Isolation of Isoquinoline Alkaloids from the Tuber of Corydalis turtschaninovii and their Inhibition Activity on Low Density Lipoprotein Oxidation

Article J. Korean Soc. Appl. Biol. Chem. 52(6), 646-654 (2009) Isolation of Isoquinoline Alkaloids from the Tuber of and their Inhibition Activity on Low Density Lipoprotein Oxidation Corydalis turtschaninovii Jong-Ki Lee , Jin-Gyeong Cho , Myoung-Chong Song , Jong-Su Yoo , Dae-Young Lee , Hye-Joung Yang , Kyung-Min Han , Dong-Hyun Kim , Young-Jun Oh , Tae-Sook Jeong , and Nam-In Baek * 1 2 2 2 2 2 2 3 2 2 2 Department of Food Science and Technology, Youngnam University, Gyongsan 712-749, Republic of Korea 2 Graduate School of Biotechnology and Institute of Life Science & Resources, Kyung Hee University, Yongin 446-701, Republic of Korea 3 National Research Laboratory of Lipid Metabolism & Atherosclerosis, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea Received September 21, 2009; Accepted October 15, 2009 1 The dried and powdered tubers of ( ) were extracted with 80% aqueous MeOH, and the concentrated extracts were partitioned using the acidity and polarity of the solvents. From the alkaloid fraction, nine alkaloids were isolated through repeated SiO column chromatography. Based on NMR, MS and IR spectroscopic data, the chemical structures of the compounds were determined to be (±)-demethylcorydalmine (1), (+)-isocorydine (2), (+)-stylopine (3), (-)-α- -methylcanadine (4), pseudoprotopine (5), tetrahydroprotopapaverine (6), allocryptopine (7), berberine (8), and pseudocoptisine (9). This was the first report in which compounds 1, 2, 4, 6, and 7 were isolated from the tuber of . And because the NMR data for some compounds have been incorrectly or incompletely identified in previous literature, they were revised on the basis of 2D NMR experiments. Compounds 1, 2, and 3 showed inhibition activity on LDL oxidation with IC values of 2.1±0.2, 2.4±0.1, and 2.0±0.2 µM, respectively, which are higher activities than that of the positive control, probucol (6.8±0.1 µM). Key words: Corydalis turtschaninovii, isoquinoline alkaloid, LDL oxidation, nuclear magnetic Corydalis turtschaninovii C. turtschaninovii 2 N C. turtschaninovii 50 resonance spectrometry (NMR) Corydalis tuber, the tuber of C. turtschaninovii, C. fumariaefolia, C. ambigua, C. filistipes, and C. ternate [Jung and Shin, 1990], has been used not only as an *Corresponding author Phone: +82-31-201-2661; Fax: +82-31-201-2157 E-mail: nibaek@khu.ac.kr Abbreviations: c.c., column chromatography; C. turtschani- DEPT, distortionless enhancement by polarization transfer; EI-MS, electron ionization-mass spectrometry; EtOAc, ethyl acetate; HMBC, heteronuclear multiple-bond connectivity; HSQC, heteronuclear single-quantum correlation spectroscopy; IR, infrared spectroscopy; LDL, low density lipoprotein; MDA, malondialdehyde; m.p., melting point; MS, mass spectrometry; NMR, nuclear magnetic resonance spectrometry; PBS, phosphate-buffered saline; SiO2, silica gel; TBARS, thiobarbituric acid reactive substances; TLC, thin layer chromatography; Ve/Vt, elution volume/total volume novii, Corydalis