Human Brain GABA-T ( γ-aminobutyric acid transaminase) Inhibitory Alkaloids from Corydalis Tuber
Soo Young Choi1, Myun-Ho Bang, Eun Jeong Lee, Oh-Shin Kwon2, Tae-Cheon Kang3, Youn-Hyung Lee, Yeong-Deok Rho and Nam-In Baek*
Graduate School of Biotechnology & Plant Metabolism Research Center, Kyunghee University, Suwon 449-701, Korea
1Department of Genetic Engineering, Division of Life Sciences, Hallym University, Chunchon 200-702, Korea
2Department of Biochemistry, Kyungpook National University, Taegu 702-701, Korea
3Department of Anatomy, College of Medicine, Hallym University, Chunchon 200-702, Korea Received April 28, 2003; Accepted June 18, 2003
The alkaloid fraction obtained from 80% MeOH extracts of Corydalis tuber showed a significant inhibitory effect on brain GABA-transaminase (GABA-T). Each alkaloid fractions using SiO2 and ODS column chromatography were isolated in seven isoquinoline alkaloids. They were identified as R-(+)- stylopine, S-(−)-corydaline, R-(+)-canadine, R-(+)-tetrahydropalmatine, protopine, S-(+)-glaucine and dehydrocorydaline, based on the interpretation of several spectral data and in comparison with those of literatures. All they inhibited the activity of GABA-T.
Key words: Corydalis, anticonvulsant, isoquinoline alkaloid, aporphine alkaloid, brain GABA-T.
GABA (γ-aminobutyric acid) has been known as a major inhibitory chemical neurotransmitter1) in the central nervous system (CNS) of mammalians. The release of GABA by nerve terminals and its subsequent binding to its receptor must be followed by a rapid inactivation of the neurotransmitter.
When the concentration of GABA in the brain diminishes to below a threshold level, various neurological disorders including epilepsy, seizures, convulsions, Huntington’s disease and Parkinsonism may start to occur2-4).
The concentration of GABA in the brain is controlled by two pyridoxal-5’-phosphate (PLP) dependent enzymes, i.e., glutamate decarboxylase (GAD; EC 4.1.1.15) and GABA transaminase (GABA-T; EC 2.6.1.19). The first enzyme catalyzes the synthesis of GABA, whereas the second enzyme catalyzes the conversion of GABA to succinic semialdehyde by a transamination reaction. Succinic semialdehyde is then primarily oxidized to succinate by succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.24); thus the carbon skeleton of GABA enters the tricarboxylic acid cycle in the form of succinate. The observation that the activation of GAD or the inactivation of the GABA-T and SSADH in brain tissues increases the concentration of neurotransmitter GABA supports that these enzymes exert a controlling influence on GABA levels. Therefore, the irreversible inhibition of GABA- T by a chemical analog of GABA is the basic action mechanism of drugs used in the treatment of convulsive disorders.5)
Most plants of genus Corydalis (Papaveraceae) are perennial herbs broadly distributed in shaded areas under trees in Korea and the tubers have been traditionally used as an analgesic.6) Even though some experiments concerning several biological activities6-9) including the effect on central nervous system were carried out and several alkaloids have been isolated from the plants of genus Corydalis,9-12) the principal component of the drugs manifesting antispasmodic has not yet been reported so far.
The present paper deals with the isolaton and identification of the principal components manifesting sedative or antispasmodic effects of Corydalis tuber.
Materials and Methods
Materials. The tuber of Corydalis was purchased at a market in Seoul and was identified by Dr. Hyeong-Kyu Lee, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea. The voucher specimen are preserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Suwon (KH01092).
Bovine brains were obtained from the Majangdong Packing Company in Seoul, Korea. Sodium pyrophosphate, 2- mercaptoethanlol, NAD+, GABA, α-ketoglutarate, and potassium phosphate were purchased from Sigma Chemical Co. (St. Louis, USA). Superose-6, CM-Sepharose, Blue- Sepharose, CM-Sephadex, DEAE-Sephadex and hydroxyapatite were obtained from Pharmacia (Uppsala, Sweden).
Instruments. Melting points were determined on a Fisher-John apparatus and uncorrected. 1H- (400 MHz) and
*Corresponding author
Phone: 82-31-201-2661; Fax: 82-31-204-8116 E-mail: [email protected]
13C-NMR (100 MHz) spectra were measured with Varian Unity-INOVA spectometer. EI mass spectra were taken on a JEOL JMS-AX505WA spectrometer. Optical rotations were measured on a JASCO DIP-370 digital polarimeter. IR spectra were taken with a Perkin-Elmer 599B spectrometer.
