LEE YONG SUP (KR)
KIM HYOUNG JA (KR)
JUNG SUH YUN (KR)
CHO JUNGSOOK (KR)
JIN CHANGBAE (KR)
LEE YONG SUP (KR)
KIM HYOUNG JA (KR)
JUNG SUH YUN (KR)
CHO JUNGSOOK (KR)
WO2003037324A1 | 2003-05-08 | |||
WO2005041994A1 | 2005-05-12 |
DOK-GO H. ET AL.: "Neuroprotective effects of antioxidative flavonoids, quercetin, (+)-dihydroquercetin and quercetin 3-methyl ether, isolated from Opuntia ficus-indica var. saboten", BRAIN RES., vol. 965, no. 1-2, March 2003 (2003-03-01), pages 130 - 136, XP002493220, DOI: doi:10.1016/S0006-8993(02)04150-1
SALEEM M. ET AL.: "Secondary metabolites from Opuntia ficus-indica var. saboten", PHYTOCHEMISTRY, vol. 67, no. 13, July 2006 (2006-07-01), pages 1390 - 1394, XP028059908, DOI: doi:10.1016/j.phytochem.2006.04.009
[CLAIMS]
[Claim 1]
A pharmaceutical composition for treating and/ or preventing cranial nerve
diseases, cerebrovascular diseases and cardiovascular diseases comprising a butanol
extract of Opuntia ficus-indica as an active ingredient.
[Claim 2]
A pharmaceutical composition for treating and/ or preventing cranial nerve
diseases, cerebrovascular diseases and cardiovascular diseases comprising an acid
hydrolysate of a butanol extract of Opuntia ficus-indica as an active ingredient.
[Claim 3]
The pharmaceutical composition according to claim 1 or 2, which is useful
for treating and/ or preventing a disease selected from the group consisting of stroke,
concussion, Alzheimer's disease, Parkinson's disease, cell death and myocardial
infarction.
[Claim 4]
The pharmaceutical composition according to claim 1 or 2, wherein the
butanol extract of Opuntia ficus-indica is obtained by a process comprising:
1) adding 0.1 to 10 L of a C 1 -C 4 lower alcohol solution per 1 kg of the stem or
fruit of Opuntia ficus-indica and performing extraction at 20 to 90 0 C under reflux;
2) filtering and evaporating the extract obtained in the step 1) under reduced pressure to obtain an alcohol extract; and
3) adding 0.1 to 10 L of water per 1 kg of the alcohol extract and performing
extraction with 1 to 5 L of butanol (n-BuOH).
[Claim 5]
In claims 1 and 2, the butanol extract of Opuntia ficus-indica is obtained by:
adding 0.1 to 10 L of butanol per 1 kg of the stem or fruit of Opuntia ficus-
indica and performing extraction at 20 to 90 0 C under reflux.
[Claim 6]
In claim 2, the acid hydrolysate is a filtrate obtained by:
acid hydrolyzing the butanol extract of Opuntia ficus-indica with dioxane and
hydrochloric acid, neutralizing to pH 6 to 8 using an alkali, and filtering using a C 1 -
C 4 lower alcohol.
[Claim 7]
The pharmaceutical composition according to claim 4, wherein the lower
alcohol solvent used in step 1) is selected from the group consisting of an absolute
alcohol and an aqueous alcohol solution with a concentration 50 % or higher.
[Claim 8]
The pharmaceutical composition according to claim 1 or 2, which is prepared
into an oral administration drug form.
[Claim 9] The pharmaceutical composition according to claim 8, which is prepared into
an oral administration drug form selected from the group consisting of tablet, pill,
powder, sachet, elixir, suspension, emulsion, solution, syrup, aerosol, and soft or
hard gelatin capsule.
[Claim 10]
The pharmaceutical composition according to claim 1 or 2, which is
prepared into an injection solution or injection suspension.
[Claim 11]
The pharmaceutical composition according to claim 1 or 2, wherein the
butanol extract comprises an active ingredient selected from the group consisting of:
isorhamnetin-3-O-(6'-O-E-feruloyl) neohesperidoside (1), 2,3,4-
trihydroxybenzoic acid (2), 4-hydroxybenzoic acid (3), ferulic acid (4),
isorhamnetin 3-O-glucoside (5), 2,3-dihydroquercetin (6), cinnamic acid (7),
kaempferol 7-O-glucopyranoside (8), zataroside-A (9), 4-O-
glucopyranosylsinapinic acid (10), isorhamnetin 3-O-rutinosyl-4'-O-β-D-glucoside
(11), isorhamnetin 3-O-(2,6-dirhamnosyl)glucoside (12), isorhamnetin 3-O-
rutinoside (nacissin) (13), 2,3-dihydrokaempferol (14), quercetin 3'-O-β-D-glucoside
(15), quercetin 3-O-methyl ether (16), isorhamnetin 3-O-neohesperidoside (17), and
n-butyl-β-D-f ructopyranoside (18) .
[Claim 12] The pharmaceutical composition according to claim 1 or 2, wherein the
butanol extract exhibits an HPLC analysis pattern as shown in Figure 1.
[Claim 13]
A pharmaceutical composition for treating and/ or preventing cranial nerve
diseases, cerebrovascular diseases and cardiovascular diseases, comprising at least
one active ingredient selected from the group consisting of:
isorhamnetin-3-O-(6'-O-E-feruloyl) neohesperidoside (1), 2,3,4-
trihydroxybenzoic acid (2), 4-hydroxybenzoic acid (3), ferulic acid (4),
isorhamnetin 3-O-glucoside (5), 2,3-dihydroquercetin (6), cinnamic acid (7),
kaempferol 7-O-glucopyranoside (8), zataroside-A (9), 4-O-
glucopyranosylsinapinic acid (10), isorhamnetin 3-O-rutinosyl-4'-O-β-D-glucoside
(11), isorhamnetin 3-O-(2,6-dirhamnosyl)glucoside (12), isorhamnetin 3-0-
rutinoside (nacissin) (13), 2,3-dihydrokaempferol (14), quercetin 3'~O-β-D-glucoside
(15), quercetin 3-O-methyl ether (16), isorhamnetin 3-O-neohesperidoside (17), and
n-butyl-β-D-fructopyranoside (18).
[Claim 14]
Isorhamnetin-3-O-(6'-O-E-feruloyl) neohesperidoside. |
[DESCRIPTION]
[Invention Title]
PHARMACEUTICAL COMPOSITION COMPRISING AN EXTRACT
FROM OPUNTIA FICUS-INDICA
[Technical Field]
The present invention relates to a pharmaceutical composition comprising an
extract from Opuntia ficus-indica, more particularly to a pharmaceutical composition
comprising a butanol extract from Opuntia ficus-indica or an acid hydrolysate of the
extract as an active ingredient, and effective in treating or preventing cranial nerve
diseases, cerebrovascular diseases or cardiovascular diseases, for example, stroke,
concussion, Alzheimer's disease, Parkinson's disease, cell death, myocardial
infarction, and so forth.
[Background Art]
When the cerebral artery or coronary arteries are obstructed by thrombosis
or arteriosclerosis and the blood flow to the brain decreases to a threshold value, the
brain cells and heart cells are damaged by ischemia, resulting in cell death, cell death
and myocardial infarction. Hypoxia caused by ischemia reduces oxidative
phosphorylation and ultimately leads to the stoppage of anaerobic glycolysis,
resulting in the depletion adenosine triphosphate (ATP) which is an energy source
of cells. When the energy falls below a critical threshold value, cell activities
essential for the survival of a cell are inhibited and various degenerative processes
resulting therefrom lead to the cell death. Cell death may occur not only during
ischemia but also during reperfusion.
In particular, cerebral ischemia is the commonest clinical condition found in
cardiac arrest and stroke. It occurs mostly in aged people and is a very serious
medical problem since it leads to intractable brain damage. A severe damage of
brain cells may lead to brain function loss, unconsciousness, and even death.
