KR102333460B1 - Pharmaceutical composition for prevention or treatment of temporal lobe epilepsy comprising the extracts of Valeriana fauriei an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating temporal lobe epilepsy and a health functional food comprising a valerian extract as an active ingredient. It relates to therapeutic pharmaceutical compositions and health functional foods.

Description

A pharmaceutical composition for preventing or treating temporal lobe epilepsy comprising a valerian extract as an active ingredient {Pharmaceutical composition for prevention or treatment of temporal lobe epilepsy comprising the extracts of Valeriana fauriei an active ingredient}

The present invention is Valeriana fauriei Briquet ) relates to a pharmaceutical composition and health functional food for preventing or treating temporal lobe epilepsy comprising an extract as an active ingredient.

Epilepsy (epilepsy) is a group of chronic diseases in which some of the nerve cells generate excessive electricity in a short time and cause repeated seizures. It is a serious neurological disease.

It is reported that about 0.5% to 1% of the world's population suffer from epilepsy, and in Korea, it is estimated that there are about 300,000 to 400,000 epilepsy patients, and about 20,000 new epilepsy patients are is reported to occur.

Among them, temporal lobe epilepsy is the most common epilepsy seen in adults. Sclerosis of the medial temporal lobe, especially the hippocampus, medial temporal lobe tumors (cancer), and stroke ( stroke), vascular malformations, cortical dysplasia, infection, etc. can be the cause. Complex partial seizures are the most common symptoms and are accompanied by epigastric aura and automatism. Confusion usually occurs after seizures, and it often progresses to secondary generalized seizures.

Epilepsy treatments include levetiracetam, carbamazepine, ethosuximide, carbapentin and oxcarbazepine, but rash, blood disease, osteoporosis, Side effects such as hyponatremia, headache, behavior change and immune response are known. Since most of these drugs serve to control symptoms rather than cause treatment, continuous drug administration is required. can cause

However, there is still no fundamental treatment method for treating such epilepsy, and a therapeutic agent for essentially treating temporal lobe epilepsy has not yet been developed.

Accordingly, there is a need for development of an effective therapeutic agent capable of improving, preventing or treating temporal lobe epilepsy in a long-term and effective manner with relatively few side effects.

Accordingly, the present inventors confirmed that the valerian extract is effective in improving, preventing or treating temporal lobe epilepsy while conducting research to develop a therapeutic agent that can safely and effectively prevent or treat temporal lobe epilepsy. has been completed

10-2016-0132169

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating temporal lobe epilepsy comprising a valerian extract as an active ingredient.

In addition, it is an object of the present invention to provide a health functional food for preventing or improving temporal lobe epilepsy comprising a valerian extract.

Hereinafter, the present specification will be described in more detail.

As one aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating temporal lobe epilepsy comprising a valerian extract as an active ingredient.

The terms used in the present invention, common valerian (Valeriana fauriei Briq) ', also known as the road chogeun, valerianaceae (Valerianaceae) mainly as a perennial herbaceous plant belonging to the genus ballerina Ana (Valeriana) distributed in northern and temperate regions of Asia and Europe. Those with hair on the fruit are called Gwangneung Valerian ( var. dasycarpa), and those with no sawtooth on the edge of the split leaf are called Long- leaved Valerian (var. integra ).

In the present invention, the valerian may be used without limitation in the production area, and at least one selected from the group consisting of leaves, flowers, and roots may be used. Preferably, it can be used including roots.

In the present specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

As used herein, the term “prevention” refers to any action that inhibits or delays temporal lobe epilepsy by administration of the pharmaceutical composition. In addition, the "treatment" refers to any action that improves or advantageously changes the symptoms of temporal lobe epilepsy by administration of the pharmaceutical composition.

In the present specification, the extract refers to a material obtained by extracting the components therein from a natural product, and may include any form regardless of the extraction method or the type of the component.

The valerian extract of the present invention can be prepared according to a conventional method known in the art, that is, under conventional temperature and pressure conditions, using a conventional solvent.

