KR101075055B1 - Preventive and therapeutic compositions containing capsaicin for epilepsy - Google Patents

Abstract

The present invention relates to a composition for preventing and treating epilepsy containing capsaicin, and more particularly, to a composition for preventing and treating epilepsy containing capsaicin, which is a main component of red pepper, which can effectively inhibit epilepsy as an active ingredient.
According to the present invention, capsaicin as an active ingredient for preventing and treating epilepsy can effectively inhibit epilepsy through antioxidant activity, anti-inflammatory activity, anti-cell death activity and hypothermia. Therefore, the composition containing the capsaicin of the present invention as an active ingredient has a prophylactic effect on epileptogenesis, and a therapeutic effect on epileptic symptoms, and thus can be applied to the pharmaceutical and functional food fields for epilepsy prevention and treatment.

Description

Prevention and treatment of epilepsy containing capsaicin as an active ingredient {Preventive and therapeutic compositions containing capsaicin for epilepsy}

The present invention relates to a composition for preventing and treating epilepsy containing capsaicin, and more specifically, containing as an active ingredient capsaicin which can effectively inhibit epilepsy through antioxidant activity, anti-inflammatory effect, anti-cell death ability and hypothermia ability. It relates to a composition for preventing and treating epilepsy.

Epilepsy is one of the degenerative neurological disorders characterized by periodic spontaneous spasms and learning and memory disorders. It has a prevalence of about 1% of the world's population, and about 30% of all epileptic patients are reported to be resistant to conventional drugs. Moreover, existing drugs are only effective in the area of symptomatic treatment, but are not effective in the development of epilepsy, and there is a growing need for the development of new therapeutic agents that can be effective in the progression of epilepsy after seizures (Acharya). et al., 2008. Prog. Neurobiol. 84, 363-404).

Cainic acid is an analogue of the excitatory amino acid glutamic acid, which causes repeated spasms when administered to the abdominal cavity of mice and eventually causes brain neurons to die (Junyent et al., 2009. J. Neurosci. 87, 1500-1508), and its pathological findings are very similar to temporal lobe epilepsy, which has the highest incidence and high drug resistance in humans, making it the most widely used animal model for epilepsy research. (Ben-Ari et al., 2000. Trends Neurosci. 23, 580-587).

Previous studies have shown that overexpression of glutamate (Fujikawa et al., 2005. Epilepsy Behav. 7, 3-11) and pro-inflammatory cytokine may be associated with neuronal cell death caused by epilepsy. Vezzani et al., 2005. Epilepsia 46, 1724-1743; Pitkanen et al., 2002. Lancet Neurol. 1, 173-181) have been shown to play an important role. Excessive excitability of glutamate receptors causes excitatory toxicity, causing oxidative stress by rapidly increasing Ca ²⁺ influx into neurons. This change eventually leads to neuronal necrosis and apoptosis. Recent studies have also shown that dysfunction of cytokine-releasing astrocytes affects the development of epilepsy (Jabs et al., 2008. Epilepsia 49, 3-12; Schwarcz et al. , 2008. Epilepsia 49, 1-2) Moreover, the increase of inflammatory cytokines in the experimental epilepsy model has been shown to have pro-ictogenic effects and cause the death of neurons more severely. The role is attracting attention. In addition, hyperthermia increases neuronal cell death, whereas hypothermia has been reported to have neuroprotective effects (Sharma et al., 2007. Toxicol Pathol. 35 (7), 984-999). . In addition, an increase in body temperature is observed in a model of convulsions induced with carboxylic acid (Oprica M et al., Cytokine. 2006 Jul; 35 (1-2): 77-87), and glutamate-induced brain damage in animals When exposed, the extent of the damage was reported to be increased compared to animals exposed to normal temperatures (Suehiro et al., 1999. J. Neurotrauma 16, 285-297). It is considered to increase.