Drugs for treating hypertension, improving blood flow to the brain, treating
hyperlipemia, and so forth are used to prevent the condition. However, there is no
internationally, clinically approved nerve-protection medicine that can be used to
protect the brain cells from the damage caused by ischemia once stroke outbreaks,
excluding clotbusters.
Worldwide researches are actively carried out to find out the mechanisms
and treatment strategies of the process from cerebral ischemia to brain damage,
including the development of animal models, in vitro neuronal/ glial cell culture
system, and so forth. Based on such test models, the glutamate cascade hypothesis
was proposed in 1980s that ischemia causes a large amount of excitatory
neurotransmitters such as glutamate to be freed, allowing high levels of calcium ions
to enter the brain cells through the action of receptors, thereby leading to the death
of the cells through excitotoxicity [Choi, D. W. /. Neurosci. 1987, 7, 369-379]. Drugs
based on this hypothesis exhibited reduction of brain damage caused by focal
ischemia in animal model test and some of them are on clinical trial. However,
most of them exhibited side reactions or insufficient efficiency, and the development
was stopped. Also, the use of calcium channel blockers such as nimodipine,
lifarizine (Syntex; dropped out in phase I) 1 SNXlIl and isradipine was tried to block
the influx of calcium ions into the cells, but the effect was not constant and much
remains to be studied.
According to the neurotoxic cascade hypothesis of ischemic brain damage,
excessive cytotoxic calcium ions present in the cell generate reactive oxygen species
(ROS) and reactive nitrogen species (RNS) by activation of nitrogen oxide synthase
(NOS) and excessive generation of NO. In the mitochondria, the increased calcium
ions inhibit oxidative phosphorylation, thereby further reducing the energy supply
and increasing the generation of free radicals. Excessively generated free radicals
damage not only DNAs, but also cell membranes through lipid peroxidation. And,
when ischemia is recovered spontaneously or by treatment and blood flow is
restored by reperfusion, the oxygen flow may enhance the biological reactions that
produce free radicals.
Recently, reports that NO, which is a free radical that can function as signal
transmitter as well as neurotoxin, plays an important role in ischemic brain damage
are increasing [Iadecola, C. Trends in Pharmacol. Sd. 1997, 20, 132-9]. Most of former
researches focused on the development of treatment for stroke through the synthesis
of antioxidant mainly by modifying the configuration of vitamin E or C, and the
effort to develop a medicine for retarding or preventing the brain damage followed
by cerebral ischemia was insignificant.
Opuntia ficus-indica has long been used as the folk remedy for treating burn,
edema, dyspepsia, abscess, bronchial asthma, and the like. A 70 % methanol
extract of its fruit is reported to be effective in inhibiting nerve damage [Nam Ho Lee,
et al., Kor. J. Pharmacogn. 2000, 31, 412-415; Myung-Bok Wie, Yakhak Hoiji 2000, 44,
613-619]. Former researches were mainly about the activity of the alcohol extract of
Opuntia ficus-indica in in-vitro experiment indicating that the extract from Opuntia
ficus-indica may provide a pharmacological effect.
The present inventors have shown that an ethyl acetate extract of Opuntia
ficus-indica (var. saboten Makino) and quercetin 3-methyl ether, an active compound
isolated from the extract, are effective in preventing oxidation and protecting brain
cells in animal models of stroke, when administered intravenously, and have been
issued a patent thereon [Korean Patent No. 523,562]. In Korean Patent No. 523,562,
the present inventors have tested the extracts obtained from the stem or fruit of
Opuntia ficus-indica using methanol (MeOH), dichloromethane (CH2CI2), ethyl
acetate (EA) and butanol (BuOH) as extractant, as illustrated in the figure below, for
the efficiencies in radical scavenging, xanthine/ xanthine oxidase-induced
neurotoxicity prevention, and hydrogen peroxide neurotoxicity prevention. The
EA fraction exhibited the most superior nerve cell damage protection effect, and
quercetin 3-methyl ether and other substances were isolated from the EA fraction as
active compounds.
Opuntia ficus-india
MeOH CHgCI 2 EA* BuOH HfeO
However, Korean Patent No. 523,562 mentions only about the use as an
injection for intravenous administration, and does not consider the use of the EA
extract from Opuntiα ficus-indicα or an active compound isolated therefrom in oral
administration.
At present, most of the worldwide researches regarding the treatment of
stroke are focusing on the development of an injection, and an oral drug that can
protect the brain cells from the damaged caused by ischemia is almost non-existent.
According to a report, it is more advantageous to utilize a natural product in the
form of an extract rather than in the pure form of the corresponding active
ingredient [M. Kaszkin, et al., Phytomedicine 2004, 11, 585-595]. However, in that
case, the relative amount of the active ingredient decreases and, thus, it becomes
difficult to prove the significance of the related effect.
During the researches to develop an extract from Opuntia ficus-indica
adequate for an oral administration form, the present inventors found out that the
butanol extract of Opuntia ficus-indica exhibits superior nerve cell protection effect
and is adequate for oral administration.
The BuOH fraction disclosed in Korean Patent No. 523,562 was one
obtained extracting Opuntia ficus-indica with methanol, dichloromethane and ethyl
acetate, in sequence, and then extracting the residue with BuOH, and its nerve cell
protection effect was insignificant compared with that of the EA fraction. In
contrast, the BuOH extract claimed by the present invention is one obtained by
extracting Opuntia ficus-indica with butanol, and exhibits superior nerve cell
protection effect compared with the butanol fraction mentioned in the above patent.
Especially, it exhibited significant efficiency when orally administered in the animal
model. Because of the difference in the selection of extractant and extraction
method, the extract is considered as different from that of the previous invention.
Actually, the HPLC analysis carried out by the present inventors exhibited a widely
different spectral behavior, as compared with those of the butanol fraction or the EA
fraction of the patented invention. Also, a significantly different nerve cell
protection activity was confirmed by the animal model of stroke.
[Disclosure]
[Technical Problem]
A feature of the present invention is to provide a pharmaceutical
composition for treating and/ or preventing cranial nerve diseases, cerebrovascular
diseases and cardiovascular diseases, which includes a butanol extract of Opuntia
ficus-indica as an active ingredient.
Another feature of the present invention is to provide a pharmaceutical
composition for treating and/ or preventing cranial nerve diseases, cerebrovascular
diseases and cardiovascular diseases, which includes an acid hydrolysate of the
butanol extract of Opuntia ficus-indica as an active ingredient.
Still another feature of the present invention is to provide a drug for oral
administration comprising a butanol extract of Opuntia ficus-indica or an acid
hydrolysate of the extract as an active ingredient.
[Technical Solution]
At least one of the above and other features and advantages of the present
invention may be realized by providing a butanol extract of Opuntia ficus-indica or an
acid hydrolysate of the extract, which exhibits an antioxidative activity and nerve
cell protection activity.
[Advantageous Effects]
[Description of Drawings]
Figure 1 compares the HPLC analysis pattern of the butanol extract of
Opuntia ficus-indica with that of standard materials.
Figure 2 compares the HPLC analysis pattern of the butanol extract of
Opuntia ficus-indica with that of the acid hydrolysate thereof.
Figure 3 shows the DPPH radical scavenging effect of the butanol extract of
Opuntia ficus-indica.
Figure 4 shows the lipid peroxidation inhibition effect of the butanol extract
of Opuntia ficus-indica.
Figure 5 shows the xanthine/ xanthine oxidase-induced neurotoxicity
inhibition effect of the butanol extract of Opuntia ficus-indica.
Figure 6 shows the hydrogen peroxide-induced neurotoxicity inhibition
effect of the butanol extract of Opuntia ficus-indica.
Figure 7 shows the NMDA-induced excitatory neurotoxicity inhibition effect
of the butanol extract of Opuntia ficus-indica.
Figure 8 shows the β-amyloid-induced neurotoxicity inhibition effect of the
butanol extract of Opuntia ficus-indica.
Figure 9 shows the effect on the corrected cortical infarct volume when the
butanol extract of Opuntia ficus-indica was orally administered fro 7 days.
