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WO2008038849A1 - Composition pharmaceutique comprenant un extrait d'opuntia ficus-indica - Google Patents

Composition pharmaceutique comprenant un extrait d'opuntia ficus-indica Download PDF

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Publication number
WO2008038849A1
WO2008038849A1 PCT/KR2006/003933 KR2006003933W WO2008038849A1 WO 2008038849 A1 WO2008038849 A1 WO 2008038849A1 KR 2006003933 W KR2006003933 W KR 2006003933W WO 2008038849 A1 WO2008038849 A1 WO 2008038849A1
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Prior art keywords
indica
isorhamnetin
opuntia ficus
acid
extract
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PCT/KR2006/003933
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English (en)
Inventor
Changbae Jin
Yong Sup Lee
Hyoung Ja Kim
Suh Yun Jung
Jungsook Cho
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Korea Institute Of Science And Technology
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Priority to PCT/KR2006/003933 priority Critical patent/WO2008038849A1/fr
Publication of WO2008038849A1 publication Critical patent/WO2008038849A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/33—Cactaceae (Cactus family), e.g. pricklypear or Cereus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to a pharmaceutical composition
  • 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.
  • cranial nerve diseases for example, stroke, concussion, Alzheimer's disease, Parkinson's disease, cell death, myocardial infarction, and so forth.
  • ischemia 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.
  • ATP adenosine triphosphate
  • 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.
  • nerve-protection medicine that can be used to protect the brain cells from the damage caused by ischemia once stroke outbreaks, excluding clotbusters.
  • NO which is a free radical that can function as signal transmitter as well as neurotoxin
  • ischemic brain damage is 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 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.
  • 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 Opunti ⁇ ficus-indic ⁇ or an active compound isolated therefrom in 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.
  • 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.
  • the extract is considered as different from that of the previous invention.
  • 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.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • the following method may be employed.
  • the stem, fruit or steamed-and-dried fruit of Opuntia ficus-indica is sliced.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pharmaceutical composition comprising a hydrolysate of the butanol extract of Opuntia ficus-indica as an active ingredient.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • Test substance was dissolved in dimethyl sulfoxide
  • IC50 concentration at which 50 % inhibition effect was obtained
  • 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 ⁇ M 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.
  • HCSS HEPES-controlled salt solution
  • 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 ⁇ M 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.
  • 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
  • the cultured cortical nerve cells were washed 3 times with HEPES-controlled salt solution (HCSS), and treated with 300 ⁇ M NMDA (N-methyl-D-aspartic acid) for 15 minutes in magnesium-free HCSS solution to induce excitatory cell damage by superoxide anion radicals.
  • HCSS HEPES-controlled salt solution
  • NMDA N-methyl-D-aspartic acid
  • 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 ⁇ -amyloid was carried out as follows.
  • the cultured cells were washed with HCSS, and treated with serum-free medium comprising 40 ⁇ M ⁇ -amyloid for 20 to 24 hours.
  • 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
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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 ⁇ m,
  • 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.
  • Example 3-1 Structure analysis of isorhamnetin-3-O-(6'-O-E- feruloyl)neohesperidoside (compound 1)
  • 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.
  • 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.
  • 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 ⁇ g/mL (IC50).
  • the lipid peroxidation inhibition effect was 80.30 ⁇ g/mL (IC5o)
  • the xanthine/ xanthine oxidase-induced neurotoxicity inhibition effect was 61.54 ⁇ g/mL (IC50)
  • the hydrogen peroxide-induced neurotoxicity inhibition effect was 94.82 ⁇ g/mL (ICso).
  • IC5o lipid peroxidation inhibition effect
  • IC50 xanthine/ xanthine oxidase-induced neurotoxicity inhibition effect
  • ICso hydrogen peroxide-induced neurotoxicity inhibition effect
  • 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
  • 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 ⁇ -amyloid-induced neurotoxicity inhibition effect of butanol extract of Opuntia ficus-indica
  • the butanol extract of Opuntia ficus-indica strongly inhibited neurotoxicity induced by ⁇ -amyloid, thereby increasing survival rate of cells.
  • Maximum cell survival rate with regard to ⁇ -amyloid-induced toxicity was about 35 % at 100 ⁇ g/mL, with reference to the control group.
  • 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 ⁇ -amyloid-induced neurotoxicity.
  • Example 7 Cranial nerve cell protection effect of subacute oral administration of butanol extract of Opuntia ficus-indica
  • TTC 2,3,5- triphenyltetrazolium chloride staining
  • 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.
  • 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
  • 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.
  • 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.
  • the longer the administration period, and the large the administration amount the nerve protection effect against brain damage was more significant.
  • Example 8 Measurement of neurobehavioral recovery effect of butanol extract of Opuntia ficus-indica
  • 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
  • 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
  • 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.
  • the groups to which the butanol extract of O ⁇ untia 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 O ⁇ untia ficus-indica exerts the neurobehavioral recovery effect.
  • the groups to which the butanol extract of O ⁇ untia 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.
  • the butanol extract of O ⁇ untia 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.
  • the butanol extract of O ⁇ untia 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.