Absorption spectroscopic measurements were carried out in Kontron UVIKON Model 930 double beam spectrophotometer. Fluorescence spectra were recorded in a Kontron SFM 25 spectrofluorometer.
Isolation of alkaloids. The tuber of Corydalis (5 kg) was milled and extracted with 80% MeOH solution (5L× 2) at room temperature. The filtration and evaporation of the extracted solution under reduced pressure yielded MeOH extracts (347 g). The aqueous solution of MeOH extracts was acidified (pH 2-3) with conc. HCl and washed with EtOAc.
The aqueous acid layer was basified (pH 11-12) with 20%
NaOH and extracted with EtOAc and n-BuOH. Two alkaloid fractions were yielded with 18 g and 23 g, respectively.
The EtOAc soluble fractions were subjected to silica gel column chromatography (350 g), which was eluted stepwise- gradiently with hexane-EtOAc-EtOH (4 : 1 : 0 3 : 1 : 0 2 : 1 : 0 5 : 5 : 1 4 : 4 : 1) and CHCl3-EtOH (10 : 1) to be divided into thirteen subfractions (HHSE-1~HHSE-13).
HHSE-3 fraction (117 mg) was successively applied to silica gel column chromatography (55 g, n-hexane-EtOAc = 5 : 1) and compound 1 was obtained as a purified compound (78 mg).
Compound 1 {R-(+)-stylopine}: colorless prisms (EtOH- CHCl3), mp 204-205oC, [α]D +309o (c = 1.5, CHCl3); IRν (CHCl3, max) 2922, 2805, 2750, 1510, 1480, 1465 cm−1, EI/
MS m/z (%) 323 (M+, 9), 174 (52), 148 (100). 1H-NMR (400 MHz, CDCl3, δ) 6.76 (1H, d, J = 8.0 Hz), 6.70 (1H, d, J = 8.0 Hz) (H-11, H-12), 6.68, 6.64 (each 1H, both s, H-1, H-4), 5.90, 5.88 (each 2H, both s, 2,3-dioxymethylene, 9,10- dioxymethylene), 4.18, 3.52 (each 1H, both d, J = 16.0 Hz, H- 8ax, H-8eq), 3.50 (1H, dd, J = 3.2, 11.0 Hz, H-14), 3.18 (1H, dd, J = 3.2, 15.8 Hz, H-13eq), 3.12, 3.09 (each 1H, both m, H- 5, H-6), 2.80 (1H, dd, J = 11.0, 15.8 Hz, H-13ax), 2.63, 2.57 (each 1H, both m, H-5, H-6). 13C-NMR (100 MHz, CDCl3);
See Table 1.
Repeated silica gel column chromatography (125 g, n- hexane-EtOAc = 6 : 1 5 : 1) for the fraction HHSE-4 (2.64 g) yielded a purified compound 2 (1.8 g).
Compound 2 {S-(−)-(corydaline}: colorless prisms (EtOH), mp 138-140oC, [α]D−299o (c = 1.2, CHCl3), IRν (CHCl3, max) 2915, 2790, 1505, 1475 cm−1, EI/MS m/z (%) 369 (M+, 12), 339 (22), 309 (24), 307 (9), 245 (100), 164 (62). 1H-NMR (400 MHz, CDCl3, δ) 6.85, 6.76 (each 1H, both d, J = 8.3 Hz, H-11, H-12), 6.66, 6.57 (each 1H, both s, H-1, H-4), 4.18, 3.45 (each 1H, both d, J = 15.9 Hz, H-8ax, H-8eq), 3.83, 3.821, 3.815, 3.79 (each 3H, all s, -OCH3× 4), 3.63 (1H, d, J = 3.1 Hz, H-14), 3.19 (1H, dq, J = 3.1, 6.8 Hz, H-13), 3.12, 3.04, 2.57, 2.52 (each 1H, all m, H-5ax, H-5eq, H-6ax, H- 6eq), 0.92 (3H, d, J = 6.8 Hz, 13-CH3). 13C-NMR (100 MHz, CDCl3); See Table 1.
The fraction HHSE-5 (217 mg) was applied to silica gel (125 g) column eluting with n-hexane-EtOAc (7 : 2-3 : 1) to yield a purified compound 3 (66 mg).
