WO2006001664A1

WO2006001664A1 - Composition for preventing or treating acute or chronic degenerative brain diseases including extract of selaginella tamariscina spring

Info

Publication number: WO2006001664A1
Application number: PCT/KR2005/001985
Authority: WO
Inventors: Byung-Hee Han; Sam-Sik Kang; Kun-Ho Son
Original assignee: Seoul National University Industry Foundation
Current assignee: Seoul National University Industry Foundation
Priority date: 2004-06-28
Filing date: 2005-06-24
Publication date: 2006-01-05
Anticipated expiration: 2006-12-28
Also published as: KR20060000252A; KR100633344B1

Abstract

Provided is a composition for preventing or treating an acute or chronic degenerative brain disease, the composition including as an effective ingredient an extract of Selaginella tamariscina Spring obtained by a process including: primarily extracting Selaginella tamariscina Spring with water or a C1 ~ C4 alcohol and secondarily extracting the primary extract with an organic solvent selected from the group consisting of hexane, chloroform, ethylacetate, and butanol, and a phar¬ maceutically acceptable carrier.

Description

COMPOSITION FOR PREVENTING OR TREATING ACUTE

OR CHRONIC DEGENERATIVE BRAIN DISEASES

INCLUDING EXTRACT OF SELAGINELLA TAMARISCINA

SPRING Technical Field [1] The present invention relates to a composition for preventing or treating acute or chronic degenerative brain diseases including an extract of Selaginella tamariscina Spring 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] Meanwhile, 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. [5] For example, Korean Patent Nos. 139,179 and 267,060 disclose that flavonoid derivatives such as isocryptomerin, cryptomerin B, and amentoflavone derived from Selaginella tamariscina Spring can be efficiently used as immunosuppressants, antiin¬ flammatory agents, anticancer agents, and analgesics. Disclosure of Invention Technical- Problem [6] While investigating the effects of various types of flavonoid derivatives on de¬ generative brain diseases, the present inventors surprisingly found that an extract of Selaginella tamariscina Spring had an excellent prevention and treatment effect for de¬ generative brain diseases, and thus completed the present invention. [7] Therefore, the present invention provides a composition for the prevention or treatment of acute or chronic degenerative brain diseases including an extract of Se¬ laginella tamariscina Spring as an effective ingredient. Technical- Solution [8] 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 an extract of Selaginella tamariscina Spring obtained by a process including: primarily extracting Selaginella tamariscina Spring with water or a C ~ C alcohol and secondarily extracting the primary extract with an organic solvent selected from the group consisting of hexane, chloroform, ethylacetate, and butanol, and a pharmaceutically acceptable carrier. [9] The extract of Selaginella tamariscina Spring contained as an effective ingredient in the composition of the present invention may be obtained by a known method, for example, a preparation process disclosed in Korean Patent No. 137,179 or 267,060. In the preparation of the extract of Selaginella tamariscina Spring, a solvent used in the primary extraction is water or a C ~ C alcohol. The C ~ C alcohol may be methanol or ethanol. The organic solvent used in the secondary extraction may be ethylacetate. [10] In the preparation of the extract of Selaginella tamariscina Spring, the primary extraction may be performed in a water bath once or more, preferably three times for 6-hour once. The primary extract may be concentrated prior to the secondary extraction. The secondary extraction may be performed as follows: the concentrate of the primary extract is suspended in distilled water, loaded onto a separating funnel, and extracted sequentially or separately with the above-exemplified organic solvents. [11] According to a disclosure in Korean Patent No. 137,179, an extract of Selaginella tamariscina Spring contains flavonoid derivatives such as amentoflavone, cryptomerin B, and isocryptomerin. However, as can be seen from the following Examples, the extract of Selaginella tamariscina Spring contained as an effective ingredient in the composition of the present invention strongly prevents the incidence of a cerebral damage relative to amentoflavone. [12] 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. [13] 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. [14] 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 [15] FIG. 1 is a graph illustrating the evaluation results for the inhibitory activity of an extract of Selaginella tamariscina Spring against nerve cell death induced by reactive oxygen species; [16] FIG. 2 is a graph illustrating the evaluation results for the inhibitory activity of an extract of Selaginella tamariscina Spring against nerve cell apoptosis; and [17] FIG. 3 is a graph illustrating the evaluation results for an effect of an extract of Se¬ laginella tamariscina Spring on caspase-3 activity in several brain sections. Best Mode [18] 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. [19] As used in the following Examples, the term 'an extract of Selaginella tamariscina Spring' refers to 'an extract fraction of Selaginella tamariscina Spring' extracted from Selaginella tamariscina Spring using methanol as a primary extraction solvent and ethylacetate as a secondary extraction solvent according to a preparation process disclosed in Example 1 of Korean Patent No. 137,179. [20] Example 1: Evaluation of inhibitory activity of extract fraction of Selaginella tamarisdna Spring against nerve cell death caused by ischemic stimuli [21] The inhibitory activity of an extract fraction of Selaginella tamariscina Spring 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 . [22] 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. [23] An extract fraction of Selaginella tamariscina Spring was dissolved in DMSO. The mixed solution was added to the cells until the final concentration of the used compound was 1, 5, and 25 D / D 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 