KR100527877B1 - Anticonvulsant composition containing phytol - Google Patents

Abstract

The present invention relates to an anticonvulsant composition containing a compound isolated from the extract of lettuce (Lactuca savita), more specifically seizures, convulsions, epilepsy, etc. are deficient in the neurotransmitter γ-aminobutyrate (γ-aminobutyrate; GABA) It is caused by increasing the amount of GABA by inhibiting the activity of GABA degrading enzyme γ-aminobutyrate transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Can be. Accordingly, the present invention relates to an anticonvulsant therapeutic and prophylactic composition which isolates and contains phytol which is effective in the above diseases in lettuce extract.

Description

A composition for the prevention and treatment of convulsive diseases containing phytol as an active ingredient. {Anticonvulsant composition containing phytol}

The present invention relates to a composition for the prevention and treatment of convulsive diseases containing phytol as an active ingredient, and more particularly to the deficiency of γ-aminobutyrate (GABA), a neurotransmitter such as seizures, convulsions, and epilepsy. The present invention relates to a therapeutic and prophylactic composition containing phytol isolated from lettuce extract against the convulsive disease caused by the drug and containing the same.

As a highly industrialized society develops, various neuropsychiatric diseases appear. Representative neurological diseases include epilepsy, seizures and convulsions due to signaling system defects of the inhibitory neurotransmitter GABA (γ-aminobutyrate), and Parkinson's disease due to neurological defects of dopamine. Parkinsonism) and schizophrenia. The main causes of neurobiochemistry in epilepsy, seizures, and convulsions are the breakdown of the balance between excitatory and inhibitory neurotransmission when neurotransmitters occur between nerve cells and are involved in neurotransmission. Treatment is possible by regulating the metabolic processes of neurotransmitters. Therefore, it is possible to develop therapeutics for these diseases by developing drugs that increase the concentration of GABA.

GABA is distributed in many tissues of mammals and is a major inhibitory neurotransmitter in the central nervous system. As shown in Figure 1, the concentration of GABA is a GABA metabolism-related enzyme glutamate dehydrogenase (GDH), glutamate decarboxylase (GAD), GABA transaminase (GABA transaminase; GABA-T ), Succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR).

Therefore, drugs to increase the level of GABA are used to alleviate epilepsy and seizure symptoms. This is a GABA synthase, glutamate dehydrogenase (GDH) or glutamate dicarboxylase (GAD). It is possible to search for an active agent or inhibitors of GABA-T, SSADH, and SSAR involved in GABA degradation metabolism. Currently, studies on substances having such efficacy from natural materials with few side effects are being actively conducted.

It is an object of the present invention to provide a therapeutic and preventive composition for convulsive diseases such as convulsions, seizures and epilepsy with phytol as an active ingredient.

In addition, another object of the present invention is to provide a therapeutic agent that is effective in alleviating diseases such as convulsions, seizures and epilepsy by separating phytol from lettuce extract and having fewer side effects.

In order to achieve the above object, the present invention confirms that the phytol isolated from the lettuce extract has an anticonvulsant effect on the convulsive disease, and prepared a light-protective composition for the prevention of convulsive disease containing the same. It relates to a composition for the prevention and treatment of convulsive diseases containing as an active ingredient.

In addition, the present invention is characterized in that the composition inhibits the activity of succinic semialdehyde dehydrogenase (SSADH).

In addition, the present invention is characterized in that the phytol is dissolved in ethyl acetate in the fraction extracted by partitioning the lettuce extract with water, ethyl acetate, n-butanol.

The succinic semialdehyde dehydrogenase (SSADH) inhibitory component was isolated by repeated column chromatography on the ethacetate fraction of Lactuca savita, which was based on various spectral data. It was identified as phytol, a nodoid. Assuming that the modification of the enzyme is irreversible, the reaction of phytol with SSADH decreased with time depending on the enzyme activity. Inactivation is performed according to the pseudo first order kinetics with the second order rate constant of 6.15x10-2 mM ^-1 min ^-1 . Complete protection against inactivation was obtained by the coenzyme NAD +, whereas the substrate succinic semialdehyde did not prevent enzyme inactivation. It can therefore be seen that phytol covalently binds at or near the active site of the enzyme. It is expected that phytol can increase the level of GABA, a neurotransmitter in the central nervous system, through the inhibitory action of SSADH, one of GABA inhibitors. Characterized in that it is used as a pharmaceutical composition for the treatment of one or more diseases of convulsions, epilepsy (Epilepsy).

