KR20040035209A - Isolated anticonvulsant material from the root of Angelica dahurica Benth. et Hook. - Google Patents

Abstract

PURPOSE: Provided is an anticonvulsant material isolated from the root of Angelica dahurica Benth. et Hook to inhibit the activity of GABA-T. It is used for a therapeutic agent for seizures, convulsions, epilepsy and the like. CONSTITUTION: An anticonvulsant material is isolated from the extract of the root of Angelica dahurica Benth. et Hook, and inhibits the activity of GABA-T. The extract is obtained by using ethylacetate, n-butanol and water, and the anticonvulsant material is imperatorine and/or falcarindiol. A pharmaceutical composition for treatment of seizures, convulsions, epilepsy and the like contains the anticonvulsant material as an active ingredient.

Description

Isolated anticonvulsant material from the root of Angelica dahurica Benth. et Hook.}

The present invention relates to an anticonvulsant substance isolated from Angelica dahurica root extract. More specifically, seizure, convulsions, epilepsy, etc., γ-aminobutyrate (γ-aminobutyrate; GABA) acts as an inhibitory neurotransmitter in the brain. As a disease caused by the deficiency of, the present invention finds that the substance isolated from copper root extract affects the level of GABA and is effective in treating the above diseases and provides the substance.

As a highly industrialized society develops, various neuropsychiatric diseases are emerging. 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 neurobiochemicals in epilepsy, seizures, and convulsions are the breakdown of the balance between excitatory and inhibitory neurotransmitters when neurotransmitters occur between nerve cells and are involved in neurotransmission. Treatment is possible by regulating the metabolic processes of neurotransmitters. Since decreased levels of GABA cause these diseases, it is possible to develop anticonvulsant drugs 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, in order to alleviate epilepsy and seizure symptoms, a drug that can increase the concentration of GABA is used, which is a GABA synthase GDH or GAD activator or GABA-T, SSADH, SSAR inhibitors involved in GABA degradation metabolism It is possible by searching, and there are active researches on substances having such efficacy from natural materials with few side effects.

An object of the present invention is to provide a substance that is effective in diseases such as convulsions, seizures and epilepsy.

In another aspect, the present invention is to provide a therapeutic agent with fewer side effects by distributing and extracting substances effective in relieving diseases such as convulsions, seizures and epilepsy in the roots of the copper.

Figure 1 shows the metabolic pathways of GABA.

Figure 2 shows the structural formula of coumarin and polyacetylene isolated from the roots of the copper bill.

Figure 3 is reacted with GABA-T (10uM) of each compound 3 (imperatorin) of 0mM, 3.5mM, 7mM, 10.5mM, 14mM concentration in potassium phosphate buffer (pH 7.4) at 25 ℃, 0.1M A graph showing the inactivation of GABA-T.

Figure 4 is reacted with GABA-T (10uM) of each of compound 4 (falcarindiol) of 0mM, 3.5mM, 7mM, 10.5mM, 14mM concentration in potassium phosphate buffer (pH 7.4) at 25 ℃, 0.1M A graph showing inactivation of GABA-T.

The present invention relates to an anticonvulsant substance isolated from Angelica dahurica Benth. Et Hook. Root extract, characterized in that the substance inhibits the activity of GABA-T.

In addition, the present invention is characterized in that the material is obtained by partitioning and extracting with ethyl acetate, n-butanol and water.

In addition, in the present invention, the anticonvulsant substance is imperatorin and / or falcarindiol.

Furthermore, the present invention provides a pharmaceutical composition comprising the above anticonvulsant substance as an active ingredient, and is used as a therapeutic agent for one or more diseases of seizures, convulsions and epilepsy. .