turtschaninovii; doi:10.3839/jksabc.2009.108 analgesic but also for the treatment of gastric disease in the Korean traditional medicinal system [Jung and Shin, 1990]. Many reported pharmacological activities include the treatment of gastric and duodenal ulcer, cardiac arrhythmia [Kamigauchi and Iwasa, 1994], rheumatism, dysmenorrhea [Tang and Eisenbrand, 1992], memory dysfunction [Houghton et al., 2006; Hung et al., 2008], and antimicrobial activity [Feng et al., 2009]. Those activities are chiefly linked to several alkaloids such as berberine, canadine, corydaline, palmatine, protopine, stylopine, and tetrahydropalamtine [Sagara et al., 1985; Matsuda et al., 1988; Jung and Shin, 1990; Choi et al., 2003; Saito et al, 2004]. C. turtschaninovii BESS. (Fumariaceae) is a perennial herb native to India, Malaysia and other tropical areas. It is distributed in the central and northern regions of Korea and occurs on mountain districts everywhere in Korea. It reaches 20 cm high with wide elliptical leaves and is 3~4 cm in length. Inhibitory activity of alkaloids from Corydalis turtschaninovii The flower blooms in April, is 10 mm long and is a light reddish purple color. The flower forms tubers about 1 cm in diameter that are yellow inside. Several roots come out of the bottom part of the tuber. The main alkaloids of the plant have been reported as berberine, coptisine, corydaline, dehydrocorydaline, glaucine, palmatine, protopine, pseudocoptisine, pseudoprotopine, stylopine, and tetrahydropalamtine [Dai-Ho and Mariano, 1988; Kubo ., 1994; Khan and Sharma, 1997; Matsuda ., 1997; Choi ., 2003; Min ., 2006; Hung ., 2008]. In continuing to search for other active components from the tuber of , we isolated nine alkaloids, five of which were isolated from this plant for the first time. This paper describes the procedure for the isolation of the nine alkaloids using SiO2 column chromatography and structure determination of the alkaloids using spectroscopic methods such as NMR, IR, and MS. The alkaloids were evaluated for inhibition activity on LDL oxidation, which plays a very important role in the formation of atherosclerotic lesions [Diaz ., 1997]. This hypothesis is supported by some observational studies that demonstrate associations between oxidized LDL cholesterol and both the presence of atherosclerotic lesions [Regnstrom ., 1992] as well the progression of carotid artery atherosclerosis [Salonen ., 1992]. et al al et et al et al et al C. turtschaninovii et al et al et al Materials and Methods Plant materials. The tubers of were collected at Kyungdong Herb Medicine Market in Seoul in 2007 and were identified by Professor Dae-Keun Kim, Woosuk University, Jeonju, Korea. A voucher specimen (KHU070123) was reserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Suwon, Korea. General experimental procedures. The organic solvents for solid extraction and solvent fractionation were first-grade reagents supplied by Daejung Chemical Ltd. (Seoul, Korea). SiO2 resin used for column chromatography was Kiesel gel 60 (Merck, Darmstadt, Germany). TLC analysis was carried out using Kiesel gel 60 F254 and RP-18 F254S (Merck) and detected using a UV lamp, Spectroline Model ENF-240 C/F (Spectronics Corporation, New York, NY) and a 10% H2SO4 solution. The melting point was determined on a Fisher-John’s apparatus and was not corrected. Optical rotation was measured on a JASCO P-1010 digital polarimeter (Tokyo, Japan). IR spectrum was obtained from a Perkin Elmer Spectrum One FT-IR spectrometer (Buckinghamshire, England). EI-MS was recorded on a JEOL JMSAX-700 (Tokyo, Japan). 