KR101493139B1 - Composition for Protecting Neuronal Cell Comprising Thymoquinone and Vitamin C - Google Patents

Abstract

The present invention relates to a composition for protecting nerve cells, a composition for preventing or treating neurological diseases, and a health functional food for preventing or ameliorating neurological diseases, which comprises thymoquinone and vitamin C as an active ingredient. The composition of the present invention comprising both thymoquinone and vitamin C has an effect of protecting nerve cells and preventing neurological diseases such as epilepsy and seizures induced by PTZ.

Description

(Composition for Protecting Neuronal Cell Comprising Thymoquinone and Vitamin C), which contains thymoquinone and vitamin C as an active ingredient,

The present invention relates to a composition for protecting nerve cells, a composition for preventing or treating neurological diseases, and a health functional food for preventing or ameliorating neurological diseases, which comprises thymoquinone and vitamin C as an active ingredient.

Epilepsy is a major symptom of recurrent seizures. It is one of the major neurological disorders in the brain and is known to cause epilepsy in about 0.5-1.0% of the world's population. Most patients suffering from epilepsy are usually suffering from focal seizures that begin and spread from one part of the brain. In general, seizures begin by spreading by affecting both the abnormal neurons and one brain hemisphere or hemisphere, with excessive abnormal electrical discharges in a large group of brain neurons. Currently, seizures are known to induce neuronal cell death in certain conditions and promote brain damage, which may affect nerve cell structure and function as well as non-fatal pathophysiological effects. Brain damage due to seizures is very complex and involves many factors that contribute to neuronal cell death. These factors include genetic factors and other biological phenomena such as excitotoxicity, oxidative stress, and significant uptake of calcium.

Pentylenetetrazole (PTZ) acts as a blocking agent for chloride ionophore (chloride ion channel) complexes against GABA _A receptor agonists and γ-aminobutyric acid (GABA) _A receptors, Model has been used. Chemically, PTZ-induced kindling is the most common and pharmacologically induced model. It can lead to damage by long-term stimulation and excitement of neurons during seizures, and the exact mechanism is not yet fully understood. Numerous rodent models of epilepsy such as kainic acid models, PTZ kindling models, and PTZ induced seizures related to oxidative stress in the central nervous system have been presented in many reports. Epileptic seizures cause brain damage through many mechanisms leading to neural defects and neurodegeneration. Oxidative stress, calcium influx, dysfunction of mitochondria, especially the reduction of GABA, an inhibitory neurotransmitter, and increased excitability of nerve cells, are among the mechanisms that cause brain damage. The neurodegeneration induced by epileptic seizures is very complex and involves many cell and molecular changes in other parts of the brain. Acute seizures, which commonly cause neurodegeneration of the hippocampus, have recently been reported by morphological approaches using TUNEL, Fluoro-Jade B and silver staining techniques. Acute PTZ induces only cytokine secretion, whereas in the case of kindling, the content of TNF-α, IL-10, PGE2 and caspase-3 was increased in the cerebral cortex.

Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitter in the brain and affects other types of receptors in GABA _A , GABA _B, and GABA _C. The GABA _B receptor (R), which has been proven to play a role in the epileptic rodent model, plays an important role during the development of the central nervous system (CNS). PTZ has anticonvulsant effects through the GABA _A receptor, affecting the major GABAergic and glutamatergic systems. Both GABA _A and GABA _B Rs are involved in the regulation of neuronal excitability and epileptogenesis, but the relevance of GABA _A Rs to control general spastic seizures is not well known. Changes in the expression level of the GABA _B receptor (R), which plays an important role in maintaining the excitation-inhibition balance in the brain, leads to the development of seizures.

Under these circumstances, the present inventors investigated the relationship between PTZ-induced interstitial seizures and GABA and neurological diseases, while naturally occurring compounds, thymoquinone and vitamin C, were effective against the above-mentioned neurological diseases, The inventors of the present invention have found that the present invention has excellent effects and that they exhibit synergistic effects particularly when they are administered in combination.