As the extraction solvent, water, an organic solvent, or a mixture thereof may be used, and the organic solvent is one selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, hexane, ethyl acetate, dichloromethane, ether, chloroform and acetone. more preferably. The lower alcohol having 1 to 4 carbon atoms is preferably at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol and n-butanol, and the lower alcohol having 1 to 4 carbon atoms includes both anhydrous or hydrous alcohols. can do.

The extraction solvent of the present invention may be an aqueous alcohol solution having 1 to 4 carbon atoms, and the aqueous alcohol solution may preferably be ethanol.

The extraction method may be performed by an extraction method known in the art, for example, cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, supercritical extraction, compression extraction, etc., but is not limited thereto.

In one embodiment of the present invention, the valerian extract can be obtained by reflux cooling (Reflux extraction method) using ethanol as an extraction solvent from dried valerian roots.

It was confirmed that the pharmaceutical composition comprising the valerian extract of the present invention as an active ingredient has a seizure inhibitory effect in an animal model of Kainic acid (KA)-induced temporal lobe epilepsy (FIG. 1).

In addition, the pharmaceutical composition comprising the valerian extract of the present invention as an active ingredient has a protective effect (FIG. 2) and immunity against apoptosis of hippocampus neurons in an animal model of caic acid-induced temporal lobe epilepsy. It was confirmed that there is an inhibitory effect on cell activity (FIG. 3).

In addition, it was confirmed that the pharmaceutical composition comprising the valerian extract of the present invention as an active ingredient has an inhibitory effect on the expression of inflammatory mediators in an animal model of caic acid-induced temporal lobe epilepsy (FIG. 4).

Therefore, the pharmaceutical composition comprising the valerian extract of the present invention as an active ingredient can exhibit an excellent effect in preventing or treating temporal lobe epilepsy.

The pharmaceutical composition of the present invention may further include a component that does not increase drug efficacy, but is commonly used in pharmaceutical compositions to improve odor, taste, vision, and the like. In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive. Pharmaceutically acceptable additives include, for example, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, Hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol and talc, but are not limited thereto. In addition, the pharmaceutical composition may include a substance having prophylactic or therapeutic activity for diabetic neuropathic pain used alone or previously used.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier and may be formulated for oral or parenteral human or veterinary use. When formulating the pharmaceutical composition of the present invention, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant and a surfactant may be used.

Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations include at least one excipient, for example, starch, calcium carbonate ( Calcium carbonate), sucrose or lactose and gelatin can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium, stearate, and talc can be used.

Liquid formulations for oral administration include suspensions, internal solutions, emulsions and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Non-aqueous solvents and suspensions may include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally according to a desired method, and when administered parenterally, external application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection It is preferable to select the injection method.

The pharmaceutical composition of the present invention can be administered to a subject to prevent or treat temporal lobe epilepsy. As used herein, the term "individual" has a seizure induced due to temporal lobe epilepsy, and includes humans, horses, sheep, and pigs with diseases whose symptoms can be improved by administering the pharmaceutical composition of the present invention. , but refers to mammals such as goats and dogs, preferably humans.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a subject by any suitable method. The administration route may be oral or parenteral administration through any general route as long as it can reach the target tissue. In addition, the pharmaceutical composition of the present invention may be administered by any device that allows it to migrate to a target cell.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term, pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the weight, sex, age, health status, and severity of the patient. , the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be administered single or multiple.

The dosage of the pharmaceutical composition according to the present invention should be a pharmaceutically effective amount. A pharmaceutically effective amount means an amount sufficient to prevent or treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level may vary depending on the formulation method, the patient's condition and weight, the patient's sex, age, and the disease. It can be variously selected by those skilled in the art according to factors such as degree, drug form, administration route and duration, excretion rate, reaction sensitivity, and the like. An effective amount will vary depending on the route of treatment, the use of excipients, and the potential for use with other agents, as will be appreciated by those skilled in the art.

