WO2006001665A1

WO2006001665A1 - Composition for preventing or treating acute or chronic degenerative brain diseases including flavonoid derivatives

Info

Publication number: WO2006001665A1
Application number: PCT/KR2005/001986
Authority: WO
Inventors: Byung-Hee Han; Sam-Sik Kang; Kun-Ho Son
Original assignee: Seoul National University Industry Foundation
Priority date: 2004-06-28
Filing date: 2005-06-24
Publication date: 2006-01-05
Also published as: KR20060000048A; KR100610562B1

Abstract

Provided is composition for preventing or treating an acute or chronic degenerative brain disease, the composition including as an effective ingredient a flavonoid derivative selected from the group consisting of 4',7-dihydroxyflavone; 3',4',7-trihydroxyflavone; 3,3'-di-O - methylquercetin; kaempferide; galangin; morin; amentoflavone; hinokiflavone; ochnaflavone; ochnaflavone 4'-O-methyl ether; kaempferol 3-0-(6'-coumaroylglucosyl)(l → 2)rhamnoside; quercetin 3-O-(6'-coumaroylglucosyl)(l → 2)rhamnoside; kaempferol 3-O-glucosyl(l → 2)rhamnoside; kaempferol 3-0-2', 6'-dirhamnosylglucoside; quercetin 3-0 - 2',6'-dirhamnosylglucoside; and kaempferol 3-0-rutinoside, and a pharmaceutically acceptable carrier.