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

Angelica dahurica inhibits the activity of GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Aqueous extracts of 80% methanol were extracted with ethyl acetate (EtOAc), n-butanol (n-BuOH) and water. Ethyl acetate (EtOAc) and n-butanol (n-BuOH) fractions were repeated by column chromatography to obtain six known coumarins; Isoimperatorin (compound 1), imperatorin (comperatorin; compound 3), pelopopterin (compound 5), oxypeucedanin hydrate (compound 6), nodakenin; Compound 9) and 3'-hydroxymarmesinin (compound 10) and two new coumarins; Oxypeucedanin hydrate-3 "-butyl ether (Compound 2), isoproeroside IV (Compound 8) and two polyacetylenes; Falcarindiol (compound 4) and octadeca-1,9-diene-4,6-dyne-3,8,18-triol (octadeca-1,9-dien-4,6-diyn- 3,8,18-triol; compound 7) was isolated. Among the pure compounds isolated, compounds 3 and 4 inactivate the activity of GABA-T depending on time and concentration. Kinetic study of the three compounds in the (kinetic) and 4 react with GABA-T and each 2.3 mM ^-1 min ^-1 and 1.5 ^-1 mM 2-order reaction rate constant of the min- ^1. It is expected that compounds 3 and 4 may inhibit GABA-T, a GABA degrading enzyme, and thus increase the amount of GABA, a neurotransmitter in the central nervous system.

Pharmaceutical compositions containing an anticonvulsant substance isolated from copper root extract as an active ingredient may be formulated orally or injectable by conventional methods in combination with a carrier generally 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>

The roots of Angelica dahurica Benth. Et Hook. Were purchased from the market in Seoul and proved by Dr. Hyung-kyu Lee of the Korea Research Institute of Bioscience and Biotechnology (Daejeon). Sample samples (KHU97246) were stored in Kyung Hee University (Suwon) natural product chemistry laboratory.

Bovine brain tissue was purchased from Majang-dong slaughterhouse in Seoul. Sodium pyrophosphate, 2-mercaptoethanol, NAD ⁺ , GABA, α-glutarate, succinic semialdehyde and potassium phosphate were purchased from Sigma (USA). Rose-6, CM-Sepharose, Blue-Sepharose, CC-Sephadex, DEAE-Sephadex and hydroxyapatite were purchased from Pharmacia (Sweden).

Example 1 Separation of Coumarin and Polyacetylene

The ground plant material (1 kg) was extracted with an aqueous 80% methanol solution (4 L × 2) at room temperature overnight. After removal of the solvent, to obtain a fraction dissolved in ethyl acetate (EtOAc, 42 g) and n-butanol (n-BuOH, 55 g), EtOAc (700 mL x 2) and n-BuOH (500). 2 ml) and water (500 ml) was added to the residue.

EtOAc fraction (36 g) was chromatographed on a silica gel (250 g) column and n-hexane with increasing proportion of EtOAc (n-hexane-EtOAc = 5: 1 → 3: 1 → 1: 1 → 1: 3, 2 L) Concentration gradient elution with -EtOAc, 12 small fractions (ADE-1 ~ ADE-12) was confirmed by TLC. Small fraction ADE-5 (1.89 g) was purified by silica gel (100 g) column (4 × 30 cm) chromatography, eluting with n-hexane-carbon trichloride-ethanol (20: 20: 1, 800 mL) to give compound 1 (isoimperatorin). , 628 mg, CHCl ₃ -EtOH = 12: 1 gave Rf: 0.39) on silica gel TLC. Isoimperatorin (compound 1): colorless prism (CHCl ₃ -EtOH), mp 109-110 ° C. Lit. [12] mp 109.5-110.5 ° C.

The small fraction ADE-7 (2.75 g) was purified by silica gel chromatography, eluting with n-hexane-carbon trichloride-ethanol (30: 10: 1, 1,200 mL) and separated into four small fractions (ADE-7-1 to ADE-). 7-4), of which the third subfraction (ADE-7-3, 540 mg) was compound 2 (Oxypeucedanin hydrate-3 "-butyl ether, 143 mg, Rf: 0.36 carbon trichloride-ethanol on silica gel TLC = 10 To produce: 1) eluted with n -hexane-CHCl ₃ -ethanol (12: 12: 1, 550 mL) and chromatographed on a silica gel (75 g) column.