Compound 3 {R-(+)-canadine}: colorless needles (MeOH), mp 128-129oC, [α]D +288o (c = 1.7, CHCl3), IRν (CHCl3, max) 2920, 2880, 1470, 1452 cm−1, EI/MS m/z (%) 339 (M+, 42), 308 (11), 174 (30), 164 (100), 149 (80); 1H-NMR (400 MHz, CDCl3) 6.83 (1H, d, J = 8.3 Hz, H-12), 6.75 (1H, d, J = 8.3 Hz, H-11), 6.70 (1H, s, H-4), 6.56 (1H, s, H-1), 5.88 (2H, s, 2,3-dioxymethylene), 4.21, 3.50 (each 1H, both d, J = 15.9 Hz, H-8ax, H-8eq), 3.83, 3.82 (both 3H, each s, 9-OMe, 10- OMe), 3.49 (1H, dd, J = 3.4, 11.1 Hz, H-14), 3.19 (1H, dd, J = 3.4, 15.8 Hz, H-13b), 3.15, 3.07 (both 1H, each m, H-5, H- 6), 2.79 (1H, dd, J = 11.1, 15.8 Hz, H-13a), 2.62, 2.57 (both 1H, each m, H-5, H-6); 13C-NMR (100 MHz, CDCl3); See Table 1.
1.1 g of a purified compound 4 was obtained through silica gel (150 g) column chromatography for the fraction HHSE-7 (1.7 g) eluting with n-hexnae-EtOAc (5 : 3-4 : 3).
Compound 4 {R-(+)-tetrahydropalmatine}: colorless prisms (EtOH), mp 118-119oC; [α]D +288o (c = 1.2, CHCl3); IRν
(CHCl3, max) 2919, 2795, 1495, 1475 cm−1, EI/MS m/z (%) 355 (M+, 23), 190 (33), 164 (67), 144 (100), 121 (36); 1H- NMR (400 MHz, CDCl3) 6.85 (1H, d, J = 8.3 Hz, H-12), 6.76 (1H, d, J = 8.3 Hz, H-11), 6.73 (1H, s, H-4), 6.60 (1H, s, H-1), 4.22, 3.51 (each 1H, both d, J = 15.6 Hz, H-8ax, H-8eq), 3.86, 3.84, 3.83, 3.81 (all 3H, each s, OMe× 4), 3.52 (1H, dd, J = 3.2, 15.6 Hz, H-14), 3.25 (1H, dd, J = 3.2, 11.2 Hz, H- 13b), 3.18, 3.13 (both 1H, each m, H-5, H-6), 2.82 (1H, dd, J = 15.6, 11.2 Hz, H-13a), 2.65, 2.58 (both 1H, each m, H-5, H-6); 13C-NMR (100 MHz, CDCl3); See Table 1.
The fraction HHSE-10 (620 mg) was further separated by silica gel (100 g) column chromatography eluting with CHCl3-EtOH (15 : 1 7 : 1) to afford a purified compound 5 (820 mg).
Compound 5 (protopine): colorless crystals (CHCl3-EtOH), mp 208-209oC, IRν (CHCl3) 2915, 2850, 1670, 1485, 1455 cm−1, EI/MS m/z (%) 353 (M+, 20), 338 (10), 322 (6), 281 (35), 267 (70), 252 (65), 163 (40), 148 (100). 1H-NMR (400 MHz, CDCl3, δ) 6.89 (1H, s, H-1), 6.69, 6.65 (both 1H, each d, J = 7.8 Hz, H-11, 12), 6.64 (1H, s, H-4), 5.94 (2H, s, 2,3- dioxymethylene), 5.91 (2H, s, 9,10-dioxymethylene), 3.82- 3.62 (2H, br. s, H-13), 3.70-3.55 (2H, br. s, H-8), 2.55, 2.46 (each 2H, both br. s, H-5, H-6), 1.92 (3H, s, N-CH3). 13C- NMR (100 MHz, CDCl3); See Table 1.
The fraction HHSE-12 (3.6 g) was applied to silica gel (250 g) column eluting stepwise-gradiently with CHCl3-EtOH (30 : 1-25 : 1-20 : 1-15 : 1) to afford nine subfractions (HHSE-12-1~HHSE-12-9). The second fraction (HHSE-12- 2, 1.4 g) was further separated by silica gel (130 g) column chromatography using CHCl3 (20 : 1) as eluents to give a purified compound 6 (1.1 g).