Description

COMPOSITION FOR PREVENTING OR TREATING ACUTE

OR CHRONIC DEGENERATIVE BRAIN DISEASES

INCLUDING FLAVONOID DERIVATIVES Technical Field [1] The present invention relates to a composition for preventing or treating acute or chronic degenerative brain diseases including a flavonoid derivative as an effective ingredient. Background Art [2] Acute degenerative brain diseases such as ischemic stroke, together with chronic degenerative brain diseases such as dementia, are disorders causing loss of brain function due to continuous destruction of cerebral nerve cells. Degenerative brain diseases exhibit various symptoms such as memory disorder, language disorder, space- time perception disability, judgment disability, personality and emotional disorder according to the degree of brain damage and an affected site, and cause permanent loss of brain function. [3] In Korea, about 10% and 1% of people with the age of 65 or more suffer from senile dementia and Parkinson's disease, respectively. Ischemic stroke is the second leading cause of death after cancer. According to the 2001 data from the National Bureau of Statistics, stroke causes 74 deaths per 100,000 population per a year. These degenerative brain diseases are adult diseases that the frequency of occurrence rapidly increases in aged persons with 50 or more of age. These degenerative brain diseases are major diseases that represent a serious social and economical burden and lower the life quality of aged persons. Furthermore, a recent trend is toward elevation of de¬ generative brain disease patients due to an increase in aged population with increasing average lifespan. Thus, the development of preventive and therapeutic strategies for degenerative brain diseases is strongly required. [4] As a medication for the treatment of cerebral damage due to stroke, there has been a clinical trial to reduce cerebral damage by removing platelet clots in brain blood vessels using an antiplatelet drug (pTA). However, this therapy is not so good in therapeutic efficacy and may worsen cerebral damage when used in hemorrhagic stroke, and thus, its clinical use is limited. Stroke treatment agents, which have been currently developed worldwide, such as an NMDA antagonist and an antiplatelet drug are under clinical trials to stroke patients. However, most treatments with these stroke treatment agents are currently discontinued in phase II or III clinical trials due to weak efficacy or side effects. [5] Cholinergic drugs such as tacrine and donepezil are currently available as a treatment agent for dementia which is a representative disease of degenerative brain diseases. These cholinergic drugs exhibit an enhanced short-term recognition memory but wholly poor therapeutic effects. Cholinergic drugs selectively acting on the muscarinic cholinergic nerve system, such as xanomeline, are currently in clinical trials to dementia patients. In addition, dementia preventive or treatment drugs based on inhibition of betaamyloid (A β ) accumulation and neurotoxicity, such as gamma- secretase inhibitors, glycogen synthase kinase (GSK3 β ) inhibitors, cyclin-dependent kinase-5 (cdk5) inhibitors, and nonsteroidal anti-inflammatory drugs, are currently in pre-clinical trials or clinical trials. [6] Acute and chronic degenerative brain diseases are different in causes and mechanism but lead to nerve cell loss, resulting in cerebral dysfunction. In this respect, attempts to develop agents directly blocking nerve cell death have been made. For example, MAP kinase inhibitors, caspase inhibitors, calpain inhibitors, anti¬ inflammatory drugs, etc. are currently in pre-clinical trials. [7] Generation of reactive oxygen species (ROSs) in normal cells is regulated by biological antioxidants or antioxidative enzymes. When production of ROSs exceeds cellular antioxidation activity in a pathophysiological condition, an oxidative damage to proteins, lipids, and DNAs is caused. As demonstrated in various studies, ROSs participate in the incidence of degenerative brain diseases such as Alzheimer's disease and Parkinson's disease, and ischemic or hemorrhagic stroke. ROSs play an important role in exitotoxicity, betaamlyoid accumulation, or degenerative brain diseases due to ischemia or recovery of nerve growth factor. Recently, antioxidative therapy has been used in the treatment of acute or chronic degenerative brain diseases. [8] Flavonoids are naturally occurring multivalent phenol compounds present in fruits, vegetables, seeds, etc. Various types of flavonoid derivatives categorized, according to chemical structure, into flavonols, flavones, and isoflavones, are known, and their biological and pharmacological activities have been studied. Disclosure of Invention Technical- Problem [9] While investigating the effects of various types of flavonoid derivatives on de¬ generative brain diseases, the present inventors surprisingly found that specific flavonoid derivatives had an excellent prevention and treatment effect for degenerative brain diseases, and thus completed the present invention. [10] Therefore, the present invention provides a composition for the prevention or treatment of acute or chronic degenerative brain diseases including a flavonoid derivative as an effective ingredient. Technical- Solution [11] According to an aspect of the present invention, there is provided a composition for preventing or treating an acute or chronic degenerative brain disease, the composition including as an effective ingredient a flavonoid derivative selected from the group consisting of 4',7-dihydroxyflavone; 3',4',7-trihydroxyflavone; 3,3'-di-0 - methylquercetin; kaempferide; galangin; morin; amentoflavone; hinokiflavone; ochnaflavone; ochnaflavone 4'-0-methyl ether; kaempferol 3-0 - (6"-coumaroylglucosyl)(l → 2)rhamnoside; quercetin 3-O-(6"-coumaroylglucosyl)(l → 2)rhamnoside; kaempferol 3-O-glucosyl(l → 2)rhamnoside; kaempferol 3-0 - 2",6"-dirhamnosylglucoside; quercetin 3-0-2", 6"-dirhamnosylglucoside; and kaempferol 3-O-rutinoside, and a pharmaceutically acceptable carrier. [12] The flavonoid derivative contained in the composition of the present invention may be selected from compounds represented by the following formulae 1 through 6: [13] <Formula 1>

[14] (1) 4',7-dihydroxyflavone: [15] R

4=OH [16] (2) 3',4',7-trihydroxyflavone: [17] R

3=R 4=OH [18] (3) 3,3'-di-O-methylquercetin: [ [1199]] R R = =RR =OOHH,, R R =R =H, R=R =0CH 1 = 1 44 2 =F 2 5 3 3 [20] (4) kaempferide: [ [2211] ] R R==RR = =OOHH,, R R =R =R =H, R =0CH 1 2 3 5 4 3 [22] (5) galangin: [ [2233]] R R==RR = =OOKH, R =R =R =R =H 1 2 3 4 5 [24] (6) morin: [ [2255]] R R==RR 1 = =RR2 = =R 4 =OH, R 3 =R 5 =H [26] <Formula 2>

[27] (7) amentoflavone [28] <Formula 3>

[29] (8) hinokiflavone [30] <Formula 4>

[31] (9) ochnaflavone: [32] R=H [33] (10) ochnaflavone 4'-(9-methyl ether [34] R=CH 3 [35] <Formula 5>