Figure 10 shows the effect on the corrected total infarct volume when the
butanol extract of Opuntia ficus-indica was orally administered fro 7 days.
Figure 11 shows the effect on the cortical shrinkage ratio when the butanol
extract of Opuntia ficus-indica was orally administered fro 7 days.
Figure 12 shows the effect on the total shrinkage ratio when the butanol
extract of Opuntia ficus-indica was orally administered fro 7 days.
Figure 13 shows the effect on the neurobehavioral recovery when the butanol
extract of Opuntia ficus-indica was orally administered fro 7 days.
Figure 14 shows the effect on the corrected cortical infarct volume when the
butanol extract of Opuntia ficus-indica was orally administered fro 14 days.
Figure 15 shows the effect on the corrected total infarct volume when the
butanol extract of Opuntia ficus-indica was orally administered fro 14 days.
Figure 16 shows the effect on the cortical shrinkage ratio when the butanol
extract of Opuntia ficus-indica was orally administered fro 14 days.
Figure 17 shows the effect on the total shrinkage ratio when the butanol
extract of Opuntia ficus-indica was orally administered fro 14 days.
Figure 18 shows the effect on the neurobehavioral recovery when the butanol
extract of Opuntia ficus-indica was orally administered fro 14 days.
[Best Mode]
Hereinafter, the present invention is described in further detail.
The present invention provides a pharmaceutical composition for preventing
and/ or treating ischemic diseases, cranial nerve diseases or cardiovascular diseases,
which includes a butanol extract of Opuntia ficus-indica (var. saboteri).
The butanol extract of Opuntia ficus-indica of the present invention, which has
nerve cell protection activity, can be obtained by extracting the stem, fruit or
steamed-and-dried fruit of Opuntia ficus-indica with butanol. For example, the
following method may be employed. The stem, fruit or steamed-and-dried fruit of
Opuntia ficus-indica is sliced. Optionally after freeze drying, a C 1 -C 4 lower alcohol
such as methanol or ethanol, which is used as extractant, is added in an amount of
from 0.1 to 10 L, preferably from 0.5 to 7 L, per 1 kg of the Opuntia ficus-indica.
Subsequently, extraction is performed at 20 to 90 0 C, preferably at 70 to 80 0 C, for 3 to
6 hours, preferably for 4 hours under reflux. The lower alcohol may be either an
absolute alcohol or an aqueous alcohol solution comprising 50 % or more water.
The extraction procedure may be repeated for 3 or more times, if required. The
resultant extract is filtered and evaporated under reduced pressure to obtain an
alcohol extract. Per 1 kg of the alcohol extract, 0.1 to 10 L, preferably 1 to 5 L, of
water is added and extraction is carried out sufficiently using 1 to 5 L, preferably 1
to 5 L, of butanol (n-BuOH) to obtain a butanol extract.
Alternatively, the stem, fruit or steamed-and-dried fruit of Opuntia ficus-
indica may be directly extracted with butanol to obtain an extract having a similar
composition as above.
The obtained butanol extract of Opuntia ficus-indica may be loaded on a
column chromatography using silica gel, Sephadex, RP-18, polyamide, Toyopearl or
XAD resin as filler in order to isolate and purify the components having structures
similar to that of quercetin 3-methyl ether, which exhibits brain cell protection
activity. The column chromatography may be performed multiple times, selecting
adequate fillers as required. Especially, it is the most preferable to perform the
column chromatography using a properly selected combination of Sephadex, RP-18
or silica gel as filler.
Through such a column chromatography process, 18 compounds were
isolated from the butanol extract of Opuntia ficus-indica and their structures were
elucidated. As a result, a novel compound isorhamnetin-3-O-(6'-O-E-feruloyl)
neohesperidoside in which a feruloyl group is bound to a flavonoid glycoside was
isolated. Among other compounds, six were tannin-based phenolic compounds,
and among the flavonoid-based compounds, all other than three quercetin 3-methyl
ethers were the compounds first isolated from this plant. Those compounds were
identified as isorhamnetin-3-O-(6'-O-E-feruloyl) neohesperidoside (1), 2,3,4-
trihydroxybenzoic acid (2), 4-hydroxybenzoic acid (3), ferulic acid (4), isorhamnetin
3-O-glucoside (5), 2,3-dihydroquercetin (6), cinnamic acid (7), kaempferol 7-0-
glucopyranoside (8), zataroside-A (9), 4-O-glucopyranosylsinapinic acid (10),
isorhamnetin 3-O-rutinosyl-4'-O-β-D-glucoside (11), isorhamnetin 3-O-(2,6-
dirhamnosyl)glucoside (12), isorhamnetin 3-O-rutinoside (nacissin) (13), 2,3-
dihydrokaempferol (14), quercetin 3'-O-β-D-glucoside (15), quercetin 3-O-methyl
ether (16), isorhamnetin 3-O-neohesperidoside (17) and n-butyl-β-D-
fructopyranoside (18).
Figure 1 compares the HPLC analysis pattern of the butanol extract of
Opuntia ficus-indica with that of standard materials.
The present invention also encompasses a pharmaceutical composition
comprising a hydrolysate of the butanol extract of Opuntia ficus-indica as an active
ingredient. As seen in the HPLC pattern in Figure 2, the content of quercetin 3-
methyl ether increases when the butanol extract of Opuntia ficus-indica is hydrolyzed
by an acid. Therefore, it is expected that acid hydroly sates of the butanol extract of
Opuntia ficus-indica obtained under various conditions can also be utilized to protect
the cranial nerve cells. The acid hydrolysis is performed as follows. The butanol
extract of Opuntia ficus-indica is acid hydrolyzed with hydrochloric acid. Then, after
neutralizing with an alkali, filtration is performed using a C 1 -C 4 lower alcohol and
the filtrate is collected. More specifically, acid hydrolysis is performed using
dioxane and 2 N hydrochloric acid. Then, after neutralizing to pH 6 to 8 using 5 N
NaOH, Ci-C 4 filtration is performed using a lower alcohol. The filtrate is collected
as acid hydrolysate.
The butanol extract of Opuntia ficus-indica and the acid hydrolysate thereof
exhibits excellent cranial nerve protection effect when orally administered in an
animal model of ischemic brain damage test very similar to the clinical test of stroke.
Accordingly, the butanol extract Opuntia ficus-indica and the acid hydrolysate thereof
may be useful for the prevention and/ or treatment of various cerebrovascular
diseases, myocardial infarction and cardiovascular diseases including stroke and
dementia.
Although the formerly developed NMDA receptor antagonists exhibited
reduced brain damage in the animal model, the development of most of them was
stopped because adverse reactions or efficacy were not verified. Besides, the
conventional treatments are mostly for injection, and the development of oral drugs
for retarding nerve cell death in patients who got stroke surgeries is almost non¬
existent. In this regard, the butanol extract of Opuntia ficus-indica according to the
present invention, whose nerve cell protection effect was confirmed when orally
administered in animal model of cerebral ischemia, is expected to provide superior
effects in preventing, treating and alleviating the cranial nerve diseases such as
Alzheimer's disease, stroke, Parkinson's disease, etc., and ischemic diseases such as
myocardial infarction and cell death.
The fact that the active ingredient quercetin 3-methyl ether and various
similar compounds exist in the butanol extract of Opuntia ficus-indica further
corroborates the present invention.
As described above, the butanol extract of Opuntia ficus-indica according to
the present invention exhibits outstanding cranial nerve protection effect when
orally administered in an animal model of ischemic brain very similar to the clinical
test for stroke. Thus, the pharmaceutical composition of the present invention
comprising the same can be useful for the prevention and treatment of brain cell
damage caused by cranial nerve diseases such as stroke, concussion, Alzheimer's
disease and Parkinson's disease, prevention and treatment of nerve cell and tissue
damage, particularly damage of brain cells and brain tissues, caused by ischemia,
and prevention and treatment of other cardiovascular cell damage caused by
ischemia such as ischemic myocardial infarction. Further, it can be used to protect
cranial nerves or the heart.