Compound 6 {S-(+)-glaucine}: colorless crystals (CHCl3- EtOH), mp 120-121oC; [α]D +117o (c = 1.2, EtOH); IRν (CHCl3, max) 2985, 2892, 2754, 1525, 1466 cm−1, EI/MS m/z
(%) 355 (M+, 45), 340 (100), 338 (24), 324 (49), 312 (37), 308 (22), 297 (46), 281 (45), 266 (15); 1H-NMR (400 MHz, CDCl3) 8.01 (1H, s, H-11), 6.70 (1H, s, H-3), 6.50 (1H, s, H- 8), 3.83, 3.82, 3.78 (all 3H, each s, 2-OMe, 9-OMe, 10-OMe), 3.57 (3H, s, 1-OMe), 3.12 (1H, ddd, J = 16.4, 5.6, 12.6 Hz, H- 4ax), 3.07 (1H, br. dd, J = 5.6, 14.0 Hz, H-5a), 3.00 (1H, dd, J = 3.6, 5.2 Hz, H-6a), 2.93 (1H, dd, J = 3.6, 13.6 Hz, H-7eq), 2.64 (1H, br. dd, J = 2.8, 16.4 Hz, H-4eq), 2.47 (1H, dd, J = 5.2, 13.6 Hz, H-7ax), 2.45 (3H, s, N-Me), 2.43 (1H, ddd, J = 2.8, 12.6, 14.0 Hz, H-5b); 13C-NMR (100 MHz, CDCl3);
See Table 1.
The n-BuOH soluble fractions were subjected to a silica gel column chromatography (160 g), which was eluted stepwise- gradiently with CHCl3-EtOH (10 : 1 5 : 1) and CHCl3- MeOH (3 : 1) to yield eight subfractions (HHSB-1~HHSB-8).
HHSB-2 fraction (2.0 g) was successively applied to a silica gel column chromatography (100 g, CHCl3-MeOH = 10 : 1) to afford a purified compund 7 (1.4 g).
Compound 7 (dehydrocorydaline): white powder (EtOH), mp 178-180oC, IRn (KBr, max) 2910, 2800, 1505, 1480 cm−1, EI/MS m/z (%) 366 (M+, 15), 351 (24), 336 (20), 227 (65), 164 (100). 1H-NMR (400 MHz, CDCl3, ) 10.51 (1H, s, H-8), 7.96, 7.91 (each 1H, both d, J = 9.3 Hz, H-11, 12), 7.18, 6.94 (each 1H, both s, H-1, 4), 5.24 (2H, br. t, J = 6.0 Hz, H-6), 4.28, 4.07, 4.00, 3.95 (each 3H, all s, 2-OMe, 3-OMe, 9-OMe, 10-OMe), 3.24 (2H, br. t, J = 6.0 Hz, H-5), 2.98 (3H, s, 13- CH3); 13C-NMR (100 MHz, CDCl3); See Table 1.
Purification of GABA-T and activity assay. Human brain GABA-T was expressed and purified by a method as described in previous papers.13,14) A coupled assay system containing of two purified enzymes, i.e. GABA transaminase and succinic semialdehyde dehydrogenase was used to study the catalytic conversion of GABA to succinic semialdehyde.
Enzymatic assays were performed in 0.1 M sodium pyrophosphate buffer, pH 8.4, containing 1 mM 2- mercaptoethanol, 5 mM NAD+, 30 mM GABA, and 10 mM Table 1. 13C-NMR data of alkaloids from Corydalis tuber (100 MHz, CDCl3)
No of C 1 2 3 4 5 6 7
1 105.33 108.55 105.43 108.40 107.99 143.99 110.68
1a, 1b 126.62
126.55
2 144.92 146.93 145.82 147.12 146.18 151.66 147.70
3 146.60 147.41 146.05 147.18 147.98 110.10 150.39
3a 128.48
4 107.23 110.97 108.30 111.09 110.31 28.79 113.87
4a 127.72 128.16 127.69 127.38 135.70 129.04
5 29.49 29.03 29.48 28.71 29.61 52.94 28.05
6 51.83 51.11 51.29 51.15 57.58 57.24
6a 62.20
7 34.13
7a 128.92
8 53.21 54.18 53.83 53.63 50.93 110.58 145.39
8a 127.71 128.10 127.61 126.43 117.47 121.45
9 146.74 149.73 150.18 149.89 145.95 147.71 151.23
10 143.22 144.58 145.00 144.71 145.88 147.16 145.81
11 109.90 110.73 110.93 110.66 106.73 111.32 120.16
11a 124.15
12 121.79 123.72 123.78 123.50 124.88 125.58
12a 128.43 134.64 128.51 128.24 128.65 133.64
13 36.27 38.00 36.33 35.91 46.07 131.93
14 59.90 62.71 59.53 58.93 194.20 136.29
14a 130.81 128.22 130.74 129.38 132.33 119.16
2,3-dioxy-methylene 100.82 100.65 101.15
9,10-dioxy-methylene 100.64 100.83
1-methoxy 59.83
2-methoxy 55.54 55.47 55.61 56.49
3-methoxy 55.49 55.46 55.46 56.20
9-methoxy 59.74 60.05 59.74 55.44 64.19
10-methoxy 55.82 55.79 55.72 56.97
13-methyl 18.07 18.00
N-methyl 41.43 43.54
α-ketoglutarate. The progress of the reaction was monitored by measuring absorbance changes at 340 nm due to the reduction of NAD+. A unit of enzyme activity is defined as the amount of enzyme that produces 1µmolÁmin−1 of succinic semialdehyde at 25oC.