[36] (11) kaempferol 3-O-(6"-coumaroylglucosyl)(l → 2)rhamnoside:

[37] (12) quercetin 3-O-(6"-coumaroylglucosyl)(l → 2)rhamnoside:

[38] (13) kaempferol 3-O-glucosyl(l → 2)rhamnoside: [39] R=R'=H [40] <Formula 6>

[41] (14) kaempferol 3-0-2",6"-dirhamnosylglucoside:

[42] (15) quercetin 3-0-2",6"-dirhamnosylglucoside:

[43] (16) kaempferol 3-0-rutinoside: [44] R=R₁=H [45] The composition of the present invention includes a pharmaceutically acceptable carrier, and may be administered orally or parenterally to human beings or animals for the prevention or treatment of acute or chronic degenerative brain diseases including dementia and stroke. [46] A composition for oral administration according to the present invention may be in any form including tablets, capsules, powders, granules, liquids, suspensions, gels, etc., and may include a conventional excipient such as a diluent, a disintegrating agent, a lubricant, etc. The excipient includes a conventional diluent such as syrup, Arabic gum, gelatin, sorbitol, lactose, sugar, corn-starch, calcium phosphate, glycine, magnesium stearate, talc, polyethyleneglycol, silica, potato starch, or sodium lauryl sulfate, and a conventional flavorant or colorant. A composition for parenteral admin¬ istration according to the present invention (e.g., for injection) may be an isotonic solution or a sterile isotonic solution, and/or may include a conventional excipient such as a preservative or a stabilizer. [47] A pharmaceutical composition of the present invention can be administered in the form of a daily dosage of 100 mg-lg for average 70 kg adult for the prevention or treatment of acute or chronic degenerative brain diseases. However, an adequate dosage is determined depending on the type of disease and the degree of disease severity. In this regard, for typical adult patients, a unit dosage form includes about 100 mg to 1 g of the extract according to the present invention in combination with a pharmaceutically acceptable carrier. Description Of Drawings [48] FIG. 1 shows the morphological analysis results for the inhibitory effects of flavonoid derivatives (kaempferide and morin) against nerve cell death; and [49] FIGS. 2A and 2B show the inhibitory effect of a flavonoid derivative (amentoflavone) against cerebral damage. Best Mode [50] Hereinafter, the present invention will be described more specifically with reference to the following Examples. The following Examples are for illustrative purposes and are not intended to limit the scope of the invention. [51] Example 1: Evaluation of inhibitory activity of flavonoid derivatives against nerve cell death caused by increased reactive oxygen species [52] The inhibitory activity of flavonoid derivatives against nerve cell death induced by ischemia was evaluated. SH-SY5Y cells (Korean Cell Line Bank (KCLB), No. 22266) were used as nerve cell lines. The nerve cells were deposited in a volume of 5X10 cells/well in a 48-well plate. Then, DMEM media containing 5% fetal bovine serum and 10% horse serum were added thereto and the cell cultures were incubated at 37 ⁰C

[53] To induce nerve cell death by ischemic stimuli, the cell culture media were replaced with serum-free DMEM media. Then, 0.5 mM of a hydrogen peroxide water solution was added thereto and the cell cultures were incubated for 24 hours. To measure the degree of cell death, MTT (methylthiazoletetrazolium) was added to the cell culture media and incubated for 3 hours. The culture media were removed, 100 D dimethyl- sulfoxide (DMSO) was added and mixed, and absorbance was measured at 595 nm. [54] Each flavonoid derivative was dissolved in DMSO. The mixed solution was added to the cells until the final concentration of the used compound was 0.4, 2, 10, and 50 uM, and treated with a hydrogen peroxide water solution at one hour after the addition of the mixed solution to induce cell death. At this time, the final concentration of DMSO was adjusted to up to 0.5% to eliminate an influence of DMSO on cell death. Assuming that cell viability in serum-containing conditions was 100%, relative cell viability (%), i.e., degree of inhibition of cell death (%) was calculated. All ex¬ periments were repeated three times. The degree of inhibition of cell death at each con¬ centration of each flavonoid derivative is presented in Table 1 below. [55] Table 1