The pharmaceutical composition of the present invention can be prepared
into pharmaceutical formulations by a conventional method. In such preparations,
it is preferable that the active ingredient is mixed with a vehicle, diluted with a
vehicle, or sealed in a capsule, sachet or other type of vehicle. Accordingly, the
pharmaceutical composition of the present invention may be prepared into
formulations for oral administration, such as tablet, pill, powder, sachet, elixir,
suspension, emulsion, solution, syrup, aerosol, and soft or hard gelatin capsule.
Also, it can be prepared into an injection form such as solution, suspension, etc., or
into a transdermal administration such as ointment, cream, gel, lotion, etc.
Examples of suitable vehicle, excipient or diluent include lactose, dextrose,
sucrose, sorbitol, mannitol, calcium silicate, cellulose, methylcellulose, amorphous
cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl
hydroxybenzoate, talc, magnesium stearate and mineral oil. The formulations may
further include a filler, an antiagglutinant, a surfactant, a wetting agent, a fragrance,
an emulsifier, an antiseptic, etc. The composition of the present invention may be
formulated by a method well known in the art such that, after administered to a
mammal, the active ingredient is released in an instant, sustained or delayed manner.
The pharmaceutical composition of the present invention can be
administered orally. For clinical propose, the butanol extract of the present
invention and the acid hydrolysate thereof may be administered to an adult singly
or separately at a dose of from about 200 mg to about 20 g, preferably from 500 mg
to 10 g, a day. This range will be sufficient, but a higher or lower dosage may be
needed, depending on the conditions. And, the patient-specific dosage may be
varied depending on the particular compound used, body weight, age, sex, physical
conditions, diet, administration time, administration method, excretion rate, mixing
of ingredients, and seriousness of disease.
When the pharmaceutical composition of the present invention is
administered for clinical purpose to treat a targeted ischemic disease, the butanol
extract of Opuntia ficus-indica may be administered combined with at least one
known nerve protecting agent. Drugs that may be administered combined with the
compounds of the present invention include N-acetylcysteine, which increase the
concentration of glutathione, nimodipine, which is a calcium antagonist, vitamins C
and E, which are antioxidants, a tissue plasminogen activator, which is also a
clotbuster, and other drugs that protect cranial nerves and heart vessels.
However, the formulations of the pharmaceutical composition of the present
invention for treating ischemic diseases are not limited to those described above.
Any formulation useful for the prevention and treatment of nerve cell and tissue
damage, particularly damage of brain cells and brain tissues, caused by ischemia, or
prevention and treatment of cardiovascular cell damage caused by ischemia will be
encompassed in the present invention.
[Mode for Invention]
The embodiments of the present invention are further illustrated by the
examples below. The examples serve only to illustrate the invention and should
not be interpreted as limiting since further modifications of the disclosed invention
will be apparent to those skilled in the art. All such modifications are deemed to be
within the scope of the invention as defined in the claims.
Reference Example 1: Measurement of DPPH radical scavenging effect
DPPH radical scavenging effect was measured by modifying Blois' method
[Blois, Nature, 181: 1199 (1958)]. Test substance was dissolved in dimethyl sulfoxide
(DMSO) and diluted to an adequate concentration. 10 μL of this solution was
reacted at 37 0 C for 30 minutes with 190 μL of a 150 μM DPPH (1,1-diphenyl 2-
picrylhydrazyl) solution in methanol. Then, absorbance was measured at 520 ran
using VERS Amax microplate reader (Molecular Devices, Sunnyvale, USA). Radical
scavenging effect was evaluated as the decrease of absorbance compared to the
control group. Non-linear regression was carried out using Prism (Graphpad
Software Inc., USA). The concentration at which 50 % inhibition effect was
obtained (IC50) was calculated.
Reference Example 2: Measurement of lipid peroxidation inhibition effect
Effect on the lipid peroxidation induced in the brain homogenate obtained
from a male white rat (Sprague-Dawley) was measured according to Cho and Lee's
method [Cho J and Lee H-K, Eur. J. Pharmacol. 485: 105 (2004)]. A reaction solution
comprising an amount of brain homogenate, 10 μM Fe 2+ , 100 μM ascorbic acid, and
the test substance dissolved in DMSO was reacted at 37 0 C for 1 hour. Then,
trichloroacetic acid and 2-thiobarbituric acid (TBA) were sequentially added, and,
after mixing and 15 minutes of heating at 100 0 C for 15, centrifuge was carried out.
The absorbance of the supernatant was measured at 532 ran using VERSAmax
microplate reader (Molecular Devices, Sunnyvale, USA). Lipid peroxidation
inhibition by the test substance was evaluated as the decrease of absorbance
compared to the control group. Non-linear regression was carried out using Prism
(Graphpad Software Inc., USA). The concentration at which 50 % inhibition effect
was obtained (IC50) was calculated.
Reference Example 3: Primary culturing of white rat cortical nerve cells
Nerve cells taken from the cerebrum of the fetus of white rat was cultured
according to Cho, et al/s method [Life Sd., 68: 1567 (2001)]. The cortical area of the
cerebrum of a 16- to 18-day-old fetus of white rat (Sprague-Dawley) was taken and
the meninges were removed using a dissecting microscope. Single cells were
separated in MEM (available from Gibco BRL) comprising 25 mM glucose, 5 % fetal
bovine serum, 5 % horse serum and 2 mM L-glutamine, using a Pasteur pipette the
tip size of which had been adjusted using an alcohol lamp. The cells were
transferred to a 24-well cell culture plate on which poly-L-lysine and laminin were
coated, at a density of 4 to 5X10 5 cells/well. They were cultured under the
condition of 37 0 C and 95 % air/ 5 % CO2. Part of the culture medium was replaced
2 times a week. On days 7 to 9, 10 μM cytosine arabinoside was treated for 24 to 72
hours, in order to inhibit the growth of cells other than nerve cells. The cultured
cells were used for test on days 10 to 14.
Reference Example 4: Inducement of nerve cell damage by oxidative stress
The cultured cortical nerve cells were washed 3 times with HEPES-controlled
salt solution (HCSS), and treated with HCSS comprising xanthine (0.5 niM) and
xanthine oxidase (10 mU/mL) for 10 minutes to induce cell damage by superoxide
anion radicals. After washing with HCSS again, the culture medium was replaced
by serum-free MEM (MEM comprising 25 mM glucose and 2 mM glutamine), and
culturing was performed under the condition of 37 0 C and 95 % air/ 5 % CO2 for 18
to 20 hours. Inducement of nerve cell damage by hydrogen peroxide was carried
out as follows. The cultured cells were washed with HCSS, and treated with HCSS
comprising 100 μM hydrogen peroxide for 5 minutes. After washing again, the
culture medium was replaced by serum-free MEM, and culturing was performed for
18 to 20 hours. In order to measure the effect on the nerve cell damage, the damage
inducing substances and the test substances at various conditions were added and
treated for a predetermined time as described above. After culturing for 18 to 20
hours, the degree of nerve cell damage was measured as described in Reference
Example 6.
Reference Example 5: Inducement of excitatory nerve cell damage by
NMDA and inducement of nerve cell damage by β-amyloid
The cultured cortical nerve cells were washed 3 times with HEPES-controlled
salt solution (HCSS), and treated with 300 μM NMDA (N-methyl-D-aspartic acid)
for 15 minutes in magnesium-free HCSS solution to induce excitatory cell damage
by superoxide anion radicals. After washing with HCSS again, the culture medium
was replaced by serum-free MEM, and culturing was performed under the condition
of 37 0 C and 95 % air/5 % CO 2 for 18 to 20 hours. Inducement of nerve cell damage
by β-amyloid was carried out as follows. The cultured cells were washed with
HCSS, and treated with serum-free medium comprising 40 μM β-amyloid for 20 to
24 hours. In order to measure the effect on the nerve cell damage, the damage
inducing substances and the test substances at various conditions were added and
treated for a predetermined time as described above. After culturing for 20 to 24
hours as describe above, the degree of nerve cell damage was measured as described
in Reference Example 6.