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Pharmaceutical compositions containing phytol as an active ingredient can be formulated orally or injectable by conventional methods in combination with a carrier that is conventionally acceptable in the pharmaceutical art. Oral compositions include, for example, tablets and gelatin capsules, which, in addition to the active ingredient, may contain diluents (e.g., lactose, textose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants, if necessary : Silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols, the tablets also contain a binder (e.g. magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or Polyvinylpyrrolidone), and preferably if desired, it contains a disintegrant (e.g. starch, agar, alginic acid or its sodium salt) or a boiling mixture and / or absorbents, colorants, antiseptics and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and the compositions mentioned are sterile and / or contain auxiliaries such as preservatives, stabilizers, wetting or emulsifier solution promoters, salts and / or buffers for controlling osmotic pressure. In addition, they may contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be administered orally as desired, or may be parenteral, ie, intravenous, subcutaneous, intraperitoneal, or topically applied. The dosage may be from 0.001 μg to 100 mg / kg of daily dose. The amount can be administered in one to several portions. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

Hereinafter, the configuration of the present invention will be described in detail by examples. However, the scope of the present invention is not limited to the following examples.

<Material>

Lettuce was purchased from Suwon Agricultural and Fisheries Market, and NAD ⁺ and SSA were purchased from Sigma.

Example 1 Purification and Assay of Enzyme

The purification of cerebellar GABA-T was performed by a method developed in our laboratory. For detailed kinetic data, NADH was found to have a molar extinction coefficient of 6.22 × 10 ³ M ⁻¹ cm ⁻¹ and the production of NADH was measured with increasing absorbance at 340 nm. All assays were performed in duplicates and initial rate data were standard assays containing 0.1 mM succinic semialdehyde and 5 mM NAD ⁺ in 0.1M sodium pyrophosphate at pH 8.4 at 25 ° C. Correlated with the mixture. Protein concentrations were determined by the Bradford method using bovine serum standard albumin (Bradford, 1976).

Example 2 Separation of Phytol as SSADH Inhibitory Component from Lettuce

Lettuce (20 kg, raw material) was chopped and extracted with 100% methanol (72 L) and 80% aqueous methanol solution (25 L) at room temperature. Filtration of the extract solution and evaporation under reduced pressure yielded a methanol extract. The extract was dissolved in 1 L of water and extracted successively with 2 L of ethyl acetate (EtOAc) and 1.6 L of n-butanol to obtain an ethyl acetate fraction (56 g) and an n-butanol fraction (35.7 g).

The ethyl acetate fraction (56 g) was gradually gradient to CHCl ₃ -MeOH (10: 1 → 7: 1 → 5: 1 → 3: 1) to divide into 8 small fractions (LSE-1 to LSE-8). It was introduced into silica gel column chromatography (750 g) while eluting. The second fraction (LSE-2, 16.6 g) was subjected to silica gel chromatography (250 g, n- hexane: EtOAc = 5: 1 → 3: to obtain 11 small fractions (LSE-2-1 to LSE-2-11). 1 → CHCl ₃ -MeOH = 10: 1). To supply purified Compound 1 (81 mg) 100 mg of the fourth fraction (LSE-2-4, 4.2 g) was subjected to ODS (50 g) column chromatography (acetone-methanol-water = 3: 7: 1 → 9: 3: 1 ).

Compound 1: a colorless oil; [a] _D = + 0.2 ° (c = 1.2, CHCl ₃ ); EI / MS m / z : 296 (M ⁺ ), 278, 263, 196, 182, 126, 123, 71, 57; IR (CHCl ₃ ), 3334, 2954, 2923, 2868, 1669 cm ⁻¹ ; ¹ H-NMR (400 MHz, CDCl ₃ , δ), 5.40 (1H, dq, J = 6.8, 1.4 Hz, H-2), 4.14 (2H, d, J = 6.8 Hz, H-1), 1.99 (2H , t, J = 7.0 Hz, H-4), 1.66 (3H, br.s, H-20), 1.00-1.66 (methine & methylene), 0.87 (6H, d, J = 6.3 Hz, H-16, 17), 0.85 (3H, d, J = 6.1 Hz, H-18), 0.84 (3H, d, J = 6.6 Hz, H-19); ¹³ C-NMR (100 MHz, CDCl ₃ , δ _c ) 140.14 (C-3), 123.11 (C-2), 59.34 (C-1), 39.85 (C-4), 39.34 (C-5), 37.40 ( C-9), 37.34 (C-6), 37.26 (C-8), 36.65 (C-10), 32.76 (C-11), 32.66 (C-7), 27.95 (C-15), 25.12 (C -12), 24.77 (C-13), 24.45 (C-14), 22.69 (C-19), 22.59 (C-20), 19.72 (C-18), 19.68 (C-16), 16.13 (C-) 17).

