Therapeutic composition for kainic acid-induced temporal lobe epilepsy

专利摘要:
The present invention relates to a composition for treating temporal lobe epilepsy with kainate (Kainic acid), the composition of the present invention containing the cheonma ether fraction obtained by extracting the cheonma with methanol and then fractionated with ether, the composition of the hippocampus (hippocampus) Inhibiting neuronal damage in the CA1 and CA3 regions, it can be used as a useful therapeutic for temporal lobe epilepsy.
公开号:KR20030071036A
申请号:KR1020020010555
申请日:2002-02-27
公开日:2003-09-03
发明作者:김현주；이성룡
申请人:씨.에프. 주식회사；
IPC主号:

专利说明:

Therapeutic composition for kainic acid-induced temporal lobe epilepsy}
[17] The present invention relates to a composition for treating temporal lobe epilepsy with chinate. More specifically, the present invention relates to a composition for treating temporal lobe epilepsy containing a cheonma ether fraction obtained by extracting cheonma with methanol and then fractionating with ether.
[18] Cainic acid (Kainic acid) is an excitatory neurotransmitter that binds to ions, which are a type of non-NMDA receptor, or an alpha-amino-3-dydroxy-5-methyl-isoxaxole-4-propionate (AMPA) receptor It is known to excite nerve cells by inducing cations such as sodium or calcium to induce polarization between nerve cell membranes.
[19] Because of these properties, kainic acid has been used to study a variety of central nervous system diseases, including excitability, excitatory toxicity, and acute cell loss. Specifically, administration of kainate selectively causes neuronal death in the CA1 and CA3 regions of the hippocampus (Young et al., Trends Pharmacol. Sci., 11: 126-133 (1990)). The phosphate has been used as a drug to induce temporal lobe epilepsy in laboratory animals (Lassman et al., Neuroscience 13, 691-704, 1984).
[20] On the other hand, Gastrodia elata Blume is a medicinal plant belonging to the orchid family, has no roots and leaves, and has a brown color. There is no greenery is a plant that not only do not support a special feature of photosynthesis and chlorophyll done does not absorb the nutrients necessary for the growth of roots depends on the energy supply of nutrients to produce a mold (Armillarea Mellea) parasitic on their own. For centuries, Asian countries have used cheon horses as herbal remedies for dizziness, general paralysis, and tetanus.
[21] Previous studies have shown that the components of cheonma inhibits glutamic acid apoptosis in neurons (Lee et al., Arch. Pharm. Res., 22: 404-409 (1999)), and ether fractions of methanol extracts of cheonma It has been reported to reduce the decrease of γ-aminobutyric acid (GABA) and increase the content of glutamic acid in this pentylentetrazole-induced epilepsy (Ha et al., J. Ethnopharmacol., 73: 329-333 (2000)., Huh et al., Life Sci., 65: 2071-2082 (1999).
[22] However, as mentioned above, although the conventional chemistry has been reported to be effective in diseases caused by cerebral blood flow disorders, nervous breakdown, pentyltetrazole-induced epilepsy, fractions obtained by fractionating ethanol methanol extract with ether are present in the hippocampus. No studies have been reported to block the neurotoxic effects of kainate, known to kill vertebral nerve cells.
[23] The inventors of the present invention, while studying the composition for treating temporal lobe epilepsy, the ether fraction obtained by fractionation of methanol extract from ether with ether has the effect of inhibiting nerve damage in CA1 and CA3 regions of hippocampus, thus preventing and treating temporal lobe epilepsy. The present invention has been completed by confirming that it can be usefully used.
[24] Accordingly, it is an object of the present invention to provide a composition for treating temporal lobe epilepsy by kainic acid, characterized in that it contains cheonmachu as an active ingredient.
[25] Hereinafter, the configuration and operation of the present invention.
[1] Figure 1 is a result of measuring the antiepileptic effect by administering the ether fraction of the cheonma methanol extract after inducing temporal lobe epilepsy in mice treated with kainate.
[2] A: Graph showing the initiation time of neurobehavioral change after cyanate treatment.
[3] B: A graph showing the grade of temporal lobe epilepsy in mice after cyanate treatment.
[4] FIG. 2 shows the results of investigating the effect of ether fractions of methanol extracts of Chunma by observing the morphology of hematoxylin eosin CA1 and CA3 regions of hippocampus after 4 days of kinate treatment in mice.
[5] (Bars indicate 50 μm, arrows indicate damaged cells in CA1 and CA3 regions.)
[6] A: CA1 region of mice treated with the same amount of saline and vehicle