Reference Example 6: Measurement of nerve cell damage
The degree of damage of nerve cells treated in Reference Example 4 and
Reference Example 5 was measured according to Hansen et al.'s method [Hansen
MB, Nielsen SE and Berg K, /. Immunol. Methods 119: 203 (1989)] by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; available from Sigma)
reduction. Morphologic change of the cells was observed using a phase contrast
microscope. The result was presented as the percentage of damaged cells as
compared to the MTT reducing activity of the control group that had been treated
with the solvent. 3 to 4 measurements were made repeatedly, two groups at once.
The obtained data were averaged, and the concentration at which 50 % inhibition
effect was obtained (IC50) was calculated by non-linear regression using Prism
(Graphpad Software Inc., USA).
Reference Example 7: Design of white rat model for middle cerebral artery
occlusion (MCAO) and transient focal cerebral ischemia by reperfusion
Male white rats (Sprague-Dawley, Samtako) weighing 250 to 300 g were used
as test animal. On the day of surgery, the rat was anesthetized by inhaling 1.5 %
isoflurane (Forane, Choongwae Pharma) in nitrous oxide (N2O, 70 %) and oxygen
(O2, 30 %). Surgery was carried out according to Nagasawa and Kogure's method
[Stroke 20:1037, 1989], while maintaining the body temperature of the test animal at
about 37 ± 0.5 0 C using a warming pad and a warming lamp.
Specifically, the middle neck was incised under isoflurane inhalation
anesthesia, and the right common carotid artery, the internal carotid artery, and the
external carotid artery were carefully separated, paying attention not to damage the
vagus nerve. The common carotid artery and the external carotid artery were
ligated, and a 17-mm probe was inserted in the internal carotid artery at the
bifurcation of the internal and external carotid arteries to ligate the area just above
the insertion, thereby occluding the middle cerebral artery. The probe had been
prepared by rounding the tip of a 4-0 nylon suture (Nitcho Kogyo Co., Ltd., Japan)
by heating, cutting it 17 mm long, and coating a length of about 7 to 9 mm of the
other end with a blend mixture of silicone (Xantopren, Bayer Dental) and a curing
agent (Optosil-Xantopren Activator, Bayer Dental) to a thickness of 0.3 to 0.4 mm.
About 25 to 30 minutes after the inducement of cerebral ischemia, neurological
deficit of the white rat was measured. Only the rats which showed conditions were
included in the ischemic group. Neurological deficit was evaluated as follows.
The white rat was fully lifted by the tail in the air, and it was observed if left
forelimb flexion occurs and if the animal spontaneously rotates to the left side.
After 120 minutes of transient middle cerebral artery occlusion, the sutured area was
opened again under isoflurane inhalation anesthesia. The spherical probe tip was
taken out by about 10 mm to provide reperfusion (white rat model of transient focal
cerebral ischemia). Next, the surgical area was stitched again, and neurological
deficit was measured about 7 or 14 days later. The rats were sacrificed, and
histologic staining was performed on the brain tissue.
0.5 % carboxymethyl cellulose (3 mL/kg) as an excipient (vehicle), or a
butanol extract of Opuntia ficus-indica was orally administered first at 2 hours after
the reperfusion and then twice a day, starting from the next day, for 6 days (unit
dosage 100, 200, 300, 500 mg/kg) or 13 days (unit dosage 100, 200, 300 mg/kg).
Example 1: Preparation of extract from Opuntia ficus-indica and
hydrolysate thereof
Example 1-1: Preparation of butanol extract from alcohol extract of Opuntia
ficus-indica
To Opuntia ficus-indica dried by hot air, 7 volume equivalents of 50 % ethanol
with respect to the dried herb was added. Reflux extraction at 80 0 C for 4 hours
was repeated two times, and the extract was concentrated and dried. The extract
was dissolved in distilled water to about 5 % concentration, and extraction was
repeated two times using the same volume of butanol solution. The fraction was
concentrated under reduced pressure to obtain a butanol extract of Opuntia ficus-
indica.
Example 1-2: Preparation of butanol extract of Opuntia ficus-indica
To Opuntia ficus-indica dried by hot air, 7 volume equivalents of 50 % ethanol
with respect to the dried herb was added. Reflux extraction at 80 0 C for 4 hours
was repeated two times, and the extract was concentrated and dried to obtain a
butanol extract of Opuntia ficus-indica.
Example 1-3: Preparation of acid hydrolysate of butanol extract of Opuntia
ficus-indica
To 1 g of the butanol extract obtained in Example 1-1, each 40 mL of dioxane
and 2 N HCl was added, and reaction was performed at 100 0 C for 30 minutes. The
resultant solution was cooled with ice water and neutralized by adding about 12.5
mL of 5 N NaOH (pH = 7.3).
The neutralized solution was completely concentrated, and 20 mL of
methanol was added (This procedure was repeated for 3 times). The formed solid
was filtered, and a hydrolysate was obtained from the filtrate.
Figure 2 compares the HPLC analysis pattern of the butanol extract of
Opuntia ficus-indica prepared in Example 1-1 with that of the acid hydrolysate
obtained in Example 1-3.
<HPLC analysis condition>
- HPLC instrument: Waters Delta 600
- Column: J'sphere ODS-H80 (250X4.6 mm LD, S-4 μm, YMC)
- Temperature: 30 0 C
- Detection: UV 210 ran
- Flow rate: 1 mL/min
- Injection volume: 20 μL
- Eluent (gradient system):
Example 2: Isolation of brain cell protecting component from butanol
extract of Opuntia ficus-indica
The components included in the butanol extract of Opuntia ficus-indica
obtained in Example 1-1 were isolated and their structure was identified.
Nonpolar compounds soluble in dichloromethane were removed from 50 g
of the butanol fraction, and 35.9 g of residue was obtained. 50 g of the butanol
extract was suspended in 1 L of distilled water, and 12.9 g of nonpolar compounds
comprising chlorophylls, fatty acids, etc., using dichloromethane (1.6 L X 3). 35.9
of a polar solvent fraction comprising flavonoid glycoside, etc., was obtained.
Column chromatography (6.5 X 38 cm) was performed on 14.5 g of the 35.9
g of butanol polar solvent fraction, using methanol as an eluent and Sephadex (Cat.
No. LH-20-100, Sigma) as a stationary phase. The obtained fractions were observed
by normal-phase silica gel TLC (eluent: dichloromethane/ methanol/ water =
4/1/0.1, v/v/v) and reverse-phase silica gel TLC (eluent: water/ methanol = 40/60,
v/v). Compounds having like polarities were combined and grouped into 8
subfractions (Fr.l to Fr.8). Subfraction Fr.4 (2.25 g) was purified by column
chromatography (2.5 X 35 cm) using silica gel. Gradually more polar eluents were
used, starting from dichloromethane/ methanol (95/5, v/v), by increasing the
volume of methanol. The subfraction was further grouped into 18 subfractions
(Fr.4.1 to Fr.4.18), based on polarity.
Of those subfractions, reverse-phase silica gel column chromatography (2 x
18 cm) was performed on the 14th (Fr.4.14, 860.0 mg) and 18th (Fr.4.18, 260.0 mg)
fractions, in which pure compounds exist intensively, using 30 % methanol as an
eluent. And, preparative reverse-phase TLC (eluent: 30 % CH3CN) was performed
on the 2nd subfraction (Fr.4.14.2, 155.6 mg) to obtain pure compound 1
(isorhamnetin-3-O-(6'-O-E-feruloyl)neohesperidoside, 12.34 mg).