[Prior Art Literature Information]
1. BMC Neurosci. 2012 Jan 19; 13: 11 (January 19, 2012)
2. Biomedicine & Preventive Nutrition 2 (2012) 223-227 (2012.10)
3. Synapse. 2010 Jun; 64 (6): 467-77 (2010.06)

One object of the present invention is to provide a composition for protecting a nerve cell, which comprises thymoquinone and vitamin C as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating neurological diseases, which comprises thymoquinone and vitamin C as an active ingredient.

It is still another object of the present invention to provide a health functional food for preventing or ameliorating neurological diseases, which comprises thymoquinone and vitamin C as an active ingredient.

In one aspect of the present invention, the present invention provides a composition for protecting a nerve cell, comprising as an active ingredient a thymoquinone and a vitamin C.

In the present invention, the term "thymoquinone" refers to fenugreek ( Nigella sativa ) is an active quinone which is known as a main active ingredient of seed oil and has various properties such as pharmacological agents, strong antioxidant activity against free radical formation factors such as antiinflammation, doxorubicin induced cardiac toxicity. In the present invention, it has been confirmed that the above-mentioned thymoquinone is able to protect nerve cells by inhibiting the death of nerve cells and prevent neurological diseases through the experiment of pretreatment of thymoquinone in treatment with pentylenetetrazole In particular, it was confirmed that the combination of vitamin C and vitamin C had a synergistic effect.

In the present invention, the term "vitamin C (vitamin C)" is one of the trace elements for maintaining the function and health of the human body, and is also known as ascorbic acid and is known as a powerful antioxidant. In the present invention, it has been confirmed through experiments that vitamin C is pretreated upon treatment with pentylenetetrazole that the vitamin C can protect nerve cells by inhibiting the death of nerve cells and prevent nerve diseases, Especially, when it is used together with the above-mentioned thymorquinone, it is confirmed that it has a synergistic effect.

In the present invention, the term "nerve cell" is a main cell that constitutes the nervous system, and controls the action of other cells including muscles. Unlike general cells, it transmits signals by an electrical method. Cells that do not divide. As such, neurons are almost impossible to recover from damage due to the restriction of the number of cleavage, and thus it is important that the cleavage is completed normally so that normal functions can be performed. In particular, the protection of nerve cells exposed to pentylenetetrazole is important because it causes seizure seizures. It is also known that the use of the combination of thymoquinone and vitamin C protects nerve cells exposed to pentylenetetrazole Was first identified.

The protective effect of the nerve cell using the composition of the present invention can protect all nerve cells regardless of the location and size of the nerve cell, and is preferably a hippocampal neuronal cell existing in the hippocampus of the fetal brain. Lt; / RTI >

The term "pentylenetetrazole (PTZ)" in the present invention is also known as pentetrazol or pentamethylenetetrazole, and is also used as a circulatory system and respiratory stimulant. These PTZs are known to cause seizures-induced neuronal death by reducing neurons in the hippocampus and inducing neuronal death by oxidative stress. PTZ-induced seizures in the brain of mature rat by PTZ are involved in neuronal cell death Lt; RTI ID = 0.0 > of caspase-3 < / RTI >

In a specific example of the present invention, when monotherapy with either thymoquinone or vitamin C was used, the mortality was reduced in an animal model of epileptic seizure induced by pentylenetetrazole (PTZ), nerve cell death was inhibited, Protection effect.

Specifically, in the PTZ-administered rats, the EEG EEG EEG showed extreme ultrafast and extramolecular complex forms, but the abnormal EEG was markedly reduced when pretreated with thymoquinone and vitamin C (Fig. 1) In the case of pretreating quinone and vitamin C, the content of GABA _B1 receptor rapidly decreased by PTZ treatment (FIG. 2), and it was confirmed that the content of rapidly increasing PKA protein was normalized (FIG. 3).