1H-NMR (400 MHz) and 13C-NMR (100 C. turtschaninovii on low density lipoprotein oxidation 647 MHz) spectra were recorded on a Varian Unity Inova AS400 FT-NMR spectrometer (Palo Alto, CA). Isolation of alkaloids from the tuber of . The dried and coarsely powdered plant C. turtschaninovii materials (3 kg) were extracted with 80% aqueous MeOH (4 L×2) at room temperature overnight and were then filtered through filter paper. The filtrate was evaporated giving a dark brownish residue. The resultant methanolic extract was poured into acidic water (pH 2.5, 1.5 L), the acidity of which was adjusted using 30% HCl, and washed twice with EtOAc (1.5 L×2). The aqueous layer was alkalized to pH 11.5 using a 20% NaOH solution and extracted with EtOAc (1.5 L×2). The concentrated organic layer (CTE, 9.3 g) was subjected to column chromatography (6×8 cm) over SiO2 (70-230 mesh, 250 g) using CHCl3-MeOH (15:1→13:1→10:1→ 7:1→5:1→3:1, each 1.5 L) and MeOH (1 L) as eluting solvents in increasing order of polarity to give 12 fractions (CTE1~CTE12), the fourth one [1, CTE4, 290 mg, Ve/Vt 0.25-0.45 in CHCl3-MeOH=10:1, TLC (Kieselgel 60 F254) Rf 0.7, CHCl3-EtOH=1:1] of which was identified as (±)-demethylcorydalmine. The third fraction (CTE3, 900 mg, Ve/Vt 0.70-0.80 in CHCl3-MeOH=13:1) was applied to SiO2 column chromatography (80 g, 3×6 cm) using hexane-EtOAc (1:4, 2 L) as the eluent to yield five fractions (CTE3-1~CTE3-5) including a pure (+)-isocorydine [2, CTE3-5, 290 mg, Ve/Vt 0.80-0.95, TLC (Kiesel gel 60 F254) Rf 0.3, -hexane-EtOAc=1:5]. Fraction CTE3-1 (411 mg, Ve/Vt 0.05-0.22) was subjected to chromatography on a SiO2 column (80 g, 3×6 cm) eluting with -hexaneEtOAc (5:1, 3 L) giving seven fractions (CTE3-1-1~ CTE3-1-7) including the third (3, CTE3-1-3, 65 mg, Ve/ Vt 0.28-0.37, TLC (Kiesel gel 60 F254) Rf 0.42, -hexaneEtOAc=3:1) and fourth (4, CTE3-1-4, 125 mg, Ve/Vt 0.38-0.52, TLC (Kiesel gel 60 F254) Rf 0.35, -hexaneEtOAc=3:1) fractions, which were identified as (+)stylopine and (-)-α- -methylcanadine, respectively. Fraction CTE6 (705 mg, Ve/Vt 0.40-0.65 in CHCl3-MeOH=7:1) was applied to a SiO2 column (150 g, 5×10 cm) using CHCl3-MeOH (5:2, 2 L) as the eluent resulting in nine fractions (CTE6-1~CTE6-9), the seventh [5, CTE6-7, 155 mg, Ve/Vt 0.70-0.80, TLC (Kieselgel 60 F254) Rf 0.30, CHCl3-MeOH=1:1] of which was identified as pseudoprotopine. Fraction CTE7 (675 mg, Ve/Vt 0.650.80 in CHCl3-MeOH=7:1) was applied to a SiO2 column (100 g, 4.5×10 cm) using CHCl3-MeOH (2:1, 1.5 L) as the eluent and resulted in eight fractions (CTE7-1~ CTE7-8), the fifth [6, CTE7-5, 338 mg, Ve/Vt 0.35-0.45, TLC (Kieselgel 60 F254) Rf 0.35, CHCl3-MeOH=2:1] and the seventh [7, CTE7-7, 153 mg, Ve/Vt 