Preparative reverse-phase TLC was performed on subfraction Fr.4.6 (56.5
mg) using 25 % CH3CN as an eluent. Compound 2 (2,3,4-trihydroxybenzoic acid,
8.32 mg), compound 3 (4-hydroxybenzoic acid, 4.71 mg) and compound 4 (ferulic
acid , 16.52 mg) were obtained in pure form. Reverse-phase silica gel column
chromatography was performed on subfraction Fr.4.12 (188.06 mg) using 30 %
methanol as an eluent. Compound 5 (isorhamnetin 3-O-glucoside, 65.7 mg) was
obtained, and preparative reverse-phase TLC was performed on the residue to
obtain compound 6 (2,3-dihydroquercetin, 27.65 mg) and compound 7 (cinnamic
acid, 6.21 mg). Subfraction Fr.4.14.1 (450.0 mg) was isolated by preparative reverse-
phase HPLC. Gradually more polar eluents were used, starting from 13 %
methanol to 43 % methanol. Compound 7 (cinnamic acid, 12.5 mg) was further
obtained and compound 8 (kaempferol 7-O-glucopyranoside, 12.13 mg) was isolated.
Subfraction Fr.l (5.35 g) was divided into 7 subfractions (Fr.1.1 to Fr.1.7) by
performing reverse-phase column chromatography (LiChroprep RP-18, 40-63 μm,
4.5 X 30 cm). Gradually more polar eluents were used, starting from 25 %
methanol to 65 % methanol, by increasing the volume of methanol by 5 %. From
the 2nd subtraction (Fr.1.2) 180.0 nig, compound 9 (zataroside-A, 6.23 mg) and
compound 18 (n-butyl-β-D-fructopyranoside, 7.2 mg) were isolated by reverse-
phase silica gel and preparative HPLC. From subfraction Fr.1.5 (180.5 mg),
compound 10 (4-O-glucopyranosylsinapinic acid, 7.05 mg) was isolated by
preparative HPLC using 22 % methanol. From subfraction Fr.1.7 (165.7 mg),
compound 11 (isorhamnetin 3-0-rutinosyl-4'-0-β-D-glucoside, 12.2 mg) and
compound 12 (isorhamnetin 3-O-(2,6-dirhamnosyl)glucoside, 695 mg) were isolated
by preparative HPLC using 38 % methanol.
Subfractions Fr.2 (4.74 g) and Fr.3 (3.5 g) were divided into 8 subfractions
(Fr.2.1 to Fr.2.8) by performing Sephadex column chromatography (6.5 X 38 cm)
using 70 % methanol as an eluent. Of these subfractions, reverse-phase silica gel
column chromatography was performed on the 2nd fraction Fr.2.2 (1.13 g) using
35 % methanol as an eluent. As a result, compound 4 (ferulic acid), compound 6
(2,3-dihydroquercetin) and compound 7 (cinnamic acid) were further added, and
compound 13 [isorhamnetin 3-O-rutinoside (nacissin)] and compound 17
(isorhamnetin 3-O-neohesperidoside) were isolated. From subfraction Fr.5 (364.8
mg), compound 14 (2,3-dihydrokaempferol), compound 15 (quercetin 3'-O-β-D-
glucoside) and compound 16 (quercetin 3-O-methyl ether) were obtained by a
combination of silica gel column chromatography and preparative reverse-phase
silica gel TLC.
Example 3: Structure determination of components included in butanol
extract of Opuntia ficus-indica
For each of the components obtained in Example 2, 1 H-NMR (300 MHz) and
13 C-NMR (75 MHz) spectra were analyzed. Chemical shift of each peak was
represented as relative value with respect to the chemical shift of the methanol
solvent (3.3 ppm, 49.8 ppm).
Example 3-1: Structure analysis of isorhamnetin-3-O-(6'-O-E-
feruloyl)neohesperidoside (compound 1)
Compound 1 was yellow amorphous powder and exhibited yellow color
when the TLC plate was sprayed with 10 % sulfuric. In 1 H-NMR, the peaks at δ
6.03 (IH, br s) and 6.20 (IH, br s) are those of H-6 and H-8 of the flavonoid A-ring.
The peak at δ 6.88 (IH, d, / = 8.5 Hz) is that from H-5 1 and is ortho-coupled with the
peak at δ 7.50 (IH, dd, / = 8.5, 2.0Hz), which is that of H-6 1 . The peak at δ 7.87
(H-6 1 ) is meta-coupled with the peak at δ 7.63 (IH, d, / = 2.0 Hz), which is that of H-
2'. As a result, it can be seen that the aromatic ring of the compound is substituted
as an ABX system. The peaks at δ 6.99 (d, J = 1.7 Hz), 6.85 (dd, / = 8.1, 1.7 Hz) and
6.78 (d, / = 8.2 Hz) shows that another aromatic ring of the compound is substituted
as an ABX system. The coupling constants of δ 7.33 (d, / = 15.9 Hz) and 6.05 (d, / =
15.7 Hz) indicate that the peaks are those of a trans-vinyl group. Each of the single
peaks at δ 3.98 and 3.88 are those of three hydrogens. As can be seen from the
chemical shift, they are those of the methoxy groups from the flavonoid and feruloyl
groups. Therefore, it can be seen that the compound has a structure in which the
quercetin nucleus is substituted by a methoxy group and the isorhamnetin backbone
is substituted by a feruloyl group. The peaks at δ 5.80 (d, / = 7.3 Hz) and 5.20 (d, /
= 1.1 Hz) are those from the anomeric protons of sugars. Their coupling constants
indicate β- and α-type bonding, respectively. Thus, it can be seen that this
compound is a disaccharide. From 13 C-NMR, it can be seen that the sugars bound
to the compound are glucose and rhamnose. The peak at δ 63.9 from C-6 of
glucose indicates that the glucose has a substituent at C-6. The peak at δ 78.8 is
from C-2 of glucose, and is shifted by about 5.5 ppm to lower magnetic field when
compared with C-2 of other glucose. This indicates that the glucose has a
substituent at C-2 [Markham et al., Tetrahedron 34:1978, 1389]. 2D NMR HMBC
experiment was carried out in order to identify exact position of the substituents.
As a result, C-2 of glucose at δ 80.1 and the anomeric proton of rhamnose at δ 5.20
showed correlation, and H-6 of glucose at δ 4.36 and 4.29 had correlation with the
carbonyl carbon of the feruloyl group. The anomeric proton of glucose showed
correlation with C-3 of glucoside (δ 134.2). The position of the methoxy groups
could be identified accurately because each of them showed correlation with the
ABX system aromatic ring. Hence, compound 1, which has a feruloyl group at C-6
of glucose, rhamnose at C-2 of the glucose, and the feruloyl disaccharide at C-3 of
isorhamnetin, was identified as isorhamnetin-3-O-(6'-O-E-feruloyl)neohesperidoside /
first isolated from the natural material.
Yellow amorphous powder; UV υ ma χ (MeOH): 332, 300 (sh), 267(sh), 252 ran;
IR max cm- 1 : 3401, 2925, 1655, 1605, 1514, 1453, 1430, 1357, 1284, 1205, 1179, 1126, 1055,
1031, 811; HRFABMS (positive ion mode): m/z 823.2086 (C 3 SH 40 Oi 9 Na, calcd.
823.2061); 1 H NMR (CD 3 OD, 300 MHz): δ 7.87 (IH, d, / = 2.0 Hz, H-2 1 ), 7.50 (IH, dd,
/ = 8.5 and 2.0 Hz, H-6 1 ), 7.33 (IH, d, / = 15.9 Hz, H-3"), 6.99 (IH, d, / = 1.7 Hz, H-5"),
6.88 (IH, d, / = 8.5 Hz, H-5 1 ), 6.85 (IH, dd, / = 8.1 & 1.7 Hz, H-8"), 6.78 (IH, d, / = 8.2
Hz, H-9"), 6.20 (IH, d, / = 2.1 Hz, H-8), 6.05 (IH, d, / = 15.7 Hz, H-2"), 6.03 (IH, d, / =
1.8 Hz, H-6), 5.80 (IH, d, / = 7.3 Hz, H-I'), 5.20 (IH, d, / = 1.1 Hz, H-I"), 4.36 (IH, dd,
/ = 11.9 and 6.1 Hz, H a -6'), 4.29 (IH, dd, / = 12.0 and 2,.8Hz, H b -6'), 4.03 (IH, m, H-
5"), 4.01 (IH, dd, / = 3.0, 1.6 Hz, H-2"), 3.98 (3H, s, OMe), 3.88 (3H, s, OMe), 3.77 (IH,
dd, / = 9.6, 3.4 Hz, H-3"), 3.68 (IH, dd, / = 8.3, 6.3 Hz, H-2'), 3.63 (IH, t, / = 9.1 Hz, H-
3'), 3.57-3.50 (IH, m, H-5'), 3.30-3.36 (2H, m, H-4', 4"), 0.90 (3H, d, / = 6.0 Hz, H-6").