In addition, Bax expression and Bcl-2 expression were decreased in the PTZ-treated group in relation to the apoptosis-inducing pro-apoptotic Bax protein and the anti-apoptotic Bcl-2 protein expression associated with apoptosis and programmed cell death, In the pretreatment of thymoquinone and vitamin C, it was confirmed that the change in the expression level was restored (Fig. 4). In addition, the expression of caspase-3, which is a marker of apoptosis, and the experiment using FJB staining / And vitamin C inhibited neuronal cell death and loss.

In addition, several PTZ - induced seizure models showed deaths in some experimental animals, but survival rates were increased when thymoquinone and vitamin C were administered.

Accordingly, in another aspect, the present invention provides a pharmaceutical composition for preventing or treating neurological diseases, which comprises thymoquinone and vitamin C as an active ingredient.

The term "neurological disease" in the present invention refers to a nervous system related disease occurring in the central nervous system, peripheral nervous system and muscles, and may be a brain-related neurological disease for the purpose of the present invention.

More preferably the neurological disorder is selected from the group consisting of epilepsy, seizure, epileptic seizure, schizophrenia, panic disorder, and neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's disease , And even more preferably it may be epileptic, seizure or epileptic seizure.

As described above, the above pharmaceutical composition has an effect of preventing or treating neurological diseases by inhibiting neuronal cell death and protecting neurons. In the present invention, pretreatment of thymoquinone and vitamin C provides a preventive effect Respectively.

The composition comprising the thymoquinone and the vitamin C of the present invention as an active ingredient may further comprise a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier, a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a fragrance and the like can be used for oral administration. In case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used. Formulations of the compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, they may be formulated in unit dosage ampoules or in multiple dosage forms have. Other solutions, suspensions, tablets, herbal medicines, capsules, sustained-release preparations, and the like. Examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltoditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

In addition, the composition of the present invention can be administered in various ways such as oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, transdermal, and the dose varies depending on the age, sex, And can be readily determined by one skilled in the art. Preferably, the composition of the present invention may be administered orally or parenterally. Also, since the dosage of the composition can be determined by those skilled in the art depending on the kind of the active ingredient, along with various related factors such as route of administration, degree of disease, sex, body weight and age, Is not limited.

In another aspect, the present invention provides a health functional food for preventing or ameliorating neurological diseases, which comprises thymoquinone and vitamin C as an active ingredient.

Thymoquinone and vitamin C according to the present invention may be formulated in the same manner as the above pharmaceutical composition and added to foods or beverages for the purpose of preventing or improving neurological diseases such as epilepsy and seizure. The type of food is not particularly limited, For example, various foods such as confectionery, bread, and noodles, water, soft drinks, fruit drinks such as drinks, gum, tea, vitamin complex, seasoning, and health functional food.

The beverage composition of the present invention is not particularly limited to liquid ingredients except that it contains thymoquinone and vitamin C as essential ingredients, and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrin and the like; And sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings (taumakin, stevia extract, etc.) and synthetic flavorings (saccharin, aspartan, etc.) can be advantageously used as flavorings other than those described above.

The health functional food of the present invention can be prepared by adding the above-described step of adding the above-mentioned thymoquinone and vitamin C to the food of the base material, Quinone and vitamin C to the mixture. At this time, a taste and odor corrector may be added as needed.

In addition to the above, the health functional food of the present invention may contain flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid concentrating agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. Such components may be used independently or in combination.

The composition of the present invention comprising both thymoquinone and vitamin C has an effect of protecting nerve cells and preventing neurological diseases such as epilepsy and seizures induced by PTZ.

