ten-month old SHR rats were anaesthetized by intraperitoneal injection of
urethane. The hearts of the anaesthetized rats were removed under assisted
respiration.
1 S The hearts were immediately inserted with an arterial catheter at the
cutting end of the
aorta and perfused with 37°C Krebs-Henselein (KH) buffer at the
pressure of 80 cmH20.
The buffer contained 118 mM NaCI, 24.0 mM NaHC03, 4.7 mM KCI, 1.2 mM KHzP04,
1.2 mM MgS04, 1.7 mM CaClz, and 10.0 mM glucose continuously gassed with the
mixture of 95% OZ and 5% CO2.
The isolated hearts were perfused for 15 minutes with the KH buffer and then
with the KH buffer containing the herbal compositions of the present invention
(STC) for
15 minutes, reaching a steady state. The coronary flow and coronary perfusion
pressure
(CPP) were measured (1) before the perfusion of STC, (2) after the perfusion
of STC, and
38
CA 02466234 2004-05-04
(3) after reperfusion with STC for 60 minutes post L-phenylephrine-induced
ischemia.
The results indicated that perfusion with the herbal compositions of the
present invention
as described in the below Example 4 could improve the reduction in the
coronary flow
that was induced by 10 mM L-phenylephrine from 12.6 mL/min to 13.2 mL/min.
This
suggested the herbal pharmaceutical compositions of the present invention was
effective
in improving cardiovascular conditions.
STUDY 5
Effects of the Herbal Compositions in Inhibiting the Enzyme Formations
and Activities of iNOS and COX-2
The mouse macrophage cell line RAW 264.7 was used in this study. The mouse
macrophage cells were treated with lipopolysaccharide (LPS) and various
amounts of the
herbal compositions of the present invention for 12 hours. LPS is known to
induce the
syntheses of the enzymes, iNOS and COX-2 in the mouse macrophage cells. The
treated
macrophage cells were first detached from the cultural plates by repeated
flush using
micropipett. The cell mixtures were then harvested and centrifuged at 2000
rpm. The
clear supernatants were collected and assayed for the content of nitrile using
the Griess
Test Reagent and for the content of PGEZ using the ELISA method. The pellets
formed
after the centrifugation were mixed with the Lysis buffer and homogenized. The
resultant mixtures were assayed for protein content. A 30-~g aliquot of the
homogenized
mixture was placed on the polyacrylamide gel to separate the proteins by
electrophoresis.
Proteins on the developed polyacrylamide gel were then transferred onto the
39
CA 02466234 2004-05-04
polyvinylidene difluoride (PVDF) membrane and detected with the antibodies for
iNOS
and COX-2.
The results showed that the herbal compositions of the present invention was
effective in inhibiting the formation of the nitrile (Figure 5) by inhibiting
the protein
synthesis of iNOS. The herbal compositions of the present invention inhibited
the
formation of PEG2, the end product of COX-2 (Figure 6), but not the protein
synthesis of
the enzyme itself, suggesting that the herbal compositions of the present
invention might
possess anti-inflammatory activity by inhibiting the activity of COX-2.
STUDY 6
Effect of the Herbal Compositions on Inhibition of
iNOS and COX-2 Protein Biosynthesis
Sprague Dawley (SD) rats and the spontaneous hypertensive (SHR) rats were
used in this study. The hearts from lipopolysaccharide-treated SD rats and the
lungs from
SHR rats treated with the herbal compositionss of the present invention and
other control
groups were removed from the animals. Small portions of the isolated organs
were first
ground in mortars containing liquid nitrogen and then homogenized in the Lysis
buffer.
Aliquots of the resultant mixtures were assayed for protein contents. Another
30-~.g aliquots of the resultant mixtures were placed on the polyacrylamide
gel to
separate the proteins by electrophoresis. Proteins on the developed
polyacrylamide gel
were then transferred onto the polyvinylidene difluoride (PVDF) membrane and
detected
with the antibodies for iNOS and COX-2.
The results showed that intraperitoneal treatment of 20 mg/kg LPS induced
inflammatory responses in the SD rats. The SD rats showed symptoms of
abdominal
CA 02466234 2004-05-04
pain and writhing. As shown in Figures 7a and 7b, formations of the COX-2 and
iNOS
proteins, respectively, were highest in the hearts of the SD rats (Column 1 ),
then followed
by the lungs of the untreated SHR rats (Column 3). STC (Example 3) clearly
inhibited
the formation of the COX-2 and iNOS proteins in the SHR rats (Column 2).
STUDY 7
Effect of the Herbal Compositions on
Inhibition of iNOS Gene Expression
The spontaneous hypertensive (SHR) rats were treated with (1) the
pharmaceutical compositions of the present invention, STC (S); (2) western
medicine; or
(3) control. Lungs from the animals were removed and tissue RNA was extracted
using
Trizol. The concentrations and ratios of the extracted RNA were determined
using the
spectrometer. The cDNA was then prepared from 1-~g aliquot of RNA by reverse
transcription. A 2-p,L of the cDNA solutions was mixed with the iNOS primer
and
multiplied using the polymerise chain reaction (PCR) method. The resultant DNA
mixtures were run through the agarose gel for DNA separations.
As indicated in 8a, detection of the (3-actin cDNA on the agarose gel
indicated the
RNA extraction process was properly carned out. As shown in Figure 8b, the DNA
bands was clearly seen in the western medicine group (A), less obvious in the
control
(untreated) group (C) and not observed for the SHR rats treated with the
herbal
compositions of the present invention (S). This suggested that the herbal
compositions of
the present invention could inhibit the expression of iNOS in SHR rats.
41
CA 02466234 2004-05-04
cTT my R
Effect of the Herbal Compositions on Reduction of CRP
The spontaneous hypertensive rats were treated with 48 mg/kg of STC (Example
3) for 2 weeks. After the conclusion of the oral dose administration, blood
samples were
collected from both the control (untreated) rats and the STC-treated rats.
Plasma samples
were prepared from the blood samples by high-speed centrifugation and then
assayed for
the CRP contents using ELISA.
As indicated in Figure 9, the herbal pharmaceutical compositions of the
present
invention was effective in reducing CRP formation in the spontaneous
hypertensive rats.
While the invention has been described by way of examples and in terms of the
preferred embodiments, it is to be understood that the invention is not
limited to the
disclosed embodiments. On the contrary, it is intended to cover various
modifications as
would be apparent to those skilled in the art. Therefore, the scope of the
appended claims
should be accorded the broadest interpretation so as to encompass all such
modifications.
42
