WO2008038849A1

WO2008038849A1 - Pharmaceutical composition comprising an extract from opuntia ficus-indica

Info

Publication number: WO2008038849A1
Application number: PCT/KR2006/003933
Authority: WO
Inventors: Changbae Jin; Yong Sup Lee; Hyoung Ja Kim; Suh Yun Jung; Jungsook Cho
Original assignee: Korea Institute Of Science And Technology
Priority date: 2006-09-29
Filing date: 2006-09-29
Publication date: 2008-04-03

Abstract

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.

Description

[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 CHgCI₂ 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 C₁-C₄ 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 90⁰C, preferably at 70 to 80⁰C, 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).

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 C₁-C₄ 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-C₄ 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 nonexistent. 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 ⁰C 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 Fe²⁺, 100 μM ascorbic acid, and the test substance dissolved in DMSO was reacted at 37 ⁰C for 1 hour. Then, trichloroacetic acid and 2-thiobarbituric acid (TBA) were sequentially added, and, after mixing and 15 minutes of heating at 100 ⁰C 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 5X10⁵ cells/well. They were cultured under the condition of 37⁰C 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 ⁰C 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 37⁰C and 95 % air/5 % CO₂ 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 ⁰C 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 ⁰C 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 ⁰C 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 ⁰C 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 instrument: Waters Delta 600

- Column: J'sphere ODS-H80 (250X4.6 mm LD, S-4 μm, YMC)

- Temperature: 30⁰C

- 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, ¹H-NMR (300 MHz) and ¹³C-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 ¹H-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-5¹ and is ortho-coupled with the peak at δ 7.50 (IH, dd, / = 8.5, 2.0Hz), which is that of H-6¹. The peak at δ 7.87

(H-6¹) 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 ¹³C-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_maχ (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 (C₃SH₄₀Oi₉Na, calcd. 823.2061); ¹H NMR (CD₃OD, 300 MHz): δ 7.87 (IH, d, / = 2.0 Hz, H-2¹), 7.50 (IH, dd, / = 8.5 and 2.0 Hz, H-6¹), 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-5¹), 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"). 1³C NMR (CD₃OD, 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-4¹, 7"), 148.4 (2C, C-3¹, 6"), 146.9 (CH, C-3"), 134.2 (C, C-3), 127.6 (C, C-4"), 124.3 (CH, C-6¹), 123.7 (CH, C-9"), 123.4 (C, C-I¹), 116.4 (CH, C-8"), 116.0 (CH, C-5¹), 114.8 (CH, C-2"), 114.6 (CH, C-2¹), 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 (CH₂, C-6'), 57.0 (CH₃, OMe), 56.5 (CH₃, OMe), 17.5 (CH₃, 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 ⁰C 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 (mm²) 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 (mm³) 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 (mm³) of the ischemia-induced hemisphere and B is the volume (mm³) 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.

Claims

[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 C₁-C₄ lower alcohol solution per 1 kg of the stem or fruit of Opuntia ficus-indica and performing extraction at 20 to 90⁰C 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 90⁰C 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 C₁- C₄ 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.