Figure 1 shows the electroencephalographic (EEG) recording of rats after one week of drug administration and 15 minutes after PTZ final injection. (A) Control group, (B) PTZ group, (C) TQ 40 + PTZ group, (D) vitamin C + PTZ group and (E) TQ + vitamin C + PTZ group.
Figure 2 shows the effect of TQ and vitamin C on GABA _B1 receptor expression in the cerebral cortex (A) and hippocampus (B).
Fig. 3 shows the results of TQ and vitamin C effects on PKA-a expression in the cerebral cortex (A) and hippocampus (B).
FIG. 4 shows the results of TQ and vitamin C effects on expression of Bax proteins (A, B) and Bcl-2 proteins (C, D) in cerebral cortex (A, C) and hippocampus (B, D)
Figure 5 shows the effect of TQ and vitamin C on the activation of caspase-3 in the cerebral cortex (A) and hippocampus (B).
FIG. 6 shows photographs of FJB staining of nerve cells of the neurodegenerated cerebral cortex and hippocampus induced by PTZ.
7 shows photographs of cresyl violet staining of nerve cells of the neurodegenerated cerebral cortex and hippocampus induced by PTZ.

Hereinafter, the present invention will be described in more detail with reference to the following examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  1: Preparation of reagent

TQ (thymoquinone) and vitamin C (ascorbic acid) were purchased from Sigma Aldrich. Fluoro-Jade B was purchased from Chemicon (Chemicon International, USA) and all other reagents were purchased from commercial suppliers with the highest analytical grade.

Example  2: Animal model and drug administration

Mature SD (Sprague-Dawley) rats (250-300 g) were used as experimental animals. Mature SD rats were housed in temperature and humidity chambers, allowed free access to food and water, and maintained a 06: 00-20: 00 h day / night cycle.

The rats were randomly divided into 8 groups of 6 rats per each group as described in Table 1 below.

Pentylenetetrazole (PTZ) was dissolved in 0.9% physiological saline solution before administration and administered by intraperitoneal injection. Thymoquinone (TQ, thymoquinone) was orally administered 2 hours before PTZ abdominal injection and vitamin C (ascorbic acid) was abdominally injected 2 hours before PTZ abdominal injection. All drugs were prepared immediately prior to each experiment, freshly used, and administered for one week.

Example  3: Seizure behavior and EEG electroencephalography , EEG ) Seizures through seizure ) Form evaluation

After daily PTZ injection, the rats were separated from the cage and observed for at least 10-30 minutes daily. All groups of experimental animals were carefully monitored for daily generalized seizure evaluation. Seizures induced by PTZ injection were scored and classified, and their triggering time, seizure response time, and mortality were recorded. The behavior of spasticity was initiated within 3-5 minutes by intraperitoneal injection of the maximum sub-convulsive dose of PTZ (50 mg / kg). This dose caused seizures in 90% of the rats, and repeated injections further exacerbated the symptoms. The rats used in the present invention showed seizures corresponding to steps 4 and 5.

Seizures induced by repeated PTZ scans were scored according to the scale of Racine, which graded the spasticity, in addition to some changes. The resulting seizures by PTZ administration were scored as such:

Step 0, no reaction; Stage 1, ear and facial contractions; Stage 2, spikes in the axial direction through the body soaring; Step 3, rearing with myoclonic jerks and standing with my hips or standing with my hind legs; Step 4, sideways collapse with intermittent cramps; Step 5, repeatedly severe severe spastic seizures or fatal convulsions; Step 6, Death occurred.

Seizure response time was defined as the average length of time between drug administration and seizure onset. Common seizures are characterized by symmetrical forelimbs and hind leg tension, intermittent spasm of the hind legs and reversed movement. Often the laboratory animals have another form of seizure because the seizures occur first or after the first few seizures, so the duration of the seizure is the sum of multiple seizures for each animal Respectively. Researchers injecting mice and observing seizures were unaware of the PTZ dosing conditions in each rat, and recorded the initial seizure onset response time, total seizure duration, and seizure episodes for each subject.