Capsaicin (N- (4-hydroxy-3-methoxybenzyl) -8-methyl-trans-6-nonenamide) is the main ingredient that makes peppers taste spicy and utilizes its unique color and unique taste in the food sector. It is widely used. In the medical aspect, it is applied to various diseases, and among them, it shows effective analgesic effect through skin coating, and many studies have been conducted as local analgesics. In addition, many studies on the antioxidant and anti-inflammatory effects of dietary capsaicin have been reported. Restores antioxidant enzymes lost in erythrocytes and liver and reduces increased lipid peroxides (Kempaiah et al., 2004. Ann. Nutr. Metab. 48 (5), 314-320; Materska et al., 2005. J. Agric.Food Chem. 53 (5), 1750-1756), inhibiting the peroxidation of various lipids (Kogure et al., 2002. Biophys. Acta. 1573 (1), 84-92), Antioxidant effects have been reported, such as inhibition of production (Joe et al., 1994. Biochim. Biophys. Acta. 1224 (2), 225-263), and excellent anti-inflammatory effects have been reported in various inflammation-related animal disease models. (Kim et al., 2003. Cell.Signal. 15, 299-306; Demirbilek et al., 2004. Anesth.Analth. 99, 1501-1507).

Accordingly, the present inventors have made intensive studies to overcome the problems of the prior art, and as a result, the epilepsy prevention and treatment effect of capsaicin was confirmed using the epileptic model mouse induced with carboxylic acid, and the present invention was completed.

Therefore, a main object of the present invention is to provide a composition for preventing and treating epilepsy, which contains capsaicin as an active ingredient, which suppresses convulsions due to epilepsy and an increase in body temperature and has antioxidant activity and anti-inflammatory activity.

Another object of the present invention to provide a food composition for preventing and improving epilepsy containing the capsaicin as an active ingredient.

According to one aspect of the present invention, the present invention provides a composition for preventing and treating epilepsy containing capsaicin as an active ingredient.

The capsaicin (capsaicin) is a spicy ingredient contained in the fruit juice of the red pepper family (eg capsicum frutescens or capsicum annum) is well known to exhibit an effective analgesic action. In particular, capsaicin is known to be very effective for analgesic of the musculoskeletal system, such as arthritis, musculitis, sprains, tendonitis and bursitis. In addition, red blood cells, liver, lungs, kidneys, and muscles have been reported to have antioxidant effects, and inflammation-related animal disease models have been reported to have anti-inflammatory effects.

However, there has been no study on the effect of capsaicin on epilepsy, one of the neurodegenerative diseases. Accordingly, the present inventors have shown that capsaicin can suppress neuronal seizures and body temperature increase caused by epilepsy and prevent, improve and treat epilepsy through oxidative damage of neural tissues and suppression of inflammatory cytokines in brain tissues. Proved for the first time.

In the composition of the present invention, the capsaicin can be easily obtained by extracting red pepper with ethanol. In addition, capsaicin can be purchased from various manufacturers (e.g. Fluka Art No. 21750, Sigma Art No. V9130, M2028 and M3404), as well as synthesized by known methods (DJ Bennet, EW Kirby). , J. Chem. Soc. C 1968, 442). Preferably, the capsaicin is N- (4-hydroxy-3-methoxybenzyl) -8-methyl-trans-6-nonenamide (C ₁₈ H ₂₇ NO ₃ ) having the following formula.

[Chemical Formula]

In the compositions of the present invention, the capsaicin can be used on its own or in the form of a pharmaceutically acceptable salt, and all salts, hydrates and solvates prepared by conventional methods are included in the scope of the present invention. That is, the capsaicin used in the composition of the present invention is not only natural capsaicin (8-methyl-N-vanylyl-6-nonenamide) but also N-vanylnononamide (pseudocapsaicin) and 8 which are commercially available as derivatives thereof. -Methyl-N-vanylyl-trans-6-nonenamide or pharmaceutically acceptable salts thereof.

In the present invention, a 'pharmaceutically acceptable salt' is prepared by reacting the stoichiometry of an appropriate base or acid in water, an organic solvent, or a mixture of two solvents. In general, pharmaceutically acceptable salts according to the present invention include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids and salts with basic or acidic amino acids. Salts with inorganic bases include, for example, alkaline metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts and ammonium salts. Salts with organic bases include trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N, N'- Examples thereof include salts with dibenzylethylenediamine. Examples of salts with inorganic acids include salts with hydrochloric acid, boric acid, nitric acid, sulfuric acid and phosphoric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid. Included. Salts with basic amino acids include, for example, arginine, lysine and ornithine. Examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid. Salts of the present invention can be prepared by conventional methods such as, for example, ion exchange. A list of suitable salts is disclosed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p1418.