0.70-0.80, TLC (Kieselgel 60 F254) Rf 0.25, CHCl3-MeOH=2:1] fractions were respectively identified as tetrahydroprotopapaverine in vacuo n n n n n N 648 Jong-Ki Lee et al. and allocryptopine. Fraction CTE8 (2.4 g, Ve/Vt 0.200.55 in CHCl -MeOH=5:1) was subjected to SiO column chromatography (250 g, 5.5×13 cm) eluting with CHCl MeOH-H O (15:3:1, lower layer, 2.5 L) to give nine fractions (CTE8-1~CTE8-9) including a pure berberine [8, CTE8-5, 124 mg, Ve/Vt 0.4-0.55, TLC (Kieselgel 60 F ) R 0.50, CHCl -MeOH-H O=12:3:1, lower layer]. Fraction CTE11 (1.7 g, Ve/Vt 0.35-0.75 in MeOH) was applied to a SiO column (200 g, 5×10 cm) eluting with CHCl -MeOH (15:1→10:1→7:1→5:1→3:1, each 500 mL) giving eight fractions (CTE11-1~CTE11-8); the sixth fraction (9, CTE11-6, 380 mg, Ve/Vt 0.25-0.75 in CHCl MeOH=5:1, TLC (Kiesel gel 60 F ) R 0.30, CHCl MeOH=5:1) was identified as pseudocoptisine. Compound 1 [(±)-demethylcorydalmine]. Amorphous powder (CHCl -MeOH); m.p. 142 C (decomposed); − [ α ] 16 D 0 (c=0.22, CH OH); IR (KBr, cm ) 3347, 2842, 1590; EI-MS m/z 327 [M] , 312, 296, 136; H-NMR (400 MHz, CD OD, δ ) 6.74 (1H, d, J=8.2 Hz, H-12), 6.65 (1H, d, J=8.2 Hz, H-11), 6.59 (1H, s, H-1), 6.48 (1H, s, H-4), 4.14 (1H, d, J=15.1 Hz, H-8a), 3.85 (3H, s, 2OMe), 3.82 (3H, s, 3-OMe), 3.46 (1H, dd, J=11.7, 3.4 Hz, H-14), 3.45 (1H, d, J=15.1 Hz, H-8b), 3.20 (1H, dd, J=15.8, 11.7 Hz, H-13a), 3.14 (1H, m, H-6a), 3.08 (1H, m, H-5a), 2.84 (1H, dd, J=15.8, 3.4 Hz, H-13b), 2.62 (1H, m, H-5b), 2.58 (1H, m, H-6b); C-NMR (100 MHz, CD OD, δ ) See Table 1. Compound 2 [(+)-isocorydine]. Colorless needles (MeOH); m.p. 185-186 C; [ α ] 16D +209.8 (c=0.27, CHCl ), +205 [Lu et al., 1989]; IR (KBr, cm− ) 3355, 1606; EIMS m/z 341 [M] , 340, 326, 298, 283, 267; H-NMR (400 MHz, CDCl , δ ) 6.71 (1H, d, J=8.2 Hz, H-8), 6.67 (1H, d, J=8.2 Hz, H-9), 6.56 (1H, s, H-3), 3.79 (1H, dd, J=12.8, 4.4 Hz, H-6a), 3.77 (3H, s, 2-OMe), 3.74 (3H, s, 10-OMe), 3.57 (3H, s, 1-OMe), 3.03 (1H, m, H-5a), 2.92 (1H, m, H-4a), 2.85 (1H, dd, J=11.0, 5.5 Hz, H-5b), 2.70 (1H, m, H-7a), 2.53 (1H, m, H-4b), 2.41 (1H, m, H-7b), 2.38 (3H, s, N-Me); C-NMR (100 MHz, CDCl , δ ) See Table 1. Compound 3 [(+)-stylopine]. Colorless prisms (EtOHCHCl ); m.p. 209-212 C; [ α ] 16D +297° (c=0.18, CHCl ), +309 [Choi et al., 2003]; IR (KBr, cm− ) 2922, 2805, 2750, 1509, 1487, 1466; EI-MS m/z 323 [M] , 308, 174, 148; H-NMR (400 MHz, CDCl , δ ) 6.70 (1H, s, H-1), 6.76 (1H, d, J=8.0 Hz, H-12), 6.60 (1H, d, J=8.0 Hz, H11), 7.57 (1H, s, H-4), 5.93 (1H, d, J=1.6 Hz, -O-CH -O-), 5.90 (1H, d, J=1.6 Hz, -O-CH -O-), 5.89 (2H, s, -O-CH O-), 4.07 (1H, d, J=15.2 Hz, H-8a), 3.54 (1H, dd, J=3.2, 11.0 Hz, H-14), 3.51 (1H, d, J=15.2 Hz, H-8b), 3.20 (1H, dd, J=3.2, 15.8 Hz, H-13a), 3.13 (1H, m, H-6a), 3.07 (1H, m, H-5a), 2.78 (1H, dd, J=11.0, 15.8 Hz, H-13b), 2.63 (1H, m, H-5b), 2.58(1H, m, H-6a); C-NMR (100 MHz, 3 2 3 2 254 f 3 2 2 3 3 254 f 3 o 3 1 3 + 3 1 H 13 3 C o o 3 o 1 + 3 1 H 13 3 C o 3 3 o 1 + 1 3 H 2 2 2 13 CDCl , δ ) See Table 1. Compound 4 [(-)-cis-α-N-methylcanadine]. Colorless 24 3 C crystals (CHCl ); m.p. 255-256 C; [ α ] D -128 (c = 0.21, MeOH), -125 [Binutu and Cordell, 2000]; IR (KBr, cm− ) 2998, 