Electroencephalography (EEG) data was recorded for 30 minutes using an amplifier (LAXTHA, LXEJ 108) and digitized at 250 Hz in an electroencephalography (EEG) recording room. The entire EGG sample was analyzed by visual inspection of the presence of epileptiform activity waves as previously defined.

Example  4: Western Blot  ( western blot ) analysis

The amount of the various proteins was measured in different groups by western blot analysis. Experimental animals were sacrificed one week after PTZ administration, and the rapidly extracted brain, cortex and hippocampus were carefully dissected and frozen in dry ice. The brain tissue was homogenized in 0.2 M phosphate-buffered saline (PBS) containing protease inhibitor cocktail. Protein concentrations were measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories, CA, USA). The same amount of total protein (30 μg per sample) was electrophoresed on a 10-15% SDS-PAGE gel under reducing conditions and transferred to polyvinylidene difluoride (PVDF, Santa Cruz Biotechnology, Santa Cruz, CA, USA) membrane. (6-175 kDa, New England Biolabs, Inc., Ipswich, Mass., USA) capable of converting to the overall range of molecular weights were simultaneously electrophoresed and the molecular weight of the detected protein was determined Respectively. Membranes were blocked with 5% (w / v) skim milk to reduce non-specific binding, and immunoblotting was performed using other primary ≪ / RTI > antibody. Immunocomplexes were visualized using enhanced chemiluminescence (Amersham ECL Advance Western Blotting Detection Kit, GE Healthcare, UK). The X-ray film was photographed and the optical density of the band was measured using a computer-based Sigma Gel program, version 1.0, SPSS, Chicago, IL , USA).

Example  5: Sample collection for tissue collection and histological staining

The experimental animals were sacrificed one week after the last PTZ injection, TQ (thymoquinone, thymoquinone) and vitamin C drug administration. Brain slices from rats with control rats and PTZ and TQ or vitamin C pretreatment were used for staining. For tissue analysis (n = 4-5 animals per group), the rats were perfused intracardially with cold 4% formalin (4% paraformaldehyde, 4% paraformaldehyde) followed by 1X phosphate- buffered saline, PBS). Brains were postfixed overnight with 4% formalin and transferred to a 20% sucrose solution until brain tissue submerged on the bottom of the tube. The brain tissue was frozen by embedding with an OCT compound (AO, USA) and the 18-μm slice cut into coronal planes using a CM 3050C cryostat (cryostat, Leica, Germany) . The sections were thaw-mounted on silicon and cation-treated slides (probe-on plus charged slide, Fisher, USA).

Example  6: Fluoro - Jade  B staining

Fluoro-Jade B (FJB) staining was performed as previously described with some changes. PTZ was administered for 1 week and 2 hours after the last injection, the animals were deeply anesthetized, perfused with 0.9% physiological saline solution into the heart and subsequently diluted to 4% in 0.1 M phosphate-buffered saline (PBS) And perfused with cold formalin (4% paraformaldehyde, 4% paraformaldehyde). Brain slices (prepared as described above) were pasted onto the slides and allowed to dry naturally overnight. Slides are immersed sequentially in 1% sodium hydroxide and 80% ethanol for 5 minutes and then in 70% alcohol for 2 minutes and then in water for 2 minutes. The slides are transferred to 0.06% potassium permanganate solution for 10 minutes, rinsed with water, and then immersed in 0.1% acetic acid and 0.01% FJB (Chemicon International, USA) for 20 minutes. The slides were washed twice with water and dried for 10 minutes. The glass cover slip was placed on the slide using a mounting medium. (Green) FJB staining was detected with a FITC filter of confocal laser scanning microscope (Fluoview FV 1000, Olympus, Japan). FJB-positive cells at various sites in each section were calculated by observers who did not know the conditions of administration.