The composition of the present invention may specifically be a pharmaceutical composition having an antiepileptic and therapeutic effect. The pharmaceutical composition may include capsaicin as an active ingredient, and may further include one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodulin solution, glycerol, ethanol and one or more of these components. And other conventional additives such as buffers and bacteriostatic agents can be added.

In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. The formulation method is not limited to a specific method, and one of ordinary skill in the art may select a preferred method according to the purpose.

The pharmaceutical compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage is based on the weight, age, sex and health of the patient. , Diet, time of administration, method of administration, rate of excretion and the severity of the disease. The daily dose of capsaicin is from 0.025 to 4 mg / kg body weight, preferably 0.1-2 mg / kg body weight, and can be administered once to several times a day. In the present invention, the composition is seizure caused by epilepsy through hypothermia with recovery of oxidative damage in the brain, inhibition of the expression of inflammatory cytokines in brain tissue, and inhibition of neuronal cell death in the hippocampus CA1 site Increasing body temperature, free radicals, inflammatory cytokines and oxidative damage and restoring hippocampal CA1 cell numbers have been shown to prevent and treat epilepsy.

In the embodiment of the present invention, the prevention and treatment effect of capsaicin for convulsions and body temperature increase due to epilepsy was confirmed (see FIGS. 1 and 2), and the antioxidant effect of capsaicin in blood and brain was confirmed using an epileptic animal model. (See Figures 3 and 5). In addition, the inflammatory cytokine inhibitory effect was confirmed in the brain tissue of the epilepsy animal model (see FIG. 4), and the inhibitory effect of capsaicin on neuronal cell death in the hippocampal CA1 region was confirmed.

Referring to the prevention and treatment of capsaicin for epilepsy as described above in more detail with reference to the embodiment of the present invention, in the present invention, the prevention and treatment effect of capsaicin in mouse epilepsy model induced by kainic acid (kainic acid) In order to confirm the seizure strength, body temperature measurement, oxidative stress-related indicator measurement, cytokine concentration measurement, and histological examination were performed.

Overexcitation of glutamate receptors is a major risk factor for excitatory toxicity and eventually neuronal death (Heinemann et al., 1994. Epilepsia 35, 10-21), and oxidative stress increases excitability. Has been demonstrated in many animal experiments (Penkowa et al., 2005. J Neurosci Res. 79 (4), 522-534). In addition, studies have shown that antioxidants such as resveratol and curcumin improve excitatory toxicity. Therefore, the application of antioxidants to an experimental epilepsy model that causes excitatory toxicity is considered. I think it will be very useful. Capsaicin is known to have an effective antioxidant activity (Kogure et al., 2002. Biochim. Biophys. Acta. 1573 (1), 84-92; Henderson et al., 1999. J. Agric. Food. Chem. 47 (7), 2563-2570) have been reported to remove free radicals from and around phospholipid membranes (Kogure et al., 2002. Biochim. Biophys. Acta. 1573 (1), 84-92), Also, according to the results of Lee et al. (Lee, CY et al., 2003. Phytother Res. 17 (5), 454-458), liver, lung, kidney, It has been shown to reduce oxidative stress by reducing lipid peroxide MDA (Malondialdehyde) in muscle. Based on these findings, the present invention was intended to confirm the epilepsy prevention and treatment effect of capsaicin in mouse epilepsy model induced by phosphate. When phosphate (30 mg / kg body weight, i.p) was administered to the mice, the mice developed cramps and elevated body temperature was observed. In particular, MDA, which is a representative indicator of oxidative stress in brain tissues, was increased, indicating that oxidative stress is related to neuronal cell death by phosphate administration. On the other hand, in animals treated with capsaicin (0.33 mg / kg and 1 mg / kg body weight), the seizure intensity decreased and the MDA levels in the brain tissues decreased. Through it was confirmed that the prevention and treatment effect on epilepsy.