Purified GABA-T (10 M) was treated with 6 mM of each isolated alkaloids for 10 min prior to performing enzyme assay. 1 mM of vigabatrin was used as a positive control.
Changes in catalytic activity were measured using the method described above. All data expressed are mean values obtained from more than two experiments carried out separately.
Results and Discussion
Since abnormally low levels of neurotransmitter GABA in the brain has been associated with a variety of neurological disorders including epilepsy, seizure and convulsant disorder,2-4) a specific inhibitory compound of GABA degradative enzyme, GABA-T, would be useful in attempts to elevate GABA levels under certain pathological conditions.5)
In preliminary experiments, the MeOH extracts of Corydalis tuber showed inhibitory effect on GABA degradative enzymes, such as γ-aminobutyric acid transaminase (GABA-T, bacterial). In addition, the reports that genus Corydalis had the pharmacological effects concerning central nervous system6) while a high content of alkaloid components9-12) led to deduction that the alkaloids might be the principal component in charge of the anticonvulsant activity of Corydalis tuber. Therefore, the isolation of alkaloids from Corydalis tuber was carried out.
A selective solvent fractionation through the adjustment of the solution’s pH afforded crude alkaloid fractions with a yield of 0.82% from dried plant sample.
The EtOAc and n-BuOH soluble fractions were revealed to contain several alkaloids through silica gel TLC which was detected by the radiation of UV absorption and the spraying of 10% aq. H2SO4 and Dragendorff’s reagents.15) The silica gel column chromatography for the fractions gave seven alkaloids, compounds 1-7 with the yield of 0.002, 0.036, 0.001, 0.022, 0.016, 0.022 and 0.028 %, respectively.
Compound 1, colorless prisms (CHCl3-EtOH) mp 218oC, [α]D +309o, showed the absorbance bands due to conjugated double bond at 1510, 1480, 1465 cm−1 in the IR spectrum (CHCl3). In the 1H-NMR spectrum (400 MHz, CDCl3), a 1,2,4,5-tetrasubstituted benzene {δ6.68, δ6.64 (each 1H, both s)}, a 1,2,3,4-tetrasubstituted benzene {δ6.76, δ6.70 (each 1H, both d, J = 8.0 Hz)}, two dioxymethylenes {δ5.90, δ5.88 (each 2H, both s)}, four methylenes {δ4.18, δ3.52 (each 1H, both d, J = 16.0 Hz}, δ3.18 (1H, dd, J = 3.2, 15.8 Hz), δ3.12, δ3.09 (each 1H, both m), δ2.80 (1H, dd, J = 11.0, 15.8 Hz), δ2.63, δ2.57 (each 1H, both m)}, and a methine (δ3.50, 1H, dd, J = 3.2, 11.0 Hz) proton signals were observed indicating that compound 1 was an isoquinoline alkaloid. In the 13C- NMR (100 MHz, CDCl3) spectrum, (Table 1) nineteen carbon signals including two dioxymethylene carbons (δC 100.82,
100.64) were observed. Four olefine-methine signals (δC
105.33, 107.23, 109.90, 121.79), four olefine-quaternary ones attached to carbons (δC 127.72, 127.71, 128.43, 130.81) and four olefine-quaternary ones attached to oxygens (δC 144.92, 146.60, 146.74, 143.22) indicated the presence of two benzenes. And four methylene carbon signals, two of which were attached to nitrogen and other two to carbon, were observed at δC 51.83, 53.21, 29.49, and 36.27, respectively.
The signal at δ59.90 was derived from a methine carbon attached to nitrogen. The molecular weight was determined to be 323 from the M+ ion peak (m/z 323) in the EI/MS spectrum. The comparison of above results to those of literatures11,16) led to identification of compound 1 as 14R-(+)- stylopine (Fig. 1).