The dosage or dosage of the pharmaceutical composition comprising valerian as an active ingredient according to the present invention may vary depending on the age, physical condition, weight, etc. of the patient, but generally 0.01 to 500 mg/kg (weight)/1 It is preferable to administer within one range. And, within the effective daily dosage range, it can be administered once a day or divided into several times a day.

As another aspect for achieving the above object, the present invention also provides a health functional food for preventing or improving temporal lobe epilepsy comprising a valerian extract.

The health functional food for preventing or improving temporal lobe epilepsy may be used by adding the valerian extract of the present invention as it is or in combination with other foods or food ingredients, and may be appropriately used according to a conventional method.

In the present specification, the functional food means a food manufactured and processed using raw materials or ingredients useful for the human body, and the function of the present specification is to control nutrients for the structure and function of the human body or to improve health through physiological action. It may mean a useful effect for the use.

There is no particular limitation on the type of the food. Examples of foods that can be added to the composition containing the valerian extract include various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, and may include all health foods in a conventional sense. In addition to the above, the composition comprising the valerian extract of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, It may contain preservatives, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination.

The pharmaceutical composition comprising the valerian extract according to the present invention as an active ingredient significantly inhibits seizures, hippocampal nerve cell death, microglia and astrocyte activity, and expression of COX-2 and iNOS, which are representative inflammatory mediators. Since the effect is excellent, it has an excellent effect in the prevention or treatment of temporal lobe epilepsy.

1 shows the inhibitory effect of valerian extract on seizure behavior in an animal model of kainic acid (KA)-induced temporal lobe epilepsy. 1A is the result of measuring the seizure level by oral administration of valerian extract (100, 200, and 400 mg/kg) 2 hours before administration of KA (55 mg/kg,ip), FIG. 1B is KA (55 mg/kg) , ip) once daily 5 days before administration (pre-5d), 2 hours before induction of animal model (pre-2h), 20 minutes after induction of animal model (post-20min) and 40 minutes after induction of animal model (post-40min) ) is the result of measuring the degree of seizure by oral administration of valerian extract (400 mg/kg), FIG. 1C shows the sum of seizure grades between 5 and 120 minutes in the graph of FIG. 1A, FIG. The graph shows the sum of the seizure grades from 5 to 120 minutes. (ANOVA test; ^* P<0.05, ^** P<0.01 vs KA group)
Figure 2 shows the protective effect of valerian extract against hippocampal neuronal cell death in an animal model of caic acid-induced temporal lobe epilepsy. 2A-N are KA (0.2 μg/4 μl) intracerebral injection once a day 5 days before (Pre-5d), 2 hours before induction of animal model (Pre-2h), 12 hours after induction of animal model (Post-12h), induction of animal model After 36 hours (Post-36h) and 60 hours after induction of the animal model (Post-60h), the valerian extract (400 mg/kg) was orally administered, and the needle staining results were shown 72 hours after KA administration (Bar = 100) μm). Figure 2O shows the degree of neuronal cell death in the CA1 region, Figure 2P is It shows the degree of neuronal cell death in the CA3 region. Figure 2Q shows the results of Western blot analysis of Caspase-3 and GAPDH protein expression in hippocampal tissue after administration of KA (0.2 μg/4 μl) once daily (Pre-5d) 5 days before injection and 72 hours after KA administration. (ANOVA test; ^## P<0.01 vs Sham group; ^* P<0.05, ^** P<0.01 vs KA group)
Figure 3 shows the inhibitory effect of valerian extract on immune cell activity in an animal model of caic acid-induced temporal lobe epilepsy. 3A-F show KA (0.2 μg/4 μl) once daily 5 days before intracerebral injection (Pre-5d), 2 hours before animal model induction (Pre-2h), and 12 hours after animal model induction (Post-12h). ) After 36 hours of animal model induction (Post-36h) and 60 hours after animal model induction (Post-60h), valerian extract (400 mg/kg) was orally administered, and Iba-1 ( Shows the results of immunohistochemical staining for calcium binding adapter molecule 1) and glial fibrillary acidic protein (GFAP) (Bar = 100 μm). Figures 3G and 3H are KA (0.2 μg/4 μl) administered once daily (Pre-5d) 5 days before intracerebral injection, and western blot analysis of the expression of CD11b and GFAP proteins in hippocampal tissue 72 hours after KA administration. The results of the implementation are shown. (ANOVA test; ^## P<0.01 vs Sham group; ^** P<0.01 vs KA group)
4 shows the results of confirming the inhibitory effect of the valerian extract on the expression of inflammatory mediators in an animal model of caic acid-induced temporal lobe epilepsy. Figures 4A and 4B show the results of western blot analysis of the expression of COX-2 and iNOS proteins in hippocampal tissue, once daily (pre-5d), 72 hours after KA administration, 5 days before intracerebral injection of KA (0.2 μg/4 μl). is shown. (ANOVA test; ^# P<0.05 vs Sham group; ^* P<0.05 vs KA group)