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Abstract

La présente invention porte sur une composition pharmaceutique comprenant un extrait d'Opuntia ficus-indica, plus particulièrement sur une composition pharmaceutique comprenant un extrait de butanol prélevé sur Opuntia ficus-indica ou un hydrolysat acide de l'extrait agissant comme ingrédient actif et efficace dans le traitement ou la prévention des affections des nerfs crâniens, des maladies cérébrovasculaires ou des maladies cardio-vasculaires telles que l'accident cérébrovasculaire, la commotion, la maladie d'Alzheimer, la maladie de Parkinson, la mort cellulaire, l'infarctus du myocarde, etc.
PCT/KR2006/003933 2006-09-29 2006-09-29 Composition pharmaceutique comprenant un extrait d'opuntia ficus-indica WO2008038849A1 (fr)

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WO2010081839A1 (fr) 2009-01-15 2010-07-22 Kaahee Research And Development Gmbh Extrait de figue de barbarie
KR101164020B1 (ko) 2006-09-29 2012-07-18 한국과학기술연구원 손바닥선인장 추출물을 함유하는 약학 조성물
CN109310688A (zh) * 2016-05-19 2019-02-05 国立研究开发法人国立循环器病研究中心 用于预防和/或治疗痴呆症的药物
CN109884222A (zh) * 2019-01-17 2019-06-14 贵阳中医学院 一种小花清风藤的hplc指纹图谱建立方法
CN111537656A (zh) * 2020-06-19 2020-08-14 劲牌有限公司 一种苦荞提取物的鉴别方法
CN111670037A (zh) * 2020-04-02 2020-09-15 广东新纪元生命科技有限公司 白花蛇舌草抗肿瘤有效组分及其制备方法和用途

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WO2003037324A1 (fr) * 2001-10-29 2003-05-08 Korea Institute Of Science And Technology Utilisation d'un extrait opuntia ficus-indica et de composes isoles de celui-ci pour proteger des cellules nerveuses
WO2005041994A1 (fr) * 2003-10-28 2005-05-12 Bioplanta Arzneimittel Gmbh Utilisation de fragments vegetaux et/ou d'extraits de figue de barbarie (opuntia) pour traiter des depressions

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WO2003037324A1 (fr) * 2001-10-29 2003-05-08 Korea Institute Of Science And Technology Utilisation d'un extrait opuntia ficus-indica et de composes isoles de celui-ci pour proteger des cellules nerveuses
WO2005041994A1 (fr) * 2003-10-28 2005-05-12 Bioplanta Arzneimittel Gmbh Utilisation de fragments vegetaux et/ou d'extraits de figue de barbarie (opuntia) pour traiter des depressions

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DOK-GO H. ET AL.: "Neuroprotective effects of antioxidative flavonoids, quercetin, (+)-dihydroquercetin and quercetin 3-methyl ether, isolated from Opuntia ficus-indica var. saboten", BRAIN RES., vol. 965, no. 1-2, March 2003 (2003-03-01), pages 130 - 136, XP002493220, DOI: doi:10.1016/S0006-8993(02)04150-1 *
KIM J.H. ET AL.: "Opuntia ficus-indica attenuates neuronal injury in in vitro and in vivo models of cerebral ischemia", J. ETHNOPHARMACOL., vol. 104, no. 1-2, March 2006 (2006-03-01), pages 257 - 262, XP025085677, DOI: doi:10.1016/j.jep.2005.09.017 *
SALEEM M. ET AL.: "Secondary metabolites from Opuntia ficus-indica var. saboten", PHYTOCHEMISTRY, vol. 67, no. 13, July 2006 (2006-07-01), pages 1390 - 1394, XP028059908, DOI: doi:10.1016/j.phytochem.2006.04.009 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101164020B1 (ko) 2006-09-29 2012-07-18 한국과학기술연구원 손바닥선인장 추출물을 함유하는 약학 조성물
WO2010081839A1 (fr) 2009-01-15 2010-07-22 Kaahee Research And Development Gmbh Extrait de figue de barbarie
US8663718B2 (en) 2009-01-15 2014-03-04 Kaahee Research And Development Gmbh Cactus fruit extract
CN109310688A (zh) * 2016-05-19 2019-02-05 国立研究开发法人国立循环器病研究中心 用于预防和/或治疗痴呆症的药物
CN109310688B (zh) * 2016-05-19 2023-09-01 国立研究开发法人国立循环器病研究中心 用于预防和/或治疗痴呆症的药物
US11925633B2 (en) 2016-05-19 2024-03-12 National Cerebral And Cardiovascular Center Drug for preventing and/or treating dementia
CN109884222A (zh) * 2019-01-17 2019-06-14 贵阳中医学院 一种小花清风藤的hplc指纹图谱建立方法
CN109884222B (zh) * 2019-01-17 2021-07-13 贵州中医药大学 一种小花清风藤的hplc指纹图谱建立方法
CN111670037A (zh) * 2020-04-02 2020-09-15 广东新纪元生命科技有限公司 白花蛇舌草抗肿瘤有效组分及其制备方法和用途
WO2021196105A1 (fr) * 2020-04-02 2021-10-07 广东新纪元生命科技有限公司 Composant antitumoral efficace de hedyotis diffusa, son procédé de préparation et son utilisation
CN111537656A (zh) * 2020-06-19 2020-08-14 劲牌有限公司 一种苦荞提取物的鉴别方法

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