Flavonoid Cell viability (%) at each concentration derivative 0.4 uM 2 uM 10 uM 5O uM

(D -0.3 + 1.4 10.4 + 0.6 55.4 + 2.7 (2) 24.5 + 1.9 61.9 + 1.5 61.4 + 0.6 (3) 14.6 + 2.0 37.2 + 2.0 122.4 + 0.1 (4) 34.0 + 3.0 47.6 + 0.4 65.0 + 4.0 (5) 9.0 + 3.1 42.4 + 3.2 15.5 + 3.4

[56] (1) 4',7-dihydroxyflavone; [57] (2) 3',4',7-trihydroxyflavone; [58] (3) 3,3'-di-0-methylquercetin; [59] (4) kaempferide; (5) galangin; (6) morin; [60] (7) amentoflavone; (8) hinokiflavone; (9) ochnaflavone; [61] (10) ochnaflavone 4'-0-methylether; [62] (11) kaempferol 3-O-(6"-coumaroylglucosyl)(l -→ 2)rhamnoside; [63] (12) quercetin 3-(9-(6"-coumaroylglucosyl)(l → 2)rhamnoside; [64] (13) kaempferol 3-0-glucosyl(l -→ 2)rhamnoside; [65] (14) kaempferol 3-0-2",6"-dirhamnosylglucoside; [66] (15) quercetin 3-O-2",6"-dirhamnosylglucoside; [67] (16) kaempferol 3-0-rutinoside [68] As shown in Table 1, the flavonoid derivatives exhibited an inhibitory activity against nerve cell death induced by reactive oxygen species at a concentration of 0.4 ~ 5O uM. [69] To morphologically evaluate the inhibitory activity of the flavonoid derivatives against nerve cell death, the nerve cell lines were cultured in serum-containing conditions and pretreated with kaempferide (10 uM) and morin (10 uM) for one hour, and then 0.5 mM of a hydrogen peroxide water solution was added thereto to induce cell death. At 8 hours after the cell death induction, the cells were stained with propidium iodide (10 uM) for 10 minutes and phase-contrast and fluorescence mi¬ croscopic analyses for the cells were performed. The analysis results are shown in FIG. 1. [70] As shown in FIG. 1, with respect to the nerve cells treated with the hydrogen peroxide water solution with no pretreatment, dead cells stained with propidium iodide were 30% of the whole cells. On the other hand, with respect to the nerve cells pretreated with kaempferide or morin, nerve cell death was remarkably reduced. [71] As described above, since flavonoid derivatives exhibit excellent pharmacological activity against nerve cell death induced by reactive oxygen species, it can be ef¬ ficiently used for the prevention or treatment of ischemic cerebral damage such as stroke. [72] Example 2: Evaluation of inhibitory activity of flavonoid derivatives against nerve cell death induced by mitochondria damage [73] It was reported that stress stimuli to cells (e.g., growth hormone removal, ischemia, low oxygen state) trigger transfer of the apoptotic signal from mitochondria, thereby leading to nerve cell death. In this Example, mitochondria-mediated cell death was induced by staurosporine and the inhibitory activities of flavonoid derivatives against the cell death were measured. [74] SH-SY5Y cells were deposited in a 48-well plate and then cultured in serum- containing media. To induce nerve cell death, the cell culture media were replaced with serum-free media. Then, 100 nM of staurosporine was added thereto and con¬ tinuously cultured for 24 hours. To measure the degree of cell death, MTT was added to the cell culture media and incubated for 3 hours. The culture media were removed, 100 D DMSO was added and mixed, and absorbance was measured at 595 nm. [75] Each flavonoid derivative was dissolved in DMSO. The mixed solution was added to the cells until the final concentration of the used compound was 0.4, 2, 10, and 50 uM, and treated with staurosporine at one hour after the addition of the mixed solution to induce cell death. At this time, the final concentration of DMSO was adjusted to up to 0.5% to eliminate an influence of DMSO on cell death. Assuming that cell viability in serum-containing conditions was 100%, relative cell viability (%), i.e., degree of inhibition of cell death (%) was calculated. All experiments were repeated three times. The degree of inhibition of cell death at each concentration of each flavonoid derivative is presented in Table 2 below. [76] Table 2

Flavonoid Cell viability (%) at each concentration derivative* 0.4 uM 2 uM 10 uM 5O uM

(D -15.1 + 1.9 -10.6 + 3.1 -16.5 + 4.2 (2) -2.7 + 0.5 -1.2 + 0.5 3.7 + 1.4

(3) -9.7 + 2.3 -1.7 + 4.3 57.9 + 9.6 (4) -8.9 + 0.3 58.4 + 0.3 37.3 + 1.7

[77] * Each flavonoid derivative is as defined in Table 1. [78] As shown in Table 2, the flavonoid derivatives effectively prevented nerve cell death induced by staurosporine. Thus, it can be seen that flavonoid derivatives can prevent mitochondria-mediated nerve cell death induced by stress stimuli. [79] Example 3: Evaluation of inhibitory activity of flavonoid derivatives against nerve cell death induced by betaamyloid [80] Reportedly, nerve cell damage in dementia patients is mainly caused by direct neu¬ rological toxicity due to betaamyloid (A β ) accumulation or inflammation due to activation of microglial cells. In this Example, nerve cell death was induced by be¬ taamyloid (A β ) peptides, and the inhibitory effect of flavonoid derivatives against the nerve cell death was evaluated. [81] The degree of nerve cell death induced by A β was measured using cell lines (PC12 cells, KCLB No. 21721) having similar characteristics to nerve cells. The PC12 cells are deposited in a volume of 5X10 cells/well in a 48-well plate. Then, DMEM media containing 10% fetal bovine serum were added thereto and the cell cultures were incubated at 37 ⁰C . [82] To induce nerve cell death, the cell culture media were replaced with serum-free DMEM media. Then, A β was added thereto and the cell cultures were incubated ¹ 25-35 for 24 hours. To measure the degree of cell death, MTT was added to the cell culture media and incubated for 3 hours. The culture media were removed, 100 D DMSO was added and mixed, and absorbance was measured at 595 nm. [83] Each flavonoid derivative was dissolved in DMSO. The mixed solution was added to the cells until the final concentration of the used compound was 2, 10, and 50 uM, and A β 25-35 was added to the cells at one hour after the addition of the mixed solution to induce cell death. At this time, the final concentration of DMSO was adjusted to up to 0.5% to eliminate an influence of DMSO on cell death. Assuming that cell viability in serum-containing conditions was 100%, relative cell viability (%), i.e., degree of inhibition of cell death (%) was calculated. All experiments were repeated three times. The results of cell death inhibitory effect at each concentration of each flavonoid derivative are presented in Table 3 below. [84] Table 3

[85] * Each flavonoid derivative is as defined in Table 1. [86] As shown in Table 3, the flavonoid derivatives exhibited inhibitory activity against nerve cell death induced by betaamyloid. [87] Example 4: Evaluation of anti-inflammatory effect of flavonoid derivatives in microglial cells [88] In dementia patients, betaamyloid (A β ) activates neighboring microglial cells by insoluble plague formation in the brain, thereby leading to inflammation. In ischemic stroke patients, nerve cell necrosis causes inflammation, thereby leading to secondary nerve cell damage. In this respect, there was reported that nonsteroidal anti¬ inflammatory drugs are effective for the treatment of stroke. Thus, it is anticipated that anti-inflammatory compounds for microglial cells can be used as treatment agents for degenerative brain diseases. [89] Microglial cells (BV-2 cell line, Pharmacology Room of the College of Medicine of Ehwa Women's Univ.) were deposited in a volume of 5X10 cells/well in a 48-well plate. Then, DMEM media containing 10% fetal bovine serum were added thereto and the cell cultures were incubated at 37 ⁰C . [90] To induce inflammation, the microglial cells were treated with 100 ng/ml of lipopolysaccharide (LPS) and cultured for 24 hours. The concentration of an in¬ flammation mediator, nitric oxide (NO) released into the cell cultures was measured. A calibration curve was prepared using a sodium nitrite solution as a standard solution. [91] Each flavonoid derivative was dissolved in DMSO. The mixed solution was added to the cells until the final concentration of the used compound was 0.4, 2, and 10 uM, and LPS was added to the cells at one hour after the addition of the mixed solution to induce inflammation. At this time, the final concentration of DMSO was adjusted to up to 0.5% to eliminate an influence of DMSO on NO production. All experiments were repeated three times. The NO production inhibition (% relative to a control) was measured and the results are presented in Table 4 below. [92] Table 4

Flavonoid NO production inhibition (%) at each concentration derivative* 0.4 uM 2 uM 10 uM

(D 17.2 + 1.8 18.0 + 0.2 43.8 + 1.0 (2) 19.8 + 0.5 12.2 + 0.8 55.1 + 0.3

(3) 24.7 + 0.7 48.0 + 0.6 91.3 + 1.4 (4) 2.4 + 1.0 9.1 + 2.4 65.5 + 0.4

(5) 4.9 + 0.4 5.5 + 0.9 30.8 + 0.9 (6) -0.9 + 1.0 1.5 + 1.4 18.9 + 1.2

(7) 3.3 + 0.6 5.7 + 0.6 25.4 + 0.3

(8) 9.6 + 0.7 62.8 + 0.3 97.2 + 0.4 (9) -7.6 + 1.4 32.2 + 0.1 88.7 + 0.8 (10) 17.5 + 0.8 25.1 + 0.5 38.2 + 0.6

(H) 17.1 + 0.8 20.5 + 1.0 17.8 + 1.6

[93] * Each flavonoid derivative is as defined in Table 1. [94] As shown in Table 4, the flavonoid derivatives effectively inhibited NO production activated by LPS. Therefore, it can be seen that flavonoid derivatives can effectively treat cerebral damage by preventing inflammation that occurs in dementia or stroke. [95] Example 5: Evaluation of therapeutic effect of flavonoid derivatives against cerebral nerve cell death in ischemic stroke animal models [96] To induce hypoxic and/or ischemic cerebral damage, Sprague-Dawley rats (7 days after birth) were subjected to left carotid artery ligation followed by suture under anes¬ thetization with isoflurane and then allowed to recover from anesthesia. The rats were exposed to a mixed gas of 8% oxygen and 92% nitrogen for 2.5 hours to induce cerebral damage. 30 mg/kg of amentoflavone was administered intraperitoneally to the Sprague-Dawley rats. At one week after the intraperitoneal administration, the brains were excised from the rat heads, fixed with paraformaldehyde, and sectioned. The brain sections were stained with cresyl violet and damaged areas were measured. The results are shown in FIGS. 2 A and 2B. [97] As shown in FIGS. 2A and 2B, amentoflavone inhibited cerebral damage at a significant level. Thus, it can be seen that the flavonoid derivatives exhibit therapeutic activity against ischemic stroke or hypoxic and/or ischemic cerebral damage. Industrial Applicability [98] The flavonoid derivative-containing composition according to the present invention can be effectively used for the prevention or treatment of acute or chronic degenerative brain diseases such as dementia or stroke.

Claims

Claims [1] A composition for preventing or treating an acute or chronic degenerative brain disease, the composition comprising as an effective ingredient a flavonoid derivative selected from the group consisting of 4',7-dihydroxyflavone; 3',4',7-trihydroxyflavone; 3,3'-di-0-methylquercetin; kaempferide; galangin; morin; amentoflavone; hinokiflavone; ochnaflavone; ochnaflavone 4'-O-methyl ether; kaempferol 3-O-(6"-coumaroylglucosyl)(l -→ 2)rhamnoside; quercetin 3-0 -(6"-coumaroylglucosyl)(l → 2)rhamnoside; kaempferol 3-(9-glucosyl(l → 2)rhamnoside; kaempferol 3-0-2", 6"-dirhamnosylglucoside; quercetin 3-0 - 2",6"-dirhamnosylglucoside; and kaempferol 3-O-rutinoside, and a pharma¬ ceutically acceptable carrier. [2] The composition of claim 1, wherein the acute or chronic degenerative brain disease is stroke or dementia.