[7] B: CA3 region of mice treated with the same amount of saline and vehicle
[8] C: CA1 area of mouse treated with kinate and vehicle
[9] D: CA3 area of mouse treated with kinate and vehicle
[10] E: CA1 region of mice treated with ether fraction (200 mg / kg dose group) and catenate of methanol extract
[11] F: CA3 region of mice treated with ether fraction of 200 mg methanol extract (200 mg / kg dose group) and catenate
[12] G: CA1 region of mice administered with ether fraction of 500 mg methanol extract (500 mg / kg) and catenate
[13] H: CA3 region of mice treated with ether fraction of 500 mg methanol extract (500 mg / kg administration group) and catenate
[14] Figure 3 is a result of examining the effect of the ether fraction of the methanol extract of cheonma by observing the number of viable cells in the hippocampus tissue of the mouse 4 days after the kainate administration.
[15] A: number of viable cells in CA1 region of hippocampus
[16] B: viable cell number in CA3 region of hippocampus
[26] The present invention provides a novel use of cheonma extract for the prevention and treatment of temporal lobe epilepsy by kainate.
[27] The cheonma extract refers to an extract obtained by pulverizing the dry cheonma, extracted with methanol, and fractionating the obtained methanol extract with ether (hereinafter, abbreviated as 'ether fraction of cheonma methanol extract').
[28] The ether fraction of the cheonma methanol extract obtained by the above method is preferably suspended in a 1% carboxymethyl cellulose solution.
[29] The composition of the present invention containing the ether fraction of the cheonma methanol extract as an active ingredient has an effect of inhibiting neuronal damage in the CA1 region and CA3 region of the hippocampus (hippocampus) by kainate, functional foods and supplements for treating temporal lobe epilepsy It can be used as a material for therapy.
[30] According to the present invention, the ether fraction of the cheonma methanol extract may be formulated into unit dosage forms or several dosage forms such as tablets, capsules, granules, suspensions, and emulsions by conventional methods, and may be used as compositions for preventing and treating temporal lobe epilepsy. have.
[31] In addition, the composition containing the ether fraction of the cheonma methanol extract as an active ingredient can be administered several times a day divided into 200 mg to 1000 mg per 1kg body weight per dose as desired, less than 200mg and can not obtain a significant effect . However, the dosage level for a particular patient may vary depending on the weight, age, sex, health condition, diet, time of administration, method of administration, severity of disease, and the like of the patient.
[32] Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
[33] EXAMPLES Preparation of Ether Fraction of Methyl Extract of Chunma
[34] Cheonma was purchased from Seokbo Cheonma Farm in Gyeongbuk and used as a material for experiments.
[35] First, fresh ginseng was washed, chopped, and cooled in a freezer at minus 70 degrees Celsius for 24 hours, and then freeze-dried and powdered. To 1 kg of dried powder of Chunma, three times the amount of methanol, ie, 3 L of methanol, was added and refluxed three times at 60 ° C. The material extracted with methanol was filtered, concentrated and suspended in 1 L of distilled water. The suspension was extracted three times with an equal volume of ether (1 L) and concentrated at low temperature under reduced pressure and then the ether fractions were suspended in 1% carboxymethyl cellulose solution.
[36] Experimental Example 1: Investigation of behavioral changes in mice according to ether fraction administration
[37] Mice were administered orally at 200 mg and 500 mg doses, respectively, using an esophageal needle for 14 days prior to kainate acid injection and 4 days after injection. It was. Control mice received oral administration of the same amount of 1% water-soluble carboxy methyl cellulose solution without the ether fraction.
[38] The mice used ICR-type mice having a weight of 25-35 g and were used under constant light and dark cycles of light from 6 am to 6 pm. Kainate (sigma co. USA) was dissolved in distilled water to a pH of 7.4 and injected intraperitoneally to 45 mg per kg. Neurobehavioral changes following the administration of kainate acid were assessed by a slightly modified criterion of Kondo et al. (Kondo et al., J. Cereb. Blood Flow Metab., 17: 241-256 (1997)). Behavioral observations were graded according to the depth of epilepsy symptoms as shown in Table 1 below.
[39] All data were expressed as mean ± standard deviation, and the significance test was performed by Student t-test. The statistical significance level was based on P value of 0.01 or less and 0.05 or less.