Hereinafter, examples will be given to describe the present specification in detail. However, the embodiments according to the present specification may be modified in various other forms, and the scope of the present specification is not to be construed as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those of ordinary skill in the art.

Example

All results were analyzed using SPSS 21 version, and the experimental results were expressed as mean±standard deviation (mean±standard deviation) values. Statistical significance was verified after analysis using ANOVA (one-way ANOVA) followed by post hoc analysis by Tukey post hoc, and the significance probability (p-value) was recognized only when p<0.05 and p<0.01.

Example 1: Preparation of valerian extract

After finely pulverizing 2 kg of dried valerian roots obtained from Ginseng Specialty Research Institute in Eumseong-gun, Chungcheongbuk-do, a total of 330.2 g of valerian extract (yield rate 16.5%) (yield 16.5%) was used by reflux cooling using pure ethanol. , which can be referred to as "VO") was obtained.

Example 2: Cainic acid inductive temporal lobe epilepsy Creation of animal models

As experimental animals, male mice of the C57BL/6 strain (Nara Biotech, Seoul, Korea) with a body weight of 22 g to 25 g at 7-8 weeks of age were acclimatized in a breeding room for 7 days and then used for the experiment. Diet and water for each group were freely ingested. The breeding room temperature was maintained at 23±2 ℃ and humidity at about 55-60%, and the lights were turned on and off in a 12-hour cycle (06:00 to 18:00). All experiments were performed in accordance with Kyunghee University's Code of Laboratory Animal Ethics.

Induction of temporal lobe epilepsy in mice was performed by intraperitoneal administration or direct intracranial injection of kainic acid (KA), which induces excitatory neuronal cell death, depending on the effect (indicator) to be confirmed.

(1) Construction of an animal model of convulsive/paroxysmal temporal lobe epilepsy

In order to prepare an animal model for caic acid-induced temporal lobe epilepsy to measure seizure and seizure suppression efficacy, KA was administered intraperitoneally (i.p.) to mice at a concentration of 55 mg/kg to prepare an animal model for temporal lobe epilepsy.

(2) Construction of an animal model of neuronal apoptotic temporal lobe epilepsy

In addition, in order to construct an animal model for caic acid-induced temporal lobe epilepsy to measure the protective efficacy against neuronal apoptosis in the hippocampus, KA at a concentration of 0.2 μg/4 μl is stereotactically directly into the lateral ventricle. An animal model of temporal lobe epilepsy was prepared by injection (intracerebroventricular (icv) injection; AP: -2.9, ML: 2.0, DV: -3.8).

Animals were observed and classified according to the following rating scale:

Grade 0: Normal;

Grade 1: Floating

Grade 2: Tension of forelimbs and tail, stiff posture

Grade 3: Head nodding, myoclonic convulsions, and constant rotation

Grade 4: Generalized convulsions, salivation, repeated standing and falling

Grade 5: Persistent generalized convulsions

Grade 6: Death

Only animals that reached at least 4 according to the above evaluation scale were considered as test subjects, and animals were divided into 5-7 animals in each group.