NMR data of compound 2, colorless prisms (EtOH), mp 138-140oC, [α]D−299o, were similar to those of stylopine (1), except for the presence of four methoxyl (δH 3.83, 3.821, 3.815, 3.79, each 3H, all s; δC 55.54, 55.49, 59.74, 55.82), one methyl (δH 0.92, 3H, d, J = 6.8 Hz; δC 18.07) and one methine (δH 3.19, dq, J = 3.1, 6.8 Hz; δC 38.00), and the absence of two dioxymethylenes and one methylene (C-13 in compound 1).
The molecular weight was 369 from the M+ ion peak (m/z 369) of the EI/MS; thus, compound 2 was identified as 14S-(−)-corydaline.17,18)
Compound 3, colorless needles (MeOH), mp 128-129oC, [α]D +288o, showed the similarity in the NMR data to stylopine (1), except for the observation of two methoxy signals (δH 3.83, 3.82, each 3H, all s; δC 55.79, 60.05), instead of one dioxymethylene signals. From the molecular weight (m/z 339), the specific rotatory degree and other physical and spectral data, compound 3 was assigned to be 14R-(+)- canadine.17,19,20)
Compound 4, colorless prisms (EtOH), mp 118-119oC; [α]D
+288o, was also very similar to stylopine (1), except for the presence of four methoxies (δH 3.86, 3.84, 3.83, 3.81, each 3H, all s; δC 55.46, 55.47, 55.72, 59.74) instead of two dioxymethylenes. The comparison of several data on compound 4 with those of literatures17,21) led to identification as 14R-(+)-tetrahydropalmatine.
Compound 5, colorless crystals (CHCl3-EtOH), mp 208- 209oC, showed the similar spectral data to compound 1, except for the additional observation of a ketone (δC 194.20;
IR 1670 cm−1) and a N-methyl (δH 1.92, 3H, s; δC 41.43), and the absence of a methine (C-14 in compound 1). The molecular weight was 353 from the M+ ion peak (m/z 353) of the EI/MS; thus, compound 5 was identified as protopine.22,23)
Compoound 6, colorless crystals (CHCl3-EtOH) mp 120- 121oC, showed the absorbance band due to conjugated double bond at 1525, 1466 cm−1 in the IR spectrum (CHCl3). In the
1H-NMR spectrum (400 MHz, CDCl3), a 1,2,4,5- tetrasubstituted benzene {δ8.01, δ6.50 (each 1H, both s)}, a 1,2,3,4,5-pentasubstituted benzene (δ6.70, 1H, s), four methoxy {δ3.83, δ3.82, δ3.78, δ3.57 (each 3H, all s)}, three methylenes {δ3.12 (1H, ddd, J = 16.4, 5.6, 12.6 Hz), δ3.07 (1H, br. dd, J = 5.6, 14.0 Hz), δ2.93 (1H, dd, J = 3.6, 13.6 Hz),
δ2.64 (1H, br. dd, J = 2.8, 16.4 Hz), δ2.47 (1H, dd, J = 5.2, 13.6 Hz), δ2.43 (1H, ddd, J = 2.8, 12.6, 14.0 Hz)}, a methine (δ3.00, 1H, dd, J = 3.6, 5.2 Hz) and one N-methyl (δ2.45, 3H, s) proton signals were observed indicating that compound 1 was a aporphine alkaloid. In the 13C-NMR (100 MHz, CDCl3) spectrum, twenty one carbon signals including four methoxy (δC 55.44, 55.46, 55.61, 59.83) and one N-methyl (δC 43.54) carbons, were observed. Three olefine-methine signals (δC
110.10, 110.58, 111.32), five olefine-quaternary ones attached to carbons (δC 126.62, 126.55, 128.48, 128.92, 124.15) and four olefine-quaternary ones attached to oxygens (δC 143.99, 151.66, 147.71, 147.16) indicated the presence of two benzenes. And three methylene carbon signals, one of which was attached to nitrogen and the other two to carbon, were observed at δC 52.94, 28.79, and 34.13, respectively. The signal at 59.90 was derived from a methine carbon. The molecular weight was determined to be 355 from the M+ ion peak (m/z 355) in the EI/MS spectrum. The comparison of the above results, including specific rotatory degree ([α]D +117o), with those of literatures24,25) led to identification of compound 1 as 6aS-(+)-glaucine.