Oxypeucedanin hydrate-3 "-butyl ether (Compound 2): light brown prism (CHCl ₃ -EtOH), mp 102-103 ° C; [a] _D : + 1.1 ° ( c = 1.1, CHCl ₃ ); UV: λ _max nm (MeOH, log ε) 252 (3.5), 269 (3.6), 315 (3.9); IR: γ _max cm ⁻¹ (CHCl ₃ ) 3465, 3015, 1725, 1620, 1520; EI-MS m / z : 360 (M ⁺ ), 345, 315, 303, 185, 174; HREI-MS m / z : 360.1577 (calcd for C ₂₀ H ₂₄ O ₆ , 360.1573); ¹ H-NMR (400 MHz, CDCl ₃ , δ) 8.14 (1H, d, J = 9.8 Hz, H-4), 7.50 (1H, d, J = 2.2 Hz, H-2 '), 7.03 (1H, br.s, H-8), 6.93 (1H, dd, J = 2.2, 0.7 Hz, H-3 ′), 6.16 (1H, d, J = 9.8 Hz, H-3), 4.53 (1H, dd, J = 10.0, 3.0 Hz, H-1 "-a), 4.31 (1H, dd, J = 10.0, 7.8 Hz, H-1" -b), 3.85 (1H, dd, J = 7.8, 3.0 Hz, H -2 "), 3.33 (2H, t, J = 6.3 Hz, H-1"'), 1.45 (2H, m, H-2 "'), 1.29 (2H, m, H-3"'), 1.19 (3H, s, H-5 "), 1.17 (3H, s, H-4"), 0.83 (3H, t, J = 7.3 Hz, H-4 "'); ¹³ C-NMR (100 MHz, CDCl ³ , δ _c ) 161.68 (C-2), 158.07 (C-7), 152.50 (C-5), 148.84 (C-8a), 144.88 (C-2 '), 139.43 (C-4), 113.85 ( C-6), 112.57 (C-3), 107.17 (C-4a), 104.90 (C-3 '), 94.22 (C-8), 76.68 (C-2 "), 75.59 (C-3"), 74.21 (C-1 "), 60.92 (C-1"'), 32.39 (C-2 "'), 21.32 (C-4"), 21.15 (C-5 "), 19.37 (C-3"') , 13.82 (C-4 "').

Compound 3 (imperatorin, 212 mg), compound 4 (falcarindiol, 502 mg) and compound 5 (phellopterin, 154 mg) {fs-0.58 (compound 3), 0.51 (compound 4), 0.42 (compound 5) on silica gel TLC with n-hexane-carbon trichloride-ethanol = 14: 14: 1 as elution solventn-Hexane-CHCl₃ADE-8 (4.0 g) was purified by silica gel (125 g, 4 × 35 cm) column chromatography using ethanol (25: 20: 1, 2,400 mL). Imperatorin (Compound 3): colorless prism (CHCl₃-EtOH), m.p. 101-102 ° C. Lit. [13] m.p. 102 [deg.] C. Falcarindiol (Compound 4): Colorless oil, [α]_D+ 318 ° (MeOH,c= 1.8). Lit. [14] [α]_D+ 321.4 ° (CH₃CN). Pelopopterin (Compound 5): Light yellow needle (n-hexane-EtOAc), m. p. 101-102 ° C. Lit. [15] m.p. 103.5-104.5 ° C.

ADE-11 (2.7 g) gave compound 6 (oxypeucedanin hydrate, 517 mg, Rf: 0.58 on silica gel TLC with n-hexane-ethylacetate-acetone = 2: 7: 2)n-Hexane-CHCl₃Silica gel (120 g) column chromatography was applied, eluting with ethanol (4: 6: 1, 1700 mL). Oxypeucedanin hydrate (Compound 6): Colorless needle (n-hexane-EtOAc), m. p. 137-138 ° C; [α]_D+ 18.9 ° (MeOH,c= 1.2). Lit. [16] m.p. 136-137 ° C .; [α]_D+ 16.9 ° (EtOH)}.