Example 3: Cainic acid inductive temporal lobe epilepsy Seizures of Valerian Extract in Animal Models deterrent effect on behavior Confirm

In order to verify the seizure inhibitory effect of the valerian extract and to select the most effective concentration, an animal model of temporal lobe epilepsy was prepared according to the method described in Example 2-(1), and the experiment was performed by dividing it into four groups as follows. said:

- KA group; KA 55 mg/kg, i.p. + saline, p.o.

- KA+VO100 group; KA 55 mg/kg, i.p. + VO 100 mg/kg, p.o.

- KA+VO200 group; KA 55 mg/kg, i.p. + VO 200 mg/kg, p.o.

- KA+VO400 group; KA 55 mg/kg, i.p. + VO 400 mg/kg, p.o.

Each valerian extract (100, 200 and 400 mg/kg) was dissolved in 0.9% saline at each ratio and administered orally 2 hours before KA injection into the abdominal cavity, and for 2 hours after KA administration, the evaluation scale described in Example 2 The severity of seizures was measured according to The results are shown in FIG. 1 .

As shown in Figure 1, in the group administered with the valerian extract, the severity of seizures was significantly reduced in a concentration-dependent manner (FIGS. 1A, C).

The most effective concentration of valerian extract was 400 mg/kg.

In order to examine the seizure inhibitory effect according to the administration time of the valerian extract, the experiment was performed by dividing it into 5 groups as follows:

- KA group; KA 55 mg/kg, i.p. + saline, p.o.

- Pre-5d group; KA 55 mg/kg, i.p. + VO 400 mg/kg, p.o.; Dosing once daily from 5 days before KA administration

- Pre-2h group; KA 55 mg/kg, i.p. + VO 400 mg/kg, p.o.; 2 hours prior to KA administration

- Post-20m group; KA 55 mg/kg, i.p. + VO 400 mg/kg, p.o.; 20 minutes after KA administration

- Post-40m group; KA 55 mg/kg, i.p. + VO 400 mg/kg, p.o.; Administered 40 minutes after KA administration

As described above, once a day from 5 days before induction of the animal model (Pre-5d), 2 hours before induction of the animal model (Pre-2h), 20 minutes after induction of the animal model (Post-20min), and 40 minutes after induction of the animal model As a result of oral administration of the post (Post-40min) extract (400 mg/kg), the Pre-5d and Pre-2h groups showed a significant seizure inhibitory effect (FIGS. 1B, D).

From the above results, it was confirmed that the valerian extract was effective in suppressing seizure behavior in temporal lobe epilepsy caused by KA.

Example 4: Cainic acid inductive temporal lobe epilepsy Confirmation of neuroprotective effect of valerian extract in animal model

In order to confirm whether the valerian extract has a neuroprotective effect on hippocampal neuronal cell death, an animal model of temporal lobe epilepsy was prepared according to the method described in Example 2-(2), and the experiment was divided into 7 groups as follows. was performed:

- Sham-kun; 4 μl of saline, i.c.v. + saline, p.o.

- KA group; KA 0.2 μg/4 μl, i.c.v. + saline, p.o.

- Pre-5d group; KA 0.2 μg/4 μl, i.c.v. + VO 400 mg/kg, p.o.; Oral administration once daily for 5 days before KA administration

- Pre-2h group; KA 0.2 μg/4 μl, i.c.v. + VO 400 mg/kg, p.o.; Oral administration 2 hours prior to KA administration

- Post-12h group; KA 0.2 μg/4 μl, i.c.v. + VO 400 mg/kg, p.o.; Oral administration from 12 hours after KA administration

- Post-36h group; KA 0.2 μg/4 μl, i.c.v. + VO 400 mg/kg, p.o.; Oral administration from 36 hours after KA administration

- Post-60h group; KA 0.2 μg/4 μl, i.c.v. + VO 400 mg/kg, p.o.; Oral administration from 60 hours after KA administration

As described above, from 5 days before direct injection of KA into the brain, once daily (Pre-5d), 2 hours before inducing the animal model (Pre-2h), and 12 hours after inducing the animal model (Post-12h) , The extract (400 mg/kg) was orally administered after 36 hours (Post-36h) and 60 hours after induction of the animal model (Post-60h) after inducing the animal model.