Previous studies have shown that pro-inflammatory cytokine is involved in excitatory neuronal death (Barone and Feuerstein, 1999. J. Cereb. Blood Flow Metab. 19, 819-834). ). Brain tissues affected by excitatory toxicity can readily find microglia that are activated, which are known to produce inflammatory cytokines that promote cell death (Penkowa et al., 2005). J Neurosci Res. 79 (4), 522-534). Therefore, in the present invention, the concentration of inflammatory cytokines IL-1β and TNF-α in the brain tissue of the animals used in the experiment was measured. As a result, the animals to which capsaicin was administered showed relatively low cytokine concentrations compared to animals to which only carboxylic acid was administered. In addition, recent studies have shown that elevated body temperature promotes neuronal cell death, while hypothermia exhibits a neuroprotective effect. More severe studies have been reported. Therefore, in the present invention was measured and compared the rectal body temperature of the animals used in the experiment. As a result, it was confirmed that the animal administered with phosphate showed a maximum body temperature of 39 ° C., but the animal administered with capsaicin showed a considerably lower body temperature. This result is consistent with the previously reported results of Sasamura et al. (Sasamura et al., 1999. Jpn. J. Pharmacol. 80, 275-280), and the mechanism is hypothalamus, where capsaicin is the thermoregulatory backbone. It appears to act directly on (Kobayashi et al., 1998. Am. J. Physiol. 275, 92-98; Osaka et al., 2000. Korean J. Intern. Med. 15, 103-108).

Taken together, the capsaicin of the present invention was able to effectively inhibit epilepsy through the antioxidant activity, anti-inflammatory activity, anti-cell death activity, and hypothermia activity in the mouse epilepsy model induced by phosphate. Therefore, the results of the present invention suggested that capsaicin has a prophylactic and therapeutic effect on epileptogenesis.

According to another aspect of the present invention, the present invention provides a food composition for preventing and improving epilepsy containing capsaicin as an active ingredient.

In the present invention, the food composition includes a capsaicin as an active ingredient, in addition to it can be used with other food or food additives. The mixing amount of the active ingredient and other additives may be appropriately determined within a conventional range depending on the purpose of use (prevention or improvement). Capsaicin may be included in 0.0001-10% by weight of the total food composition. However, the content of long-term intake for the purpose of preventing epilepsy may be below the above range.

There is no particular limitation on the kind of food to which the composition of the present invention is applied. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, etc., includes all of the health food in the conventional sense.

In particular, in the case of the health beverage composition of the present invention, various flavors or natural carbohydrates and the like may be contained as additional components, as in the usual beverage. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetener, natural sweeteners, such as tautin and a stevia extract, or synthetic sweeteners, such as saccharin and aspartame, can be used. The proportion of said natural carbohydrate is generally 0.01-0.04 g, preferably 0.02-0.03 g per 100 ml of the composition of the present invention.

The composition of the present invention, in addition to the above components, various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols And carbonation agents used in carbonated beverages. In addition, the composition of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01-0.1 parts by weight with respect to 100 parts by weight of the composition of the present invention.

As described above, according to the present invention, capsaicin can effectively inhibit epilepsy through antioxidative activity, anti-inflammatory activity, anti-cell death activity and hypothermia as an active ingredient of a composition for preventing and treating epilepsy. Therefore, the composition containing the capsaicin of the present invention as an active ingredient has a prophylactic effect on epileptogenesis, and a therapeutic effect on epileptic symptoms, and thus can be applied to the pharmaceutical and functional food fields for epilepsy prevention and treatment.

1 is a graph showing the preventive and therapeutic effects on neuronal seizure of capsaicin in a phosphate-induced epilepsy model mouse.
FIG. 2 is a graph showing that capsaicin is effectively reduced with respect to body temperature increase caused by epilepsy in a phosphate-induced epilepsy model mouse.
FIG. 3 is a graph showing that TBARS was significantly increased in oxidative damage in the brain of phosphate-induced epilepsy model mice, but TBARS was significantly decreased by capsaicin administration. The mechanism of action regarding the preventive and therapeutic effects of capsaicin against oxidative damage is described.
Figure 4 is a graph showing the inhibitory effect of capsaicin on the increase in the expression of cytokines IL-1β and TNF-α in the brain tissue of kinase-induced epilepsy model mice.
FIG. 5 is a graph showing capsaicin effectively inhibits capsaicin from the epileptic model-induced epilepsy model mice, as well as capsaicin to the normal level, and effectively inhibits capsaicin against free radicals increased by epilepsy. The mechanism of action that inhibits epilepsy is described.
6 is a photograph showing the prevention and treatment effect of capsaicin on hippocampal cell death observed in the brain tissue of phosphate-induced epilepsy model mice. Capsaicin administration proved to significantly inhibit neuronal death caused by epilepsy.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Reagents and Test Substances

The carboxylic acid used was Sigma Chem. Co. (St Louis, MO, USA) and the test substance Capsaicin (Casaicin) is Cayman Chemical Co. Inc., (Michigan, USA).