ADE-12 (2.76 g) is Compound 7 (Octadeca-1,9-dien-4,6-diyn-3,8,18-triol, 34 mg, n-hexane-ethylacetate-methanol = 7: 7: 1 Silica gel (120 g) column chromatography was applied, eluting with n -hexane-CHCl ₃ -methanol (10: 3: 1, 1,400 mL) to give Rf: 0.48) on silica gel TLC. Compound 7: colorless oil, [α] _D + 308 ° (CHCl ₃ , c = 1.2). Lit. [14] [α] _D + 318.2 ° (CH ₃ CN).

The n -butanol fraction (40 g) was CHCl ₃ -MeOH-H ₂ O = 10: 1: 0 (1,000 mL) → 7: 1: 0 (800 mL) to obtain 10 small fractions (ADB-1 to ADB-10). ) Was introduced into a silica gel (250 g) column (7 × 17 cm) while eluting and changing from 5: 1: 0 (800 mL) to 65:35:10 (1,000 mL) to 6: 4: 1 (1,000 mL). To obtain five small fractions (ADB-5-1 to ADB-5-5), the fifth fraction (ADB-5, 914 mg) was eluted with CHCl ₃ -MeOH (5: 1, 1,700 mL), followed by silica gel ( 200 g) column (7 × 15 cm) chromatography. Small fraction ADB-5-1 (572 mg) was chromatographed on silica gel (150 g) column (5 × 25 cm) using n- BuOH-EtOAc (2: 5, 1,200 mL) as the elution solvent. 1-1 to ADB-5-1-3) were obtained. A mixture of ADB-5-1-2 (165 mg), pyridine (4 ml) and acetic anhydride (4 ml) was stirred at room temperature overnight. The reaction solution was added to ice water (150 mL) and extracted with EtOAc (150 mL x 2). The organic layer was washed successively with 5% aqueous hydrochloric acid solution (150 mL), saturated aqueous sodium bicarbonate solution (150 mL) and brine (150 mL), and dried over anhydrous magnesium sulfate. The acetylated mixture was eluted with n -hexane-EtOAc (1: 1, 750 mL) to obtain compound 8a (isopraeroside IV tetraacetate; 221 mg, n -hexane-EtOAc = 1: 2, Rf: 0.42 on silica gel TLC). Purified by silica gel (75 g) column (3 × 24 cm) chromatography.

Compound 8a (Isopraeroside IV tetraacetate): pale yellow oil, [α] _D + 54 ° (MeOH, c = 1.2); IR: λ _max cm ⁻¹ (CHCl ₃ ) 1715, 1610, 1515; ¹ H-NMR (400 MHz, CDCl ₃ , δ) 7.62 (1H, d, J = 9.3 Hz, H-4), 7.25 (1H, d, J = 8.8 Hz, H-5), 6.71 (1H, d , J = 8.8 Hz, H-6), 6.20 (1H, d, J = 9.3 Hz, H-3), 5.13 (1H, dd, J = 9.5, 9.5 Hz, glc-4), 4.94 (1H, dd , J = 9.7, 8.5 Hz, glc-2), 4.84 (1H, dd, J = 9.7, 9.5 Hz, glc-3), 4.75 (1H, d, J = 8.5 Hz, glc-1), 4.69 (1H , dd, J = 2.6, 5.6 Hz, H-2), 3.93 (1H, dd, J = 5.3, 12.2 Hz, glc-6a), 3.56 (1H, dd, J = 2.4, 12.2 Hz, glc-6b) , 3.48 (1H, m, glc-5), 3.25 (1H, dd, J = 2.6, 12.0 Hz, H-1a), 3.23 (1H, dd, J = 5.6, 12.0 Hz, H-1b), 1.96, 1.95, 1.92, 1.91 (all 3H, each s, acetyl-methyl), 1.29 (3H, s, H-4), 1.22 (3H, s, H-5); ¹³ C-NMR (100 MHz, CDCl ₃ , δ _c ) 171.14, 170.52, 170.34, 169.32 (acetyl-carbonyl), 163.75 (C-2), 160.95 (C-7), 151.31 (C-8a), 143.87 (C -4), 128.65 (C-5), 113.96 (C-8), 113.12 (C-4a), 112.32 (C-3), 106.43 (C-6), 95.62 (glc-1), 90.40 (C- 2), 78.62 (C-3), 72.90 (glc-5), 71.52 (glc-3), 71.49 (glc-2), 69.46 (glc-4), 61.68 (glc-6), 60.38 (C-2 '), 27.70 (C-1), 22.75 (C-4), 22.65 (C-5), 20.59 (x2), 20.56, 20.53 (acetyl-methyl).