The mice were anesthetized, and 4% paraformaldehyde (PFA, paraformaldehyde) was perfused into the heart to fix it, and the brain was extracted. The extracted brains were immersed in 4% PFA and fixed at 4°C for one more day, then changed to 30% sucrose and stored at 4°C for more than 3 days to prevent freeze damage. The hippocampus was cut into 30 μm thick using a cryosection and stored in PBS (phosphate buffered saline).

(One) needle dye ( cresyl violet stain)

In order to check whether the valerian extract has a protective effect on hippocampal neuronal cell death, the brain containing the hippocampus cut to a thickness of 30 ㎛ is placed on a coated slide and dried, and then 10-dried in 0.5% cresyl violet (pH 3.9) solution. Soaked for 15 minutes, Nissl staining was performed. After immersion in 70% ethanol and 80% ethanol to control dyeability, and when dehydrated in 95% or 100% ethanol, immersion in xylene and cover glass were covered. According to the degree of damage or death of neurons in CA1 and CA3 regions of the hippocampus, scores were divided into 0-6 grades. The results are shown in FIG. 2 .

As shown in FIG. 2 , the pre-5d and pre-2h groups showed a significant inhibitory effect on neuronal apoptosis in CA1 and CA3 regions of the hippocampus.

(2) western blot (Western blot)

After anesthetizing the mice and collecting the hippocampus from the brain, protease-added lysis buffer (10 mM TrisCl, pH 7.4, 0.5 mM EDTA, 0.25 M Sucrose) was used and homogenized at 4 °C using a homogenizer. The dissolved protein was centrifuged (4 °C, 13,000 rpm, 15 minutes) to obtain a supernatant.

The isolated protein was quantified by the Bradford (Bio-rad, USA) method using bovine serum albumin (BSA, sigma, USA), and 25 μg of protein was subjected to 10% SDS-PAGE. It was separated by (Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis) and transferred to a PVDF (polyvinylidene difluoride) membrane (membrane, Gendepot, UK). The PVDF membrane was blocked in 5% skimmed milk (BD, USA) for 1 hour. After washing with TBST (mixture of tris-buffered saline and Tween 20), primary antibody rabbit anti-(cleaved) caspase-3 (1:1,000; Cell Signaling Technology, Beverly, MA, USA), rat anti-CD11b (1: 500; Serotec, Oxford, UK), mouse anti-GFAP (1:2,000; DACO, USA), mouse anti-cyclooxygenase-2 (COX-2; 1:1,000; BD Biosciences, San Jose, CA, USA) and rabbit Anti-inducible nitric oxide (iNOS; 1:500; Sigma-Aldrich, St. Louis, MO, USA) was diluted 1:1,000 in 3% skim milk and reacted at 4 for one day, washed 3 times with TBST for 10 minutes each 2 It was reacted with the primary antibody at room temperature for 1 hour. After the secondary antibody reaction, the band was confirmed by ECL system (Santacruz, USA) after washing in TBST. Identification and quantitative analysis of the protein was performed using an imaging device ChemiDoc XRS+ (Bio-Rad). The results are also shown in FIG. 2 .

As a result of Western blot analysis, similar to the Nissel staining result, the expression of caspase-3 protein was increased in the KA group, but significantly decreased in the pre-5h group ( FIG. 2Q ).

From the above results, it was confirmed that the valerian extract had a protective effect on hippocampal neuronal cell death in temporal lobe epilepsy caused by KA.