2805, 1525; EI-MS m/z 354 [M] , 353, 339, 174, 164, 149; H-NMR (400 MHz, CDCl , δ ) 6.82 (1H, d, J=8.4 Hz, H-12), 6.74 (1H, d, J=8.4 Hz, H-11), 6.68 (1H, s, H-1), 6.54 (1H, s, H-4), 5.86 (2H, s, -O-CH -O-), 4.20 (1H, d, J=15.6 Hz, H-8a), 3.81 (3H, s, 9-OMe), 3.80 (3H, s, 10-OMe), 3.50, (1H, d, J=15.6 Hz, H-8b), 3.49 (1H, m, H-14), 3.33 (3H, s, N-Me), 3.19 (1H, dd, J=15.8, 5.5 Hz, H-13a), 3.14 (1H, m, H-6a), 3.06 (1H, m, H-5a), 2.79 (1H, dd, J=15.8, 5.5 Hz, H-13b), 2.63 (1H, m, H-5b), 2.59 (1H, m, H-6b); C-NMR (100 MHz, CDCl , δ ) See Table 1. Compound 5 (pseudoprotopine). Colorless crystals (CHCl -MeOH); m.p. 199-200 C; IR (KBr, cm− ) 1679, 1618, 1495; EI-MS m/z 353 [M] , 338, 322, 281, 267, 252, 193, 148; H-NMR (400 MHz, CDCl , δ ) 6.88 (1H, s, H-4), 6.66 (1H, s, H-1), 6.47 (1H, s, H-12), 6.62 (1H, s, H-9), 5.93 (2H, s, -O-CH -O-), 5.90 (2H, s, -O-CH -O-), 3.67-3.80 (2H, m, H-13), 3.47-3.62 (2H, m, H-8), 2.57 (2H, m, H-5), 2.45 (2H, m, H-6); C-NMR (100 MHz, CDCl , δ ) See Table 1. Compound 6 (tetrahydroprotopapaverine). Colorless crystals (CHCl ); m.p. 154-155 C; [ α ] 24D 0 (c=0.17, MeOH); IR (KBr, cm− ) 3380, 1600, 1515; EI-MS m/z 333 [M] , 181, 151; H-NMR (400 MHz, CDCl , δ ) 6.71 (1H, d, J=1.6 Hz, H-2'), 6.69 (1H, d, J=8.4 Hz, H-5'), 6.54 (1H, dd, J=1.6, 8.4 Hz, H-6'), 6.51 (1H, s, H-5), 6.32 (1H, s, H-8), 3.81 (3H, s, 4'-OMe), 3.80 (3H, s, 3'-OMe), 3.66 (1H, dd, J=6.0, 6.0 Hz, H-1), 3.16 (1H, ddd, J=15.4, 6.7, 4.5 Hz, H-3a), 2.81 (1H, m, H-4a), 3.02 (1H, dd, J=14.4, 6.0 Hz, H-9a), 2.77 (1H, dd, J=14.4, 6.0 Hz, H-9b), 2.73 (1H, m, H-3b), 2.57 (1H, m, H-4b), 2.42 (3H, s, N-Me); C-NMR (100 MHz, CDCl , δ ) See Table 1. Compound 7 (allocryptopine). Colorless prisms (CHCl EtOH); m.p. 162-163 C; IR (KBr, cm− ) 3005, 1655, 1504; EI-MS m/z 369 [M] , 354, 338, 206, 164, 149; HNMR (400 MHz, CDCl , δ ) 6.88 (1H, s, H-1), 6.84 (1H, d, J=8.4 Hz, H-11), 6.73 (1H, d, J=8.4 Hz, H-12), 6.56 (1H, s, H-4), 5.85 (2H, s, -O-CH -O-), 3.93 (2H, br. s, H8), 3.78, 3.71 (both 3H, each s, 9-OMe, 10-OMe), 3.68 (2H, br. s, H-13), 2.68 (2H, br. s, H-5), 2.54 (2H, br. s, H6), 1.79 (3H, s, N-Me); C-NMR (100 MHz, CDCl , δ ) See Table 1. Compound 8 (berberine). Yellow crystals (CHCl EtOH); m.p. 160-161 C; IR (KBr, cm− ) 2980, 1686, 1636, 1200; EI-MS m/z 336 [M] , 321, 320, 306, 304, 292, 278; (400 MHz, CD OD, δ ) 9.75 (1H, s, H-8), 8.67 (1H, s, H-13), 8.09 (1H, d, J=9.2 Hz, H-11), 7.97 (1H, d, J=9.2 Hz, H-12), 7.63 (1H, s, H-1), 6.94 (1H, s, H-4), o o 3 o 1 + 1 3 H 2 13 3 o C 1 3 + 1 3 H 2 2 13 3 C o o 3 1 + 1 3 H 13 3 C 3 o 1 + 3 1 H 2 13 3 C 3 o 1 + 3 H Inhibitory activity of alkaloids from 6.09 (2H, s, -O-CH -O-), 4.92 (2H, t-like, J=6.4 Hz, H-6), 4.19 (3H, s, O-Me), 4.09 (3H, s, O-Me), 3.24 (2H, t-like, J=6.4 Hz, H-5); C-NMR (100 MHz, CD OD, δ ) See Table 1. Compound 9 (pseudocoptisine). Yellow crystals (CHCl -EtOH); m.p. 280-281 C (decomposed); IR (KBr, cm ) 2978, 1605, 1511; EI-MS m/z 320 [M] , 306, 193, 148; H-NMR (400 MHz, CD OD, δ ) 9.72 (1H, s, H-8), 8.71 (1H, s, H-13), 7.85 (2H, s, H-1, H-9), 7.63 (1H, s, H12), 6.94 (1H, s, H-4), 6.45 (2H, s, -O-CH -O-), 6.09 (2H, s, -O-CH -O-), 4.88 (2H, t-like, J=6.7 Hz, H-6), 3.24 (2H, m, H-5); C-NMR (100 MHz, CD OD, δ ) See Table 1. 