Example  7: Nissl body ( nissle body )of Cresyl  Violet ( cresyl violet ) dyeing

Cresyl violet was used to stain tissue sections for histological examination and measurement of neuronal cell loss. The nystagmus of the adult rat brain and the presence or absence of dead or damaged neurons were analyzed by mounting a 18-μm thick brain slice on a microscope slide. The sections prepared from eight experimental groups of adult rats were degreased with high density alcohol (70-100%), hydrated with low density alcohol (95-70%), washed with acetate buffer (pH 5.0) Gt; 0.25% cresyl violet. ≪ / RTI > The sections were washed with water and dried with ethanol. Images were taken with a fluorescent light microscope.

Example  8: Data analysis and statistical processing

In the western blot, the band was measured using a sigma gel system (SPSS Inc., Ghicago, IL) using densitometry (image density recorded on the developed photographic material was measured according to a strictly defined method) . The concentration values were expressed as mean ± standard error (SEM). Differences between groups were analyzed using Student's t-test followed by one-way analysis of variance (ANOVA). All analyzes were considered statistically significant if the p-value was less than 0.05.

Experimental Example  1: seizure seizure ) Behavioral Observation and Electroencephalography ( EEG )

The development of generalized seizures by the maximum sub-convulsive dose of PTZ-induced (50 mg / kg) was observed when PTZ was administered for 5 days. Data collected after one week administration were analyzed. PTZ-induced seizures in adult rats typically begin with a hind paw, followed by a typical quadriplegic spasm of the legs, and even a few animals have died.

On the other hand, the number of surviving experimental animals was significantly increased in the experimental group treated with TQ (thymoquinone) and vitamin C compared to the group treated with PTZ alone. Of the 6 PTZ-treated groups, 2 died and 33% died, whereas the vitamin C group had a mortality rate of 16%. No mortality was observed in the group receiving TQ (thymoquinone) and the survival rate was increased to 100% (Table 3).

The maximum effect of TQ (thymoquinone) was observed at a dose of 40 mg / kg. To demonstrate seizures, EEG (electroencephalography) was monitored for 30 minutes and started 15 minutes after PTZ injection. When pretreated with TQ of 40 mg / kg and 250 mg / kg of vitamin C, abnormal EEG decreased abnormal EEG, whereas in PTZ-administered rats, polyspike and extreme wave Complex form (Figure 1).

Experimental Example  2: Cerebral Cortex In the sea horse GABA _B1  Receptor PKA for the expression of TQ And Vitamin C

The effects of TQ and vitamin C on the content of GABA _B1 receptor and PKA-α in the cerebral cortex and hippocampus were studied in comparison with PTZ administration. Western blotting analysis showed that the content of GABA _B1 receptor decreased rapidly in the cerebral cortex and hippocampus of the PTZ group, whereas when TQ and vitamin C were pretreated, the pattern was reversed and the GABA _B1 receptor content was maintained (Fig. 2). These changes were much more pronounced in the hippocampus compared to the cerebral cortex in all experimental groups.

PKA (protein kinase A), on the other hand, is a major regulator of neurotransmission that is likely to be involved in the action of molecules and cells to the mechanism of epileptogenesis. Therefore, the experiment was performed to confirm the protein content of PKA in the PTZ induced seizure model. The PKA protein content of the PTZ group was increased in the cerebral cortex and hippocampus compared to the control group, and the increase in the hippocampus was noticeable due to the sensitivity of the site. The increased activity of PKA is due to long-term PTZ-induced seizures. In contrast, PKA content was normalized at all test concentrations pretreated with TQ and vitamin C (FIG. 3). In particular, the protein content of PKA was significantly decreased in the pretreated group of TQ and vitamin C.

Experimental Example  3: PTZ  Cerebral cortex in induced seizure model In the sea horse Bax Wow Bcl -2 expression level TQ And Vitamin C

Apoptotic Bax Protein and Apoptosis and Anti-Apoptotic Bcl-2 Protein Associated with Apoptosis and Programmed Cellular Apoptosis are members of the Bcl-2 family and are important in the mitochondrial process of apoptosis It is an adjuster.