Example 5: Cainic acid inductive temporal lobe epilepsy Confirmation of inhibitory effect on immune cell activity of valerian extract in animal model

To confirm whether the valerian extract had an effect on immune cell activity, frozen sections were subjected to immunostaining for Iba-1 and GFAP.

(1) Immunohistostaining ( Immunohistochemistry )

Tissue sections were treated with _{3% H 2} O ₂ for 30 minutes to block endogenous peroxidase. Wash with PBS 3 times for 10 min each and use a blocking solution (5 % normal goat serum, 2 % bovine serum albumin, 2 % fetal bovine serum and 0.1 % triton X-100 to reduce non-specific reactions that may occur during immunostaining). in PBS) for 1 hour. Thereafter, each tissue was prepared with a primary antibody [rabbit anti-ionized calcium binding adapter molecule 1 (Iba-1); 1: 2,000; WAKO, Japan or rabbit anti-glial fibrillary acidic protein (GFAP); 1: 2,000; DACO, USA)] was reacted for at least 18 hours. After the reaction, the tissue was washed 3 times for 10 minutes with PBS, and then reacted with secondary antibodies such as goat anti-rabbit IgG (Vector, CA, USA) and ABC complex (Vector, CA, USA) for 1 hour each at room temperature. made it After the antigen-antibody reaction has been completed, check color development in a Tris buffer (pH 7.4) solution containing DAB (3,3'-diaminobenzidine) for 3 to 5 minutes, then place the brain tissue on a slide and dry it. After dehydration and clearing, the mounting solution was dropped and the cover glass was sealed, and the immunoreactivity of each was observed under a microscope. The results are shown in FIG. 3 .

As shown in FIG. 3, the activities of Iba-1(+) microglia and GFAP(+) astrocytes in the hippocampus of the KA group increased, but were significantly decreased in the Pre-5d group (FIGS. 3A-D, HK) . Consistent with these results, the expression of proteins for CD11b and GFAP by western blot analysis was also remarkably suppressed (FIGS. 3O, P). From the above results, it was confirmed that the valerian extract had an inhibitory effect on immune cell activity in temporal lobe epilepsy caused by KA.

Example 6: Cainic acid inductive temporal lobe epilepsy Inflammatory mediators of valerian extract in animal models Inhibitory effect on expression Confirm

To confirm whether the valerian extract had a modulating effect on the expression of inflammatory mediators, the protein expression levels of COX-2 and iNOS were analyzed by Western blot with hippocampal tissue 72 hours after KA administration. The results are shown in FIG. 4 .

As shown in FIG. 4 , the protein expression levels of COX-2 and iNOS in the hippocampus of the KA group increased, but significantly decreased in the Pre-5d group ( FIGS. 4A and B).

From the above results, it was confirmed that the valerian extract had an inhibitory effect on inflammatory mediators in temporal lobe epilepsy caused by KA.

Claims (6)

As a pharmaceutical composition for preventing or treating temporal lobe epilepsy caused by hippocampal nerve cell death, comprising an ethanol extract of Valeriana fauriei Briq root as an active ingredient,
Inhibits neuronal cell death by reducing the expression of caspase-3 protein in the CA1 and CA3 regions of the hippocampus,
Inhibits inflammatory mediators by reducing the protein expression of COX-2 and iNOS in hippocampal tissue,
A composition that inhibits the activity of Iba-1 (+) microglia and GFAP (+) astrocytes. delete delete As a health functional food for preventing or improving temporal lobe epilepsy caused by hippocampal nerve cell death, comprising an ethanol extract of Valeriana fauriei Briq root as an active ingredient,
Inhibits neuronal cell death by reducing the expression of caspase-3 protein in the CA1 and CA3 regions of the hippocampus,
Inhibits inflammatory mediators by reducing the protein expression of COX-2 and iNOS in hippocampal tissue,
A composition that inhibits the activity of Iba-1 (+) microglia and GFAP (+) astrocytes.
delete delete

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