2 13 3 C o 3 -1 + 1 3 H 2 2 13 Low-density lipoprotein isolation and oxidation assay. 3 C Plasma was obtained from fasted healthy normolipidemic volunteers. The LDL was isolated by a standard procedure with a slight modification [Havel et al., 1955]. The TBARS assay of Buege and Aust [Buege and Aust, 1978] was used with a slight modification. Consequently, an LDL solution (250 µL, 50-100 µg of protein) in 10 mM PBS (pH 7.4) was supplemented with 10 µM CuSO . The oxidation was performed in a screw-capped 5 mL glass vial at 37 C in a shaking water bath. After a four h incubation, the reaction was terminated by the addition of 1 mL 20% trichloroacetic acid. Following precipitation, 1 mL of 0.67% thiobarbituric acid in 0.05 N NaOH was added and vortexed, and the final mixture was heated for five min at 95 C, cooled on ice, and centrifuged for two min at 1000×g. The optical density of the produced MDA was measured at 532 nm. Calibration was completed with an MDA standard prepared from tetramethoxypropane. 4 o o Results and Discussion The tubers of C. turtschaninovii were extracted in 80% MeOH, and the obtained extracts were partitioned using the acidity and polarity of the solvents to produce the alkaloid fraction. The repeated SiO column chromatography for the alkaloid fraction yielded nine purified alkaloids, 19. Compounds 3, 5, 8, and 9 have been previously isolated from this plant and identified as (+)-stylopine [Dai-Ho and Mariano, 1988; Choi et al., 2003], pseudoprotopine [Kahn and Sharma, 1997], berberine [Min et al., 2006], and pseudocoptisine [Hung et al., 2008], respectively, through comparison of physicochemical data with those reported in the literature. Compounds 1, 2, 4, 6, and 7 were isolated for the first time from the tuber of C. turtschaninovii. When all of the isolated compounds were developed on a SiO TLC plate, they exhibited absorbance under UV light and were visualized by spraying with a Dragendorff’s reagent, resulting in an orange spot, indicating that they were alkaloids. 2 2 on low density lipoprotein oxidation Corydalis turtschaninovii 649 Compound 1, a white amorphous powder, showed absorbance bands due to the hydroxy group (3347 cm− ) and the double bond (1590 cm− ) in the IR spectrum. The molecular weight was determined as 327 from the molecular ion [M] at m/z 327 in the EI-MS spectrum. In the H-NMR (400 MHz, CD OD) spectrum, two singlet olefin methine (δ 6.59, δ 6.48), two doublet olefin methine showing vicinal coupling (δ 6.74, J=8.2 Hz; δ 6.65, J=8.2 Hz) and two methoxy (δ 3.85, δ 3.82) proton signals were observed. In the high magnet field, one nitrogenated-methine (δ 3.46, dd, J=11.7, 3.4 Hz), two nitrogenated-methylene [(δ 4.14, 1H, d, J=15.1 Hz; δ 3.45, 1H, d, J=15.1 Hz), (δ 3.14, 1H, m; δ 2.58, 1H, m)], and two methylene [(δ 3.20, 1H, dd, J=15.8, 11.7 Hz; δ 2.84, 1H, dd, J=15.8, 3.4 Hz), (δ 3.08, 1H, m; δ 2.62, 1H, m)] proton signals were identified. The above described H-NMR data allowed the assumption that compound 1 was a protoberberine-type isoquinoline alkaloid. The C-NMR (100 MHz, CD OD) spectrum showed 