Example 1 Experimental Animal and Test Plan

Female ICR mice for epilepsy induction were supplied from Coatech (Pyeongtaek, Gyeonggi-do) and used after about 1 week of laboratory purification. Three animals were housed in a mouse cage (220 × 200 × 145 mm), and the environment of the animal laboratory was 23 ± 2 ℃, relative humidity 55 ± 10%, ventilation frequency 12 times / hour, lighting cycle 12 hours (08: 00-20:00), the illuminance was adjusted to 150 ~ 300 lux. Feed for experimental animals and drinking water were freely consumed. The experimental animals were divided into four groups, the control group, carboxylic acid alone group, capsaicin low concentration (0.33 mg / kg) group after phosphate administration, and capsaicin high concentration (1 mg / kg) group after phosphate administration. . Cainic acid was dissolved in physiological saline and administered at a concentration of 30 mg / kg into the abdominal cavity. Capsaicin was dissolved in a solution mixed with tween 80, ethanol and physiological saline at a ratio of 10:10:80 to administer phosphate. After 15 minutes subcutaneously, the control group received excipients only. Six of the 12 animals per group were necropsied 12 hours after phosphate administration and the remaining 6 were necropsied 3 days after phosphate administration. This experiment was performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of the Chungbuk National University.

Example 2 Measurement of Cramp Strength and Body Temperature

Convulsive strength was measured every 20 minutes for 200 minutes after phosphate administration and was measured according to Racine's five-stage classification (Racine et al., 1972) (Stage 1 facial clonus, Stage 2 nodding, Stage 3 forelimb clonus, Stage) 4 forelimb clonus with rearing, and Stage 5 rearing, jumping and falling). Body temperature measurements were measured at the first 30 and 60 minutes after phosphate administration using a digital thermometer (Model TH-5; Physitemp Instruments Inc., New Jersey, USA), followed by rectal for 5 hours at 1 hour intervals. Body temperature was measured. The measurement of rectal body temperature recorded the temperature of the animal when the probe was inserted into the rectum at a constant depth of 2 cm and showed a constant value for 3 seconds or more.

As a result of the measurement, in the measurement of convulsion strength, convulsive strength increased up to 80 minutes in the carboxylic acid alone group and then gradually decreased. However, in the capsaicin-administered group, the spasm intensity decreased dose-dependently. In particular, in the 1 mg / kg group, the seizure intensity decreased significantly at 40, 60, 80, 160, 180 and 200 minutes (P <0.05). There was a significant difference at 80 minutes in the 0.33 mg / kg group (P <0.05).

In Fig. 2, in the measurement of the change in body temperature, 38.2 ± 0.1 ° C, which is the body temperature 10 minutes before the administration of carboxylic acid, was determined as the basal body temperature. In all groups, body temperature was lowered 30 minutes after phosphate administration compared to the control group. In the carboxylic acid alone group, the body temperature increased in 1 to 4 hours (2h: 39.6 ± 0.2 ° C, 3h: 38.7 ± 0.2 ° C). However, in the capsaicin-administered group, body temperature was lowered at 1, 2, and 3 hours compared to the phosphate-administered group (P <0.05). The hypothermic effect of capsaicin was dose-dependent, and the high-dose group showed significant decreases in body temperature at 1, 2, and 3 hours (1h: 34.7 ± 0.6 ℃, 2h: 35.7 ± 0.3 ℃, 3h: 35.5). ± 0.3 ° C, P <0.05).

Example 3 Necropsy and Sample Preparation

Twelve hours after the administration of phosphate, zoletil50 ™ was administered to six animals in each group, followed by anesthesia (50 mg / kg body weight), followed by blood collection in a heparinized tube from the abdominal vein, and total antioxidant activity and total nitric oxide content in the blood. Brain tissue obtained after the autopsy was then frozen in liquid nitrogen and stored at −70 ° C. until analysis. Immediately before analysis, brain tissues were weighed and homogenized by mixing at a ratio of 1: 7 (w: v) with 0.01M PBS. The homogenate was centrifuged at 8000 rpm for 15 minutes to obtain supernatant, which was used for quantification of total protein, MDA and cytokine. Three days after phosphate administration, the remaining 6 animals in each group were anesthetized with zoletil50 ™, perfused with 4% paraformaldehyde, and necropsied to obtain brain tissue. The obtained brain tissue was fixed and stored in 4% NBF (neutral buffered formalin).