To confirm the effect of TQ and vitamin C on the expression of Bax and Bcl-2 proteins, the amount of Bax and Bcl-2 protein expression was determined in the control group and each treatment group. Western blot analysis showed no significant difference in expression of Bax and Bcl-2 proteins in the cerebral cortex, but in the hippocampus, the expression of Bax, a pro-apoptotic protein in the PTZ-treated group, The expression of Bcl-2 was decreased. In addition, in the group pretreated with TQ and vitamin C, the expression level of Bax was decreased and the expression of Bcl-2 was increased (FIG. 4). These results suggest that TQ and vitamin C may inhibit PTZ-induced apoptosis in rats.

Experimental Example  4: PTZ  Of the induced seizure model Caspase -3 ( caspase -3) that affect the activation of TQ And Vitamin C

Expression of caspase-3, a major marker of apoptosis, was examined to determine the effect of TQ and vitamin C on neuronal cell death.

First, in order to determine whether PTZ-induced seizures cause caspase-3 activation, Western blotting was used to observe changes in the expression level of caspase-3 in the cerebral cortex and hippocampus of rats. As a result, the expression of caspase-3 was significantly activated in the cerebral cortex and hippocampus in the PTZ-treated group compared to the control group. On the other hand, it was confirmed that the expression level of caspase-3 was decreased in the TQ and vitamin C-treated groups, and the expression level of caspase-3 was remarkably decreased in the TQ and vitamin C-administered groups (Fig. 5). These results suggest that PTZ-induced neuronal apoptosis, that is, activation of caspase-3 is inhibited by TQ and vitamin C administration, and that TQ and vitamin C may protect the brain from harmful effects of seizures .

Experimental Example  5: PTZ Induced neurodegeneration ( neurodegeneration ) For TQ And Vitamin C

FJB staining, a reliable experimental technique for neuronal vulnerability, was performed to determine whether PTZ-induced seizures lead to neuronal cell death. Anionic Fluorochrome Fluoro-Jade B (FJB) was used to stain degenerated neurons at several time points after PTZ administration.

FJB-positive cells were not observed in both the cerebral cortex and hippocampus of the control group injected with saline for one week. The number of FJB-positive cells in both cerebral and hippocampal regions increased significantly when PTZ was administered for one week, indicating that excessive nerve cell loss was induced in the PTZ injection group by Fluro-Jade B (FJB) (Fig. 6). In addition, pretreatment of TQ and vitamin C to the PTZ injection group significantly reduced the number of FJB-positive cells in both the cerebral cortex and hippocampus, respectively.

In addition, to evaluate the degree of neuronal apoptosis and the degree of nerve cell protection by TQ and vitamin C in the cerebral cortex and hippocampus of adult rat brain induced by PTZ administration after one week of drug treatment, nissl staining Respectively.

As a result, clear signs of neuronal cell death in the cerebral cortex and hippocampus of the PTZ-administered group (ie, cells with small or large condensations, splitting, dark-blue nuclei, and apoptotic bodies) . Control In the experimental animal group, neurons in both brain regions were morphologically morphologically normal, whereas in the PTZ-treated experimental animal group, the degree of neuronal loss, vacuolization, and tissue collapse in both brain regions (Fig. 7). On the other hand, pretreatment of TQ and vitamin C in the PTZ administration group resulted in much less vacuolization of the brain area and reduced nerve cell loss.

These results indicate that PTZ can induce the death and loss of neuronal cells, and that TQ and vitamin C can protect these neurons.

Claims (7)

delete delete delete delete A pharmaceutical composition for the prevention or treatment of seizure or epilepsy, comprising as an active ingredient, thymoquinone and vitamin C.
delete A health functional food for preventing or ameliorating seizure or epilepsy, which comprises thymoquinone and vitamin C as an active ingredient.

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