19 carbon signals: four oxygenated-olefine quaternary [δ 145.14, δ 144.03 (×2), δ 141.51], four olefin quaternary (δ 130.58, δ 128.22, δ 126.12, δ 121.23) four olefin methine (δ 119.42, δ 111.53, δ 110.68, δ 109.04), two methoxy (δ 56.21, δ 55.90), one nitrogenated-methine (δ 59.16), two nitrogenatedmethylene (δ 53.59, δ 51.64) and two methylene (δ 36.34, δ 29.20) carbon signals. The chemical shifts and the coupling patterns in the H- and C-NMR data corresponded to those of a protoberberine-type alkaloid, demethylcorydalmine [Haisova and Slavik, 1973; Rucker et al., 1994], which has been previously isolated from Argemone ochroleuca [Haisova and Slavik, 1973]. And the specific rotation of compound 1 was zero, suggesting that the compound was a mixture of trans- and cisconfigurations between H-14 and the non-sharing electron pairs of nitrogen. Consequently, compound 1 was identified as (±)-demethylcorydalmine. Compound 2, colorless needles, showed absorbance bands due to the hydroxy group (3355 cm− ) and the double bond (1606 cm− ) in the IR spectrum. The molecular ion peak [M] at m/z 341 indicated the molecular weight to be 341. In the H-NMR (400 MHz, CDCl ) spectrum, one singlet olefin methine (δ 6.56), two doublet olefin methine showing vicinal coupling (δ 6.71, J=8.2 Hz; δ 6.67, J=8.2 Hz) and three methoxy (δ 3.77, δ 3.74, δ 3.57) proton signals were observed. In the high magnet field, one nitrogenated-methine (δ 3.79, dd, J=12.8, 4.4 Hz), one nitrogenated-methylene (δ 3.03, 1H, m; δ 2.85, 1H, dd, J=11.0, 5.5 Hz), one nitrogenatedmethyl (δ 2.38), and two methylene [(δ 2.92, 1H, m; δ 2.53, 1H, m), (δ 2.70, 1H, m; δ 2.41, 1H, m)] proton signals were identified. The H-NMR data suggested that 1 1 + 1 3 H H H H H H H H H H H H H H H 1 13 3 C C C C C C C C C C C C C C C C C C 1 13 1 1 + 1 3 H H H H H H H H H H H H H 1 H 650 Jong-Ki Lee et al. Fig. 1. Chemical structures of the alkaloids from the tuber of compound 2 was an aporphine-type isoquinoline alkaloid. The 13C-NMR spectrum showed 20 carbon signals including three methoxy carbon signals (δC 61.63, δC 55.67, δC 55.40). In the low magnet field, four oxygenated-olefin quaternary (δC 150.67, δC 148.86, δC 143.36, δC 141.51), five olefin quaternary (δC 129.58, δC 129.53, δC 128.52, δC 125.32, δC 119.64), and three olefin methine (δC 118.62, δC 110.47, δC 110.23) carbons signals were observed. Except for them, one nitrogenatedmethine (δC 62.43), one nitrogenated-methylene (δC 52.27), one nitrogenated-methyl (δC 43.49), and two methylene (δC 35.44, δC 28.87) carbon signals were assigned. The 1H- and 13C-NMR spectra were suggestive of a tetrasubstituted aporphine alkaloid [Jackman et al, 1979]. The stereochemistry at C-6a was determined as R from the specific rotation value, +209.8o [Guinaudeau et al, 1975] and the large coupling constant of H-6a (J=12.8, 4.4 Hz) [Hasan et al., 2000]. Ultimately, compound 2 was identified as (+)-isocorydine. Compound 4, colorless crystals, showed an absorbance band of the double bond (1525 cm−1) in the IR spectrum. The EI-MS spectrum showed the molecular ion peak [M]+ at m/z 354. The 1H-NMR (400 MHz, CDCl3) and 13 C-NMR