Example 4 Determination of Malondialdehyde Concentration

Malondialdehyde (MDA) concentration measurement was performed by measuring the red color at 532 nm when MDA reacts with thiobarbituric acid (TBA) using brain homogenate as a sample. The absorbance measured is expressed as μM MDA / mg protein compared to the standard curve measured using 1,1,3,3, -tetramethoxypropane. All measurements were performed using Cayman's TBARS kit (Cayman Chemical Company, Michigan, USA).

As a result of evaluating thiobarbituric acid reactant (TBARS) as an indicator of oxidative damage of neural tissues in the brain, as shown in FIG. 3, TBARS concentration was significantly increased in the phosphate treated group compared to the control group (control group: 0.35 ± 0.02 pg / mg protein, kainic acid group: 0.29 ± 0.02 pg / mg protein, P <0.05). In the capsaicin-administered group, the dose-dependent TBARS concentration decreased, especially in the high-dose group (0.23 ± 0.01 pg / mg protein, P <0.05).

Example 5 Determination of Cytokine Concentrations

The concentration of IL-1β and TNF-α was measured using a brain homogenate sample using a mouse ELISA kit (Bender MedSystems, Burlingame, CA), and the results were expressed in pg / mg protein.

The effect of capsaicin on cytokine changes in brain tissues of epileptic mice was measured. As a result of excitatory toxicity and neuronal cell death, IL-1β (A in FIG. 4) and TNF-α (B in FIG. 4) were measured. Concentrations were measured using an ELISA kit. In FIG. 4, compared with the control group, the concentrations of the two cytokines were relatively increased in the carboxylic acid administration group (IL-1β; control group: 4.66 ± 0.33 pg / mg protein, kainic acid group: 5.61 ± 0.09 pg / mg protein) , TNF-α; control group: 1.35 ± 0.06 pg / mg protein, kainic acid group: 1.59 ± 0.06 pg / mg protein). In the capsaicin-treated group, the cytokine concentration was decreased compared to the carboxylic acid-treated group, and the concentration was similar to that of the control group (IL-1β 1 mg / kg; 4.85 ± 0.31 pg / mg protein, TNF-α 1 mg / kg). 1.21 ± 0.07 pg / mg protein).

Example 6 Blood Antioxidant Activity and Nitric Oxide Measurement

Blood antioxidant capacity and the amount of nitric oxide were measured using a FORD, FORT measurement kit (Form CR3000, Callegari, Parma, Italy). Reactive oxygen species (ROS) measurement in blood was performed by measuring the absorbance at 505 nm after reacting the sample with phenylenediamine derivative using FORT (free oxygen radicals test). The measurement results are expressed as mmol / LH ₂ O ₂ . The antioxidant activity in blood was measured by measuring the absorbance at 505 nm after reacting the sample with FeCl ₃ , a stable oxidant, using FORD (free oxygen radicals defence). The measurement results are expressed in mmol / L Trolox equivalent.

As a result of measuring the antioxidant effect of capsaicin in the blood and brain of epileptic mice, the antioxidant capacity was measured by the FORD method using heparinized whole blood. In A of FIG. 5, the antioxidant activity was significantly lower in the carboxylic acid administration group compared to the control group (1.77 ± 0.02 mmol / l Trolox equivalents). In the high dose capsaicin group, antioxidant activity was significantly increased (1.98 ± 0.06 mmol / l Trolox equivalents) compared to the phosphate group.

In addition, reactive oxygen species was measured by the FORT method using heparinized whole blood. In B of FIG. 5, the concentration of reactive oxygen species was significantly increased in the group treated with phosphate (Kainic acid group: 2.34 ± 0.24 mmol, Control group: 1.53 ± 0.13 mmol, P <0.05), and in the high dose capsaicin group It can be seen that significantly decreased compared to the carboxylic acid administration group (1.37 ± 0.01 mmol, P <0.05).