Compound 7, white powder (EtOH), mp 178-180oC, showed the absorbance band due to conjugated double bond at 1505, 1480 cm−1 in the IR spectrum (KBr). In the 1H-NMR spectrum (400 MHz, CDCl3) of compound 7, a 1,2,4,5- tetrasubstituted benzene {(δ7.18, δ6.94 (each 1H, both s)}, a 1,2,3,4-tetrasubstituted benzene {(δ7.96, δ7.91, each 1H, both d, J = 9.3 Hz)}, four methoxies {δ4.28, δ4.07, δ4.00, δ3.95
(each 3H, all s)}, one allyl methyl (δ2.98, 3H, s) one methylene (3.24, 2H, br. t, J = 6.0 Hz) proton signals were observed. Especially, one olefine methine (δ10.51, 1H, s) and one methylene (δ5.24, 2H, br. t, J = 6.0 Hz) signals observed at very low magnet field indicated the ionization of nitrogen attached to both carbons. In the 13C-NMR (100 MHz, CDCl3) spectrum, twenty two carbon signals including four methoxyl (δ56.49, 56.20, 64.19, 56.97), one methyl (δ18.00), one lowly shifted olefinic methine (δ145.39) and one methylene (δ57.24) signals were observed. The molecular weight was determined to be 366 from the M+ ion peak (m/z 366) in the EI/MS spectrum. Accordingly compound 7 was determined to be dehydrocorydaline.10)
Among seven alkaloids, stylopine (1), canadine (3) and glaucine (6) have never been so far isolated from Corydalis turtschaninovii and C. ternata, which have been mainly used as medicinal materials in Korea and China.
The multiplicity of all carbon signals were confirmed through DEPT experiment, while previous structural evidences were confirmed by several spectral data including COSY, gHMQC and gHMBC.
All purified alkaloids and a vigabatrin as a positive control were evaluated for inhibitory activity on GABA-T (Table 2).
Most alkaloids showed to have inhibitory activity on GABA-T and especially compound 5 (protopine) inhibited the activity by 64%, whereas none of purified alkaloids had any inhibitory effect on SSADH (data not shown). The significant relation between the inhibitory activity and chemical structures of the Fig. 1. Chemical structure of alkaloids from Corydalis tuber.
Table 2. The residual activity of GABA-T after the treatment with alkaloids isolated from Corydalis tuber
Compounds No 1 2 3 4 5 6 7 vigabatrin
Residual activity (%) 82± 1.6 54± 0.9 79± 1.2 54± 1.3 36± 0.4 71± 2.1 56± 1.1 63± 0.7 GABA-T was treated with 6 mM of each compound for 10 min prior to enzymes assays as described under “Materials and Methods”.
Activity of GABA-T treated with 1 mM vigabatrin is included as a positive control. The activity (%) was expressed as average of duplicate experiments within 5%.
alkaloids was not approved. Even though the inhibitory activities of the alkaloids were relatively lower than that of vigabatrin, the alkaloids seemed to be surely the principal components manifesting antispasmodic effects of Corydalis tuber. There has been little reported on naturally occurring compounds to bind to the active site of GABA-T and to behave as effective irreversible inhibitors of the enzyme.
Further studies to elucidate detailed kinetics and inhibitory mechanisms may provide a framework for designing a new class of therapeutic anticonvulsant.
Acknowledgments. This study was supported by the Brain Neuroscience Research Grant (M1-0108-00-0019) from the Ministry of Science and Technology, by Korea Health 21 R&D Project grant (02-PJ1-PG10-20706-0002) from Ministry of Health and Welfare, and by the BioGreen 21 Program from Rural Development Administration, Republic of Korea, and by a grant from the Korea Science &
Technology Foundation through Plant Metabolism Research Center, Kyung Hee University.
References
1. Fletcher, A. and Fowler, L. J. (1980) 4-aminobutyric acid metabolism in rat brain following chronic oral administra- tion of ethanolamine-O-sulfate. Biochem. Pharmacol. 29, 1451-1454.
2. Perry, T. L., Hansen, S. and Klister, M. (1973) Deficiency of 4-aminobutyric acid in the brain. New. Engl. J. Med.
288, 337-342.
3. Lloyd, K. G., Sherman, L. and Hornykiewicz, O. (1977) Distribution of high affinity sodium independent [3H]-γ-ami- nobutyrate binding in the human brain. Alteration in Parkin- son’s disease. Brain. Res. 127, 269-275.
4. De Biase, D., Barra, D., Bossa, F., Pucci, P. and John, R. H.
(1991) Chemistry of the inactivation of 4-aminobutyrate aminotransferase by the antiepileptic drug vigabatrin. J.
Biol. Chem. 266, 20056-20061.
5. Lippert, B., Metcalf, B. Y., Jung, M. J. and Casara, P.
(1977) 4-aminohex-5-enoic acid; a selective catalytic inhibi- tor of 4-aminobutyrate aminotransferase in mammalian brain. Eur. J. Biochem. 74, 441-445.