spectra were similar to those of compound 4 C. turtschaninovii. with the exception of a dioxymethylene (δH 5.86, 2H, s; δC 100.69, t) and a quaternary-N-methyl group (δH 3.33, 3H, s; δC 50.31, q). The comparison of the NMR data with those in the literature [Binutu and Cordell, 2000] led us to identify compound 4 as N-methylcanadine. Usually, quaternary-N-methyl shows a chemical shift in the 1HNMR at ca δH 2.90 for trans-configuration of the B/C fused ring and at ca δH 3.30 for cis-configuration [Binutu and Cordell, 2000], respectively, indicating compound 4 to have cis-configuration from the chemical shift of Nmethyl, δH 3.33. And the absolute stereostructure of C-14 and N-7 were determined as S and S from the optical rotation, -128o [Binutu and Cordell, 2000]. Consequently, compound 4 was identified as (-)-cis-α-N-methylcanadine. Compound 6, colorless crystals, showed a molecular ion peak [M]+ at m/z 333 in the EI-MS spectrum and absorbances due to the hydroxy (3380 cm−1) and the olefin (1515 cm−1) groups in the IR spectrum. In the 1HNMR (400 MHz, CDCl3) spectrum, five olefin methine protons, two of which were due to one 1,2,4,5tetrasubstituted benzene ring (δH 6.51, s; δH 6.32, s) and three of which due to a 1,2,4-trisubstituted benzene ring (δH 6.71, d, J=1.6 Hz; δH 6.69, d, J=8.4 Hz; δH 6.54, dd, J=1.6, 8.4 Hz), and two methoxy (δH 3.81, 3H, s; δH 3.80, Inhibitory activity of alkaloids from Corydalis turtschaninovii Table 1. C-NMR data of alkaloids from the tuber of 13 on low density lipoprotein oxidation C. turtschaninovii (100 MHz) No of C 1 2 3 4 5 6 1 111.53 141.51 105.43 105.38 106.66 64.43 2 144.03 150.67 144.82 145.84 146.19 3 145.14 110.47 145.82 146.02 147.87 46.53 4 110.68 28.87 106.67 108.30 108.10 24.82 5 29.20 52.27 29.56 29.32 32.23 110.51a 6 51.64 51.83 51.30 58.63 145.24b 7 35.44 143.21b 8 53.59 118.62 52.89 53.78 50.81 113.73 9 141.51 110.23 146.02 144.86 110.40 40.90 10 144.03 148.86 143.11 150.12 145.87 11 109.04 143.36 108.33 110.97 145.75 12 119.42 120.93 123.79 124.97 13 36.34 36.45 36.13 46.49 14 59.16 59.73 59.55 194.20 1' 132.84 2' 115.58 3' 145.11c 4' 144.98c 5' 110.39a 6' 120.74 1a 125.32 3a 128.52 4a 126.12 127.61 127.48 136.02 124.85 6a 62.43 7a 129.53 8a 121.23 127.61 128.11 117.79 129.84 11a 119.64 12a 128.22 128.39 127.34 128.87 14a 130.58 130.53 130.37 132.65 1b 129.58 N-Me 43.49 50.31 41.48 42.25 O-Me 56.21 61.63 60.13 55.83 O-Me 55.90 55.67 55.83 55.76 O-Me 55.40 100.95 100.69 101.14 O-CH2-O 100.70 100.79 O-CH2-O Compounds 1, 8, and 9 were dissolved in CD3OD, and compounds 2-7 in CDCl3. a, b, c: The chemical shifts with the same characters were exchangeable. 3H, s) proton signals were observed. In the high magnet field, one nitrogenated-methine (δ 3.66, dd, J=6.0, 6.0 Hz), one nitrogenated-methylene (δ 3.16, ddd, J=15.4, 6.7, 4.5 Hz; δ 2.73, m), one nitrogenated-methyl (δ 2.42, 3H, s), and two methylene [(δ 2.81, m; δ 2.57, m), (δ 3.02, dd, J=14.4, 6.0 Hz; δ 2.77, dd, J=14.4, 6.0 Hz)] proton signals were identified. The above mentioned proton signals led to the assumption that compound 6 was a hydrogenated-benzylisoquinoline alkaloid. The CNMR spectrum showed