[40] Grade according to the depth of epilepsy symptomsSymptom 0 stepMice do not show any epilepsy. Stage 1Mouse is staring or auto repeating copper foil with arched tail Tier 2This stage reveals myoclonic twitches of the forelimbs, as if the mice were playing the piano without dropping. Tier 3Shows systemic epilepsy or intense spastic leap.
[41] Behavioral changes in mice were observed for 3 hours after kainate (Kainic acid) administration, and the onset time of the first behavioral change (FIG. 1A) and the inducible epilepsy grade of the mouse (FIG. 1B) were examined.
[42] Experimental results showed that the onset of neurobehavioral change was significantly delayed when compared with the group administered with kainate and vehicle in the group administered with kainate acid and 500 mg of ether fraction per kg. P <0.01). Moreover, the depth of neurobehavioral change was reduced (P <0.05, FIG. 1B). However, at a dose of 200 mg per kg, ether fractions of methanol extracts of Chunma did not reduce neurobehavioral changes by kainic acid. It was.
[43] Experimental Example 2: Investigation of histological changes in mouse hippocampus (hippocampus) according to ether fraction administration
[44] Histologically, in order to measure the effect of ether fraction administration of methanol extract, the mouse of Experimental Example 1 was deeply anesthetized with chloral hydrate 4 days after the administration of kainate acid (phosphate buffered saline solution containing heparin (PBS, pH). 7.2) perfusion through the heart. It was then perfused with 10% formalin diluted in PBS.
[45] Brain tissues were immediately extracted and further fixed for 24 to 48 hours in 10% formalin fixative. The brain tissues were cut to 6-micron thickness using a rotating microtome to make coronal sections. Tissue sections were stained with hematoxylin and eosin, and histological examination was performed by a non-experimental investigator. The left and right averages were counted by counting normal CA1 vertebral neurons at 500 micrometers and normal CA3 vertebral neurons observed under an optical microscope 200 times. All data were expressed as mean and deviation, statistical analysis was performed using variance analysis and scheffe ex posttest (histology). Student t-test was performed on the interstitial depth and urethral time for initial neurobehavioral changes. The statistical significance level was based on the P value of 0.05 or less.
[46] When compared with the control group treated with kainate (Kainic acid) and the control group not administered, there was a significant decrease in the vertebral nerve cells surviving in the CA1 and CA3 region of the hippocampus (Figs. 2 and 3). In the hippocampal CA1 region (P <0.01) and CA3 region (P <0.05), the reduction of viable vertebral neurons by Cainic acid was significantly reduced by administration of ether fraction of 500 mg of methanol extract per kg. It was found that the cell damage was protected. The ether fraction of the methanol extract of Chunma did not reduce neuronal damage in the hippocampus when administered at a dose of 200 mg / kg.
[47] In view of the above results, the ether fraction of methanol extract of Chunma has the effect of reducing the hippocampal nerve damage caused by antiepileptic effect and excitatory poisoning effect by Kainic acid. It is believed to be able.
[48] As is apparent from the above examples and experimental examples, the ether fraction obtained by fractionating the methanol extract of Chunma with ether has an antiepileptic effect on temporal lobe epilepsy caused by kainate acid and prevents neuronal damage. Because it has an excellent effect on preventing and treating temporal lobe epilepsy, it is a very useful invention for the functional food and pharmaceutical industry.

权利要求:
Claims (1)
[1" claim-type="Currently amended] A composition for treating temporal lobe epilepsy by kainate, comprising pulverizing dry cheonma, extracting by adding methanol, and then fractionating with ether as an active ingredient.

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引用文献:

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法律状态:
2002-02-27|Application filed by 씨.에프. 주식회사

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2002-02-27|Priority to KR1020020010555A

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2003-09-03|Publication of KR20030071036A

优先权:

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申请号 | 申请日 | 专利标题

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KR1020020010555A|KR20030071036A|2002-02-27|2002-02-27|Therapeutic composition for kainic acid-induced temporal lobe epilepsy|