Compound 8a (50 mg) was dissolved in 5 ml 2% KOH solution (H ₂ O-MeOH = 1: 3) and stirred at room temperature for 30 minutes. The reaction mixture was neutralized by addition of Dowex 50w x 8 (H ⁺ form), filtered and evaporated to dryness, and then silica gel (50 g) column (3x12 cm) chromatography ( n -hexane-EtOAc = 8: 5) to obtain compound 8. , 450 mL) was performed. (12.2 mg, Rf: 0.61 on silica gel TLC at CHCl ₃ -MeOH-H ₂ O = 65: 35: 10).

Compound 8 (Isopraeroside IV): white powder ( n -hexane-EtOAc-MeOH), mp117-118 ° C .; [a] _D : + 66 ° (MeOH, c = 1.2); IR: λ _max cm ⁻¹ (CHCl ₃ ) 3382, 1710, 1604, 1490; EI-MS m / z : 408 (M ⁺ ), 275, 246, 228, 187, 175; HREI-MS: Found: 408.1422, Calcd. For C ₂₀ H ₂₄ 0 ₉ : 408.1420; ¹ H-NMR (400 MHz, CD ₃ OD, δ) 7.77 (1H, d, J = 9.5 Hz, H-4), 7.30 (1H, d, J = 8.6 Hz, H-5), 6.67 (1H, d, J = 8.6 Hz, H-6), 6.09 (1H, d, J = 9.5 Hz, H-3), 4.49 (1H, d, J = 7.8 Hz, glc-1), 4.12 (1H, dd, J = 2.1, 5.5 Hz, H-2), 3.05 (1H, dd, J = 2.1, 12.0 Hz, H-1a), 3.01 (1H, dd, J = 5.5, 12.0 Hz, H-1b), 1.28, 1.26 (both 3 H, each s, H-4, 5); ¹³ C-NMR (100 MHz, CD ₃ OD, δ _c ) 165.60 (C-2), 163.25 (C-7), 152.54 (C-8a), 146.33 (C-4), 130.27 (C-5), 115.53 (C-8), 114.46 (C-4a), 112.36 (C-3), 107.86 (C-6), 98.91 (glc-1), 91.90 (C-2), 79.16 (C-3), 78.13 (glc-5), 77.50 (glc-3), 75.12 (glc-2), 71.39 (glc-4), 62.35 (glc-6), 28.27 (C-1), 23.79, 22.47 (C-4, 5 ).

ADB-5-3 (29 mg) was acetylated in a similar procedure to ADB-5-1-2 and silica gel (30 mL) eluting with n -hexane-ethyl acetate (5: 6, 240 mL) to feed compound 9a. Column (3 × 9 cm) was applied. The same treatment of compound 9a as that of compound 8a and compound 9 (nodakenin, 17 mg, Rf purified by silica gel (30 g) column (3 × 9 cm) chromatography (CHCl ₃ -MeOH = 4: 1, 320 mL). : 0.52 on silica gel TLC in CHCl 3 -MeOH-H 2 O = 65:35:10). Compound 9 (Nodakenin): white powder ( n -hexane-EtOAc), mp 224-225 ° C .; [a] _D + 28.0 ° (MeOH, c = 0.7). Lit. [12] mp 217-219 ° C .; [a] _D + 24 ° (EtOH-H ₂ 0 = 1: 1, c = 0.9).