6. Soka, T. (1985) Dictionary of Chinese Drugs, Shanghai Sci- ence Technology Shogaukan (eds.) Shogakukan Press, Tokyo.
7. Loza, I., Orallo, F., Verde, I., Gil-Longo, J., Cadavid, I. and Calleja, J. M. (1993) A study of glaucine-induced relax- ation of rat aorta. Planta Med. 59, 229-231.
8. Matsuda, H., Shiomoto, H., Naruto, S., Namba, K. and Kubo, M. (1988) Anti-thrombic action of methanol extract and alkaloidal components from Corydalis tuber. Planta Med. 54, 27-33.
9. Matsuda, H., Tokuoka, K., Wu, J., Shiomoto, H. and Kubo, M. (1997) Inhibitory effects of dehydrocorydaline isolated from Corydalis tuber against type I-IV allergic models.
Biol. Pharm. Bul. 20, 431-434.
10. Slavik, J. and Slavikova, L. (1979) Alkaloids from Coryda- lis cava. Collection Czechoslov. Chem. Commun. 44, 2261- 2274.
11. Joshi, B. S., Haider, S. I. and Pelletier, S. W. (1990) Alka- loids of Corydalis ramosa. Planta Med. 56, 418-419.
12. Takao, N. (1990) On the alkaloids of genus Corydalis of the Papaveraceous plants. Kor. J. Pharmacog. 24, 1-19.
13. Jeon, S.G., Bahn, J.H., Jang, J.S., Park, J., Kwon, O.S., Cho, S.W., and Choi, S.Y. (2000) Human brain GABA tran- saminase; Genomic organization, Tissue distribution and Molecular expression Eur. J. Biochem. 267, 5601-5607.
14. Choi, S. Y., Kim, I., Jang, S. H., Lee, S. J., Song, M. S., Lee, Y. S. and Cho, S. W. (1993) Purification and proper- ties of GABA transaminase from bovine brain. Mol. Cells.
3, 397-401.
15. Krebs, K. G., Heusser, D. and Wimmer, H. (1969) Spray reagents. In Thin layer chromatography, pp. 873-874, Springer-Verlag, New York.
16. Dai-Ho, G. and Mariano, P. S. (1988) Exploratory, mecha- nistic, and synthetic aspects of silylarene-iminium salt SET photochemistry. Studies on diraidcal cyclization processes and applications to protoberberine alkaloid synthesis. J. Org.
Chem. 53, 5113-5127.
17. Hughes, D. W., Holland, H. L. and MacLean, D. B. (1976)
13C Magnetic resonance spectra of some isoquinoline alka- loids and related model compounds. Can. J. Chem. 54, 2252-2260.
18. Cushman, M. and Dekow, F. W. (1978) A total synthesis of corydaline. Tetrahedron 34, 1435-1439.
19. Iwasa, K., Gupta, Y. P. and Cushman, M. (1981) Absolute configurations of the cis- and trans-13-methyltetrahydropro- toberberines. Total synthesis of (+)-thalictricavine, (+)-cana- dine, (Û)-, (-)-, and (+)-thalictrifoline, and (Û)-, (-)-, and (+)-cavidine. J. Org. Chem. 46, 4744-4750.
20. Gentry, E. J., Jampani, H. B., Keshavarz-Shokri, A., Mor- ton, M. D., Vander, Velde, D., Telikepalli, H. and Mitscher, L. A. (1998) Antitubercular natural products: Berberine from the roots of commercial Hydrastis canadensis pow- der. Isolation of inactive 8-oxotetrahydrothalifendine, cana- dine, β-hydrastine, and two new quinic acid esters, hycandinic acid esters-1 and 2. J. Nat. Prod. 61, 1187-193.
21. Pyne, S. G. and Dikic, B. (1990) Diastereoselective addi- tions of (R)-(+)-methyl p-tolyl sulfoxide anion to imines.
Asymmetric synthesis of (R)-(+)-tetrahydropalmatine. J.
Org. Chem. 55, 1932-1936.
22. Hao, H. and Qicheng, F. (1986) Chemical study on alka- loids from Corydalis bulleyana. Planta Med. 52, 193-198.
23. Guinaudeau, H. and Shamma, M. (1982) The protopine alkaloids. J. Nat. Prod. 45, 237-246.
24. Eloumi-Ropivia, J. (1984) Isolation of glaucine from Art- abotrys lastourvillensis. J. Nat. Prod. 47, 1067.
25. Kerr, K. M., Kook, A. M. and Davis, P. J. (1986) High field and 2D-NMR studies with the aporphine alkaloid glaucine.
J. Nat. Prod. 49, 576-582.