13 C NMR (CD 3 OD, 75 MHz): δ 168.7 (C, C-I"), 179.2 (C, C-4), 165.6 (C, C-7),
163.0 (C, C-5), 158.7 (C, C-2), 158.3 (C, C-9), 150.6 (2C, C-4 1 , 7"), 148.4 (2C, C-3 1 , 6"),
146.9 (CH, C-3"), 134.2 (C, C-3), 127.6 (C, C-4"), 124.3 (CH, C-6 1 ), 123.7 (CH, C-9"),
123.4 (C, C-I 1 ), 116.4 (CH, C-8"), 116.0 (CH, C-5 1 ), 114.8 (CH, C-2"), 114.6 (CH, C-2 1 ),
111.4 (CH, C-5"), 105.8 (C, C-10), 102.8 (CH, C-I"), 100.2 (CH, C-I'), 99.8 (CH, C-6),
94.7 (CH, C-8), 80.1 (CH, C-2'), 78.8 (CH, C-3'), 75.7 (CH, C-5'), 74.0 (CH, C-4"), 72.4
(3CH, C-4', 2", 3"), 70.0 (CH, C-5"), 63.9 (CH 2 , C-6'), 57.0 (CH 3 , OMe), 56.5 (CH 3 ,
OMe), 17.5 (CH 3 , C-6").
The above analysis result of the butanol extract of Opuntia ficus-indica of the
present invention is well consistent with that of published literatures: 2,3,4-
trihydroxybenzoic acid (2), 4-hydroxybenzoic acid (3), ferulic acid (4), isorhamnetin
3-O-glucoside (Karl et al, Planta Med. 41:1981, 96), 2,3-dihydroquercetin (Nonaka et
al, Chem. Pharm. Bull. 35:1987, 1105), cinnamic acid (7), kaempferol 7-0-
glucopyranoside (Lee et al., /. Agric. Food Chem. 46:1998, 3325), zataroside-A (AIi et
al., Phytochemistry 52:1999, 685), 4-O-glucopyranosylsinapinic acid (Materska et al.,
/. Agric. Food Chem. 53:2005, 1750), isorhamnetin 3-O-rutinosyl-4'-O-β-D-glucoside
(Aquino et al., Biochem. Syst. Ecol. 15:1987, 667), isorhamnetin 3-O-(2,6-
dirhamnosyl)glucoside (Liu et al., Zhongguo Yaoxue Zazhi 33:1998,587), isorhamnetin
3-O-rutinoside (nacissin) (Nakano et al., Phytochemistry 28:1989, 301), 2,3-
dihydrokaempferol (Lee et al., Arch. Pharm. Res. 26:2003, 1018), quercetin 3'-O-β-D-
glucoside (Saito et al., Phytochemistry 35:1994, 687), quercetin 3-O-methyl ether
(Roitman et al., Phytochemistry 24:1985, 835), isorhamnetin 3-O-neohesperidoside
(Nørbεek et al., Phytochemistry 51:1999, 1113), n-butyl-β-D-fructopyranoside (Xu et al.,
Arch. Pharm. Res. 28:2005, 395).
Example 4: Identification of antioxidation effect of butanol extract of
Opuntia ficus-indica
The butanol extract of Opuntia ficus-indica obtained in Example 1-1 was tested
for DPPH radical scavenging effect, lipid peroxidation inhibition effect,
xanthine/ xanthine oxidase-induced neurotoxicity inhibition effect and hydrogen
peroxide-induced neurotoxicity inhibition effect, according to the methods described
in Reference Examples 1 to 4.
As seen in Figures 3 to 6 and Table 1, the butanol extract of Opuntia ficus-
indica exhibited outstanding antioxidative activity, showing DPPH radical
scavenging effect and lipid peroxidation inhibition effect. Also, it exhibited nerve
cell protection effect by effectively inhibiting oxidative neurotoxicity induced by
xanthine/ xanthine oxidase or hydrogen peroxide in cultured cortical nerve cells.
Specifically, the butanol extract of Opuntia ficus-indica exhibited a radical scavenging
effect of 66.90 μg/mL (IC50). The lipid peroxidation inhibition effect was 80.30
μg/mL (IC5o), the xanthine/ xanthine oxidase-induced neurotoxicity inhibition effect
was 61.54 μg/mL (IC50), and the hydrogen peroxide-induced neurotoxicity
inhibition effect was 94.82 μg/mL (ICso). Such outstanding antioxidative activity
and nerve protection effect implicate that the butanol extract of Opuntia ficus-indica
can be effective in treating or preventing various degenerative brain diseases
accompanying oxidation-related nerve cell damages such as stroke, Alzheimer's
disease and Parkinson's disease.
Example 5: Identification of excitatory neurotoxicity inhibition effect of
butanol extract of Opuntia ficus-indica
Inhibition effect of the butanol extract of Opuntia ficus-indica obtained in
Example 1-1 was measured against excitatory neurotoxicity according to the method
of Reference Example 5.
As seen in Figure 7 and Table 1, the butanol extract of Opuntia ficus-indica
strongly inhibited excitatory neurotoxicity induced by NMDA (IC50 = 50.58 μg/mL).
Accordingly, it was confirmed that, in addition to providing protection against
oxidation-related nerve cell damages, the butanol extract of Opuntia ficus-indica
further protects the nerve cells by inhibiting excitatory neurotoxicity. This result
indicates that the butanol extract of Opuntia ficus-indica may be effective in treating
or preventing such degenerative brain diseases as stroke, Alzheimer's disease and
Parkinson's disease, which are caused by excitatory neurotoxicity due to excessive
free glutamic acids.
Example 6: Identification of β-amyloid-induced neurotoxicity inhibition
effect of butanol extract of Opuntia ficus-indica
Inhibition effect of the butanol extract of Opuntia ficus-indica obtained in
Example 1-1 was measured against β-amyloid-induced neurotoxicity according to
the method of Reference Example 5.
As seen in Figure 8, the butanol extract of Opuntia ficus-indica strongly
inhibited neurotoxicity induced by β-amyloid, thereby increasing survival rate of
cells. Maximum cell survival rate with regard to β-amyloid-induced toxicity was
about 35 % at 100 μg/mL, with reference to the control group. This result indicates
that, in addition to providing nerve cell protection effect and excitatory
neurotoxicity inhibition effect through antioxidative activity, the butanol extract of
Opuntia ficus-indica may be effective in treating or preventing such degenerative
brain diseases as stroke, Alzheimer's disease and Parkinson's disease, through its
inhibition effect against β-amyloid-induced neurotoxicity.
[Table 1]
Example 7: Cranial nerve cell protection effect of subacute oral
administration of butanol extract of Opuntia ficus-indica
To evaluate ischemia-induced brain damage induced in white rat model of
120 minutes' of transient focal cerebral ischemia followed by reperfusion, TTC (2,3,5-
triphenyltetrazolium chloride) staining (Bederson et al., Stroke 17:1304, 1986) was
carried out. After middle cerebral artery occlusion followed by reperfusion, white
rats were sacrificed at 7th or 14th day and brain was immediately taken out. At
each of two 2-mm thick brain slices, 1 mm apart from the frontal pole, 2 % TTC
solution prepared using 0.9 % physiological saline was injected using a brain matrix
(ASI Instruments, Warren, MI, USA), and staining was performed at 37 0 C for 60
minutes. The TTC-stained brain slices were immersed in 10 % phosphate-buffered
formalin solution, and the rear side images of the slices were attained via a computer
using a CCD video camera. The area (mm 2 ) of the infarcted area of the cerebral
cortex and the corpus striatum (the region not stained to dark red color) was
measured using an image analysis software (Optimas, Edmonds, WA, USA). The
infarct volume (mm 3 ) was calculated by multiplying the sum of the infarct area of
the slices by the thickness of the slices. The total infarct volume was obtained as
the sum of the infarct volumes of the cerebral cortex and the corpus striatum.