ADB-7 (1.5 g) was purified using silica gel (150 g) using CHCl ₃ -EtOH (3: 1, 1600 mL) as the elution solvent to obtain five small fractions (ADB-7-1 to ADB-7-5). It was introduced into column (5 × 25 cm) chromatography. The fifth fraction (ADB-7-4, 44 mg) provided compound 10 (3`-hydroxymarmesinin, 22 mg, Rf: 0.33 on silica gel TLC in CHCl ₃ -MeOH-H ₂ O = 65:35:10) In order to do this, a silica gel (50 g) column (3 × 14 cm, n -hexane-EtOAc-EtOH = 2: 10: 3) was chromatographed. Compound 10 (3-Hydroxymarmesinin): colorless needle ( n -hexane-EtOAc-EtOH), mp 262-263 ° C .; [a] _D -24 ° (MeOH, c = 0.7). Lit. [9] mp 217-218 ° C .; [α] _D -11 ° (pyridine, c = 0.1).

The yield of each fraction in the extract is 1: 0.063%; 2: 0.017%; 3: 0.025%; 4: 0.059%; 5: 0.018%; 6: 0.060%; 7: 0.004%; 8: 0.007%; 9: 0.002%; 10: 0.003%.

Example 2 Enzyme Purification and Assay

Purification of cerebellar GABA-T was performed by a method developed in our laboratory (Choi et al. Purification and properties of GABA transaminase from bovine brain.Molecules and cells3, 397-401. 1993). Succinic semialdehyde dehydrogenase (SSADH) is a CM-Sepharose, Blue-Sepharose and hydroxyapatite chromatographic method from bovine brain tissue. methods). Protein concentrations were determined with protein assay kits from Bio-Rad using bovine serum albumin as standard.

A coupled assay system consisting of two purified enzymes, GABA-T and SSADH, is used for the study of the catalytic conversion of GABA to succinic semialdehyde.

GABA + a-ketoglutarate --- → succinic semialdehyde (SSA) + glutamic acid

SSA + NAD ⁺ + H ₂ O ---- → succinate + NADH

Enzymatic assays include 01.M sodium pyrophosphate buffer at pH 8.4 containing 1 mM 2-mercaptoethanol, 5 mM NAD ⁺ , 30 mM GABA and 10 mM α-ketoglutarate The solution was performed in sodium pyrophosphate buffer. Progress of the reaction was observed by measuring the change in absorbance at 340 nm in the amount of decrease of NAD ⁺ . In coupled assay systems, it is appropriate to measure the amino group transfer rate when the concentration of SSADH is at least 5 times higher than the concentration of GABA-T. The unit of enzymatic activity was defined as the amount of enzyme that produced 1umol · min ⁻¹ succinic semialdehyde at 25 ° C.

In order to measure the activity of SSADH, production of NADH was measured by an increase in absorbance at 340 nm. All assays were performed in duplicates and the initial rate data correlated with a standard assay mixture containing 100 μM of 0.1 mM sodium pyrophosphate (pH 8.4) at 25 ° C. and succinic semialdehyde and 1 mM NAD ⁺ . .

The isolated compounds were evaluated for their inhibitory effects on the activities of SSADH and GABA-T, but most of the compounds showed no significant effect on SSADH activity. On the other hand, compounds 3 and 4 significantly inhibited the activity of GABA-T, compound 7 showed a slight inhibitory effect. The structure of each compound is very similar but their inhibitory effect is completely different. Table 1 shows the activities of SSADH and GABA-T treated with the compounds isolated from the cups.

