[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.