The yield of compound 1 was 0.017%, and compound 1 was identified as phytol by comparing the spectral data and physical data of various documents.

Example 3: Reaction of Pitol and SSADH

SSADH was pretreated with the sample in 25 ° C., 0.05 M potassium phosphate buffer (pH 8.4). Small amounts of the culture mixture were removed to measure enzyme activity at various time intervals. A protection experiment was performed in the same manner except that the enzyme was pretreated with the substrate or coenzyme for 30 minutes before the addition of phytol.

The reaction of phytol with various concentrations of SSADH resulted in a decrease in enzyme activity over time. For different sample concentrations, log plots of residual activity over time showed the pseudo-first-order kinetics, respectively (Figure 2). Assuming that the reversible complex of SSADH and phytol exists before the irreversible complex is formed, the double inverse plot yields a straight line graph that does not cross the origin (inset in Figure 2). An overview of such inhibition can be expressed in Scheme 1.

Where E is the enzyme, I is the inhibitor, k _d is the dissociation constant of EI, a reversible enzyme-inhibitor complex, and K _inact is the rate constant of the irreversible inactivated complex E-I '.

The primary reaction rate constant, k _app , for the decrease in activity due to phytol is given by the following equation, which can be obtained from a graph of log residual activity versus pretreatment time.

k _app = {[I] × k _inact } / {[I] + k _d }

The values of k _d and k _inact obtained from the 1 / k _app and 1 / [I] linear graphs, and k _i , the second-order rate constant for inhibition, are obtained from the equation k _i = k _inact / k _d . Indicated.

k _d K _inact k _i         mM sec ^-1 mM ^-1 min ^-1        1.63 1.67 x 10 ^-3 6.15 x 10 ^-2

Inactivation of the samples was tested for the ability of the substrate to block enzymatic sites from inactivation in order to assess the induced active sites. Residual activity increased dramatically when the enzyme was pretreated with the coenzyme NAD ⁺ , whereas little or no blocking effect was observed for the substrate SSA (Table 2). This result clearly shows that the binding site of the phytol is likely to be located at or near the coenzyme binding site of the enzyme rather than the substrate SSA binding site.

            Reaction mixture      Remaining active (%)      SSADH          100      SSADH + phytol           32      SSADH / SSA + phytol           38      SSADH / NAD + + Phytol           98

Since the specific level of the neurotransmitter GABA in brain tissue is associated with various neurological diseases including epilepsy, seizures and cramps, the phyto-containing composition of the present invention can be treated by lowering the activity of SSADH, a GABA inhibitor enzyme, It can be provided as a therapeutic agent such as epilepsy, seizures and cramps.

In addition, in the present invention, lactocin (lactucine), which is known to induce drowsiness in lettuce, was not detected in the fraction showing SSADH inhibitory activity, so phytol isolated from lettuce can be used as an effective antiepileptic and antiepileptic agent.

Figure 1 shows the metabolic pathways of GABA.

2 is a structural formula of phytol isolated from lettuce.

Figure 3 is a graph showing the inactivation with time of phytol and 10uM SSADH (succinic semialdehyde dehydrogenase) concentration of 0mM, 0.4mM, 0.8mM, 1.6mM and 3.2mM.

Claims (6)

A composition for the prevention and treatment of convulsive diseases containing phytol as an active ingredient. According to claim 1, wherein the composition is characterized in that to suppress the activity of succinic semialdehyde dehydrogenase (SSADH), prevention and treatment of convulsive disease containing phytol as an active ingredient Composition. According to claim 1, wherein the phytol is partitioned lettuce extract into water, ethyl acetate, n-butanol, characterized in that the dissolved in ethyl acetate in the extracted fraction, prevention of cramping disease containing phytol as an active ingredient And therapeutic compositions. delete delete delete