One 2 3 4 5 6 7 8 9 10 Control GABA-T 80 79 15 24 75 97 50 97 76 89 100 SSADH 87 90 89 82 74 90 85 96 107 100 100

In the reaction of GABA-T with various concentrations of compounds 3 and 4, the enzyme activity decreased with time. Compound 3 inactivated the enzyme depending on time and concentration (FIG. 3). Inactivation depends on the form of the pseudo-first order kinetics in each constant concentration of sample.

Since the coupled assay system contains two enzymes, GABA-T and SSADH, the inhibitory effects of SSADH samples were independently investigated in a similar manner. Pretreatment with purified SSADH with increasing sample concentration did not provide a significant reduction in catalytic activity as shown in Table 2. This result shows that Compound 3 and Compound 4 only cause inactivation of GABA-T activity.

Reaction mixture Remaining activity (%) GABA-T 100 GABA-T + imperatorin (7mM) 24 GABA-T + falcarindiol (7 mM) 35 SSADH (10uM) 100 SSADH + imperatorin (14mM) 89 SSADH + falcarindiol (14 mM) 82

Example 3 Inactivation of Pure Compounds and Enzymes Isolated from Copper Roots

Purified GABA-T (10 uM) was treated with samples of various concentrations. The change in catalyst activity was measured by the above method. The reaction was started with the addition of a sample to 25 ℃ 0.1M potassium phosphate buffer (pH 7.4). After initiation of the inactivation reaction, a small amount was removed for activity measurement at regular intervals. Inactivation protection experiments were performed in the same manner as the previous experiment, except that the enzyme was pretreated with a substrate (GABA or α-glutarate) before the modification began with the addition of the sample.

Inhibition rate constants are calculated from data points or least squares in the Kitz & Wilson plot. All kinetic data shown here are averages from two or more experiments.

Reaction mixture Remaining activity (%) GABA-T (uM) 100 GABA-T + imperatorin (14mM) 14 GABA-T and α-ketoglutarate (10 mM) + imperatorin (14 mM) 98 GABA-T and GABA (20mM) + imperatorin 74

As shown in Table 3, inactivation by Compound 3 was completely protected when the enzyme was pretreated with α-ketoglutarate, while partial reaction with GABA was also observed. The same result for substrate blocking was obtained from inactivation by compound 4 (falcarindiol).

Reaction mixture Remaining activity (%) GABA-T (10 uM) 100 GABA-T + falcarindol (14 mM) 23 GABA-T and α-ketoglutarate (10 mM) + falcarindol 95 GABA-T and GABA (20mM) + falcarindol 98

These results indicate that inactivation is the result of selective modification of the reactor at the center of the enzyme activity rather than a nonspecific reaction mechanism.

The present invention is the first to show that the natural-derived compounds imperitorin (compound 3) and falcarindaiol (compound 4) bind to the active site of GABA-T, act as an efficient irreversible inhibitor of the enzyme, and are effective as anti-strain therapies. Is an experimental report.

In addition, the present invention relates to the specific level of neurotransmitter GABA in brain tissues associated with various neurological diseases including epilepsy, seizures and cramps, so that certain inhibitors of GABA-inhibiting enzyme GABA-T are levels of GABA in pathological conditions. Useful for raising

Claims (6)

Anticonvulsant substance isolated from the extract of Angelica dahurica Benth. Et Hook. The anticonvulsant substance according to claim 1, wherein the substance inhibits the activity of GABA-T. The anticonvulsant substance according to claim 1, wherein the substance is obtained by partition extraction with ethyl acetate, n-butanol and water. The anticonvulsant substance according to claim 1, wherein the substance is imperatorin and / or falcarindiol. A pharmaceutical composition comprising the anticonvulsant substance according to any one of claims 1 to 4 as an active ingredient. The pharmaceutical composition according to claim 5, wherein the substance is used as a therapeutic agent for at least one of seizures, convulsions, and epilepsy diseases.