Example 7. Histological Examination

Brain tissue obtained on day 3 after phosphate administration was subjected to general tissue treatment, and then paraffin-embedded and cut into 10 μm to obtain four consecutive sections, and then slides were made of cresyl violet stain. It was used to count the number of normal cells contained per 1 mm ³ in hippocampus CA1. The remaining two slides were used for TUNEL analysis. TUNEL analysis was performed using Fluorescein In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) and DAPI staining for nuclear staining. Stained slides were observed under a fluorescence microscope (Axioskop 40, Zeiss, Germany).

As a result of measuring the inhibitory effect of capsaicin on the change of apoptosis in the hippocampal CA1 region of the epileptic mouse, the hippocampus was cut out after 3 days of phosphate administration, and slides were prepared. Cresyl violet staining and TUNEL analysis were performed. The number of normal cells in the pyramidal cell layers of the CA1 region of each group was measured. As can be seen in Figures I and III, on cresyl violet staining, the number of intact neurons was significantly reduced in the phosphate treated group compared to the control group (control group: 241.3 ± 16.2 neuron / mm ² , kainic acid group: 88.2 ± 10.2 neuron / mm ² , P <0.05). In the capsaicin-administered group, the number of normal cells increased compared to the phosphate-administered group. In particular, it was significantly increased in the high dose group (157.2 ± 12.3 neuron / mm ² , P <0.05).

Example 8. TUNEL Analysis

TUNEL analysis was performed using Roflu Diagnostics' Fluorescein In Situ Cell Death Detection Kit to confirm the degree of apoptosis in the tissue sections. The paraffin fragments were deparaffinized and hydrated and washed with PBS (phosphate-buffered saline). After washing with water, the mixture was reacted with a mixture of ethanol and acetic acid 3: 1 and washed with PBS again. In order to increase cell membrane permeability, 3% Triton X-100 was used for 60 minutes at room temperature, and TdT-enzyme containing a buffer containing fluorescent UTP was reacted for 90 minutes at 37 ° C while maintaining humidity after dropping. . After completion of the reaction, the tissue sections were sealed using an encapsulant containing 4 ', 6'-diamino-2-phenylindole (DAPI; Vector) and observed at 460 nm (DAPI) and 520 nm (TUNEL) under a fluorescence microscope. .

In the TUNEL analysis, the number of TUNEL positive cells was increased in the phosphate-treated group compared to the control group, but the TUNEL-positive cells were significantly decreased in the capsaicin-concentrated group compared with the phosphate-treated group (II in FIG. 6).

Statistical analysis

The test results are expressed as mean ± standard error. One-way analysis of variance (ANOVA) and Tukey t-test were used to compare the differences between groups. All results were analyzed using SPSS and determined to be statistically significant when P <0.05.

Formulation Example 1 Preparation of Syrup

A syrup containing 1 mg of capsaicin according to the present invention was prepared by the following method. Capsaicin (Cayman Chemical Co. Inc., Michigan, USA), saccharin, sugars were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml. The components of the syrup are as follows.

Formulation Example 2 Preparation of Tablet

Tablets containing 0.5 to 1 mg of capsaicin were prepared by the following method. 250 g of capsaicin was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

Formulation Example 3 Preparation of Injection Solution

Injection solution containing 0.2 to 1 mg of the active ingredient was prepared by the following method. 1 g of capsaicin, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was bottled and sterilized. The components of the injection solution are as follows.

Formulation Example 4 Preparation of Beverage

After dissolving 0.1 to 1 mg of capsaicin in accordance with the present invention in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and oligosaccharides are added as co-agents, and an appropriate amount of sodium benzoate is added as a preservative, followed by the addition of water. To 100 ml to prepare a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

Claims (6)

A composition for preventing and treating epilepsy containing capsaicin as an active ingredient.
The composition of claim 1, wherein the capsaicin is N- (4-hydroxy-3-methoxybenzyl) -8-methyl-trans-6-nonenamide (C ₁₈ H ₂₇ NO ₃ ) having the following formula.
[Chemical Formula]

The composition of claim 1, wherein the capsaicin is extracted from red pepper.
The composition of claim 1, wherein the capsaicin as the active ingredient is administered at 0.1-2 mg / kg body weight.
The method of claim 1, wherein the epilepsy prevents and cure effects by reducing oxidative damage in the brain, suppressing the expression of inflammatory cytokines in the brain tissue, and inhibiting neuronal cell death in the hippocampus CA1 region. Composition.
Food composition for preventing and improving epilepsy containing capsaicin as an active ingredient.

Images