Corrected infarct volume was calculated to compensate for the effect of shrinkage.
For each slice, total corrected infarct area was calculated by the following equation:
corrected infarct area = left hemisphere area - (right hemisphere area - infarct area).
Total corrected infarct volume was calculated by multiplying the total corrected
infarct area by slice thickness.
Shrinkage ratio of the ischemia-induced hemisphere was calculated by the
following equation.
[Equation 1]
Contract ratio (%) = ^- x 100
In Equation 1, A is the volume (mm 3 ) of the ischemia-induced hemisphere
and B is the volume (mm 3 ) of the normal hemisphere.
Oral administration of the butanol extract of Opuntia ficus-indica was
commenced 2 hours after the reperfusion. Following the next day, the oral
administration was performed twice a day for 6 days with a unit dosage of 300
mg/kg. The oral administration group exhibited cranial nerve damage protection
effect, with significance reduction in all of corrected cortical infarct volume,
corrected total infarct volume, cortical shrinkage ratio, and total shrinkage ratio, by
27.5, 27.2, 39.2, and 35.6 %, respectively, as compared to the control group to which
0.5 % carboxymethyl cellulose was administered. The oral administration group to
which administration was performed for 13 days with a unit dosage of 200 mg/kg
exhibited significance reduction in corrected cortical infarct volume, corrected total
infarct volume, cortical shrinkage ratio, and total shrinkage ratio, by 31.4, 27.6, 44.4
and 37.4 %, respectively, as compared to the control group to which 0.5 %
carboxymethyl cellulose was administered. And, the oral administration group to
which administration was performed for 13 days with a unit dosage of 300 mg/kg
exhibited significance reduction in corrected cortical infarct volume, corrected total
infarct volume, cortical shrinkage ratio, and total shrinkage ratio, by 40.4, 34.3, 61.7
and 46.7 %, respectively, as compared to the control group to which 0.5 %
carboxymethyl cellulose was administered. To conclude, the longer the
administration period, and the large the administration amount, the nerve
protection effect against brain damage was more significant. The results are
summarized and presented in Figures 9, 10, 11, 12, 14, 15, 16 and 17 and Tables 2 and
3 (values are given in the format of average ± standard error).
[Table 2]
Effect on brain damage when butanol extract of Opuntia ficus-indica was
orally administered (twice a day) for 7 days after 120 minutes' of middle cerebral
artery occlusion followed by reperf usion
[Table 3]
Effect on brain damage when butanol extract of Opuntia ficus-indica was
orally administered (twice a day) for 14 days after 120 minutes' of middle cerebral
artery occlusion followed by reperfusion
Example 8: Measurement of neurobehavioral recovery effect of butanol
extract of Opuntia ficus-indica
Neurobehavioral recovery effect of white rats treated according to the
method of Example 7 was measured by Relton et al.'s neurological score evaluation
(Stroke 28:1430, 1997) as follows.
Specifically, forelimb flexion (left forelimb flexion when the white rat was
fully lifted by the tail in the air), duration of forelimb flexion (time of forelimb
flexion over 10-second period) and symmetry of movement (when the white rat was
made to walk only using forelimbs while being lifted by the tail and its hindlimbs
hanging in the air) were examined, and the observed scores were given as
summarized in Table 4.
[Table 4]
The final neurological scores of each group are shown in Tables 5 and 6 (each
experimental score is given in the format of average ± standard error) by adding
up the scores of each test item, and also depicted in Figures 13 and 18. 10 point
means normal (no neurological deficits), and the lower score, the larger neurological
deficit.
[Table 5]
Effect on neurobehavioral recovery when butanol extract of Opuntia ficus-
indica was orally administered (twice a day) for 7 days after 120 minutes' of middle
cerebral artery occlusion followed by reperfusion
[Table 6]
Effect on neurobehavioral recovery when butanol extract of Opuntia ficus-
indica was orally administered (twice a day) for 14 days after 120 minutes' of middle
cerebral artery occlusion followed by reperfusion
Time from
Unit dosage middle cerebral artery occlusion until Vehicle 100 mg/kg 200 mg/kg 300 mg/kg measurement
30 minutes 2.14 + 0.10 2.18 + 0.12 2.28 ± 0.12 2.25 ± 0.13
1 day 2.07 ± 0.07 2.55 + 0.37 2.45 + 0.21 2.67 + 0.22
2 days 2.14 ± 0.10 2.82 ± 0.38 * 3.18 ± 0.35 * 3.50 ± 0.26 *
3 days 2.21 ± 0.11 3.27 ± 0.41 * 3.40 + 0.41 * 4.50 ± 0.34 *
4 days 2.36 ± 0.23 3.64 ± 0.39 * 4.09 ± 0.48 * 5.25 ± 0.43 *
5 days 2.57 + 0.23 3.91 0.46 * 4.27 ± 0.52 * 5.75 + 0.49 *
6 days 3.00 ± 0.26 4.09 ± 0.56 5.00 ± 0.56 * 6.25 ± 0.63 *
7 days 3.36 + 0.27 4.27 ± 0.59 5.45 + 0.58 * 6.75 ± 0.52 *
8 days 4.21 ± 0.37 4.73 + 0.57 6.09 ± 0.53 * 7.00 ± 0.46 *
9 days 4.86 ± 0.46 4.91 ± 0.56 6.36 ± 0.47 * 7.67 + 0.47 *
10 days 5.29 ± 0.47 5.64 ± 0.73 6.91 ± 0.51 * 7.83 ± 0.46 *
11 days 5.71 ± 0.47 6.00 ± 0.69 7.36 ± 0.51 * 8.00 ± 0.44 *
12 days 6.14 + 0.43 7.09 + 0.72 7.73 ± 0.54 * 8.09 + 0.51 *
13 days 6.57 + 0.41 7.73 ± 0.78 7.91 ± 0.56 * 8.18 ± 0.46 *
14 days 6.86 ± 0.36 7.82 ± 0.74 8.00 + 0.57 8.27 + 0.45 *
* Significant difference 1 " rom control group c it same time in Duncan's 3 multiple range test (p < 0.05)
As shown in Table 5 and Figure 13, the groups to which the butanol extract
of Oγuntia ficus-indica was orally administered for 7 days at a dosage of 100 or 300
mg/kg showed significantly higher neurological scores than the control group,
showing that the butanol extract of Oγuntia ficus-indica exerts the neurobehavioral
recovery effect.
Also, as shown in Table 6 and Figure 18, the groups to which the butanol
extract of Oγuntia ficus-indica was orally administered for 14 days at a dosage of 200
or 300 mg/kg showed significantly and much higher neurological scores than the
control group.
Accordingly, it was confirmed that the butanol extract of Oγuntia ficus-indica
exhibits distinct nerve protection effect against brain damage in animal model of
cerebral ischemia and neurobehavioral recovery effect, and therefore, is effective in
preventing and treating cranial nerve diseases and cardiac ischemia.
[Industrial Applicability]
As described, the butanol extract of Oγuntia ficus-indica exhibits distinct
nerve protection effect against brain damage in animal model of cerebral ischemia,
and is expected to provide excellent prevention and treatment effect of cranial nerve
diseases such as stroke, concussion, Alzheimer's disease and Parkinson's disease,
and ischemic diseases such as myocardial infarction and cell death.
While the embodiments of the invention have been described and illustrated,
it is oblivious that various changes and modifications can be made therein without
departing from the spirit and scope of the present invention which should be limited
only by the appended claims.