experiments were repeated three times. The degree of inhibition of cell death at each concentration is shown in FIG. 1. [24] As can be seen from FIG. 1, an extract fraction of Selaginella tamariscina Spring exhibits an effective inhibitory activity against nerve cell death induced by reactive oxygen species. In FIG. 1, '*' indicates that cell death was inhibited at a significant level relative to a control (p<0.05). [25] Nerve cell death can be morphologically classified into apoptosis and necrosis, and caspase-3 is a protease playing an important role in apoptotic cell death. To evaluate the effect of an extract fraction of Selaginella tamariscina Spring on caspase-3 activity, the nerve cell lines were treated with a cell culture (as a control) or an extract fraction of Selaginella tamariscina Spring (1, 5, 25 D / D ). At one hour after the treatment, the cells were treated with 0.4 mM of a hydrogen peroxide water solution to induce cell death. At 8 hours after the treatment, the cells were collected and dissolved in a buffer (10 mM HEPES pH7.4, 100 mM NaCl, 5 mM MgCl , 1% Triton X-100, 1 mM PMSF) to separate total proteins. Caspase-3 activity was measured using a peptide substrate by fluorescent analysis and the results are shown in FIG. 2. [26] As shown in FIG. 2, the hydrogen peroxide water solution remarkably increased caspase-3 activity, thereby leading to apoptotic cell death. With respect to the cell groups treated with the extract fraction of Selaginella tamariscina Spring, caspase-3 activity was remarkably reduced in a concentration-dependent manner. In FIG. 2, '*' indicates that cell death was inhibited at a significant level relative to the control (p< 0.05). [27] As described above, since an extract fraction of Selaginella tamariscina Spring exhibits excellent pharmacological activity against nerve cell death induced by reactive oxygen species, it can be efficiently used for the prevention or treatment of ischemic cerebral damage such as stroke. [28] Example 2: Evaluation of therapeutic effect of an extract fraction of Se¬ laginella tamariscina Spring in ischemic stroke animal models [29] To induce ischemic cerebral damage, Sprague-Dawley rats (7 days after birth) were subjected to left carotid artery ligation followed by suture under anesthetization 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] The animal subjects were grouped into a control and two drug treatment groups. With respect to the control, a physiological saline was administered intraperitoneally to the Sprague-Dawley rats. With respect to the two drug treatment groups, amentoflavone known to be contained in an extract fraction of Selaginella tamariscina Spring and an extract fraction of Selaginella tamariscina Spring were respectively ad¬ ministered intraperitoneally to the Sprague-Dawley rats in a dosage of 10 mg/kg. At 24 hours after the intraperitoneal administration, the brains were excised from the rat heads. The degree of cerebral damage was evaluated by measuring Caspase-3 activity by fluorescent analysis and the results are shown in FIG. 3. [31] As shown in FIG. 3, about 13-fold higher caspase-3 activity was observed at the cerebral damage sites induced by ischemia. With respect to the two drug treatment groups, caspase-3 activity was reduced at a significant level. The extract fraction of Se¬ laginella tamariscina Spring exhibited stronger caspase-3 inhibitory activity relative to amentoflavone. Thus, it can be seen that an extract fraction of Selaginella tamariscina Spring effectively inhibits cerebral damage by a physiological synergic effect of amentoflavone and another compound(s) contained therein. In FIG. 3, '*' indicates that cell death was inhibited at a significant level relative to the control (p<0.05) and '#' indicates that cell death was inhibited at a significant level relative to the amentoflavone group (p<0.05). [32] Example 3: Evaluation of inhibitory effect of an extract fraction of Selaginella tamariscina Spring against nerve cell death induced by betaamyloid [33] Reportedly, cerebral damage in dementia patients is mainly caused by direct neu¬ rological toxicity due to betaamyloid (A β ) accumulation or inflammation due to activation of microglia cells. In this Example, nerve cell death was induced by be- taamyloid (A β ) peptides, and the inhibitory effect of an extract fraction of Se- laginella tamariscina Spring against the nerve cell death was evaluated. [34] 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 . [35] 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 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. [36] An extract fraction of Selaginella tamariscina Spring 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 D / D , 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 are presented in Table 1 below. [37] Table 1

[38] As can be seen from Table 1, an extract fraction of Selaginella tamariscina Spring exhibits an inhibitory effect of nerve cell death induced by betaamyloid. Industrial Applicability [39] A composition according to the present invention including an extract of Selaginella tamariscina Spring can effectively prevent nerve cell death induced by ischemia or be¬ taamyloid. The physiological inhibitory activity of the extract of Selaginella tamariscina Spring against nerve cell death is attributed to a synergic effect of amentoflavone known to be contained in the extract and another ingredient(s) contained in the extract. Therefore, the 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 an extract of Se- laginella tamariscina Spring obtained by a process comprising: primarily extracting Selaginella tamariscina Spring with water or a C ~ C alcohol and secondarily extracting the primary extract with an organic solvent selected from the group consisting of hexane, chloroform, ethylacetate, and butanol, and a pharmaceutically acceptable carrier. [2] The composition of claim 1, wherein the C 1 ~ C 4 alcohol is methanol or ethanol. [3] The composition of claim 1, wherein the organic solvent is ethylacetate. [4] The composition of any one of claims 1 through 3, wherein the acute or chronic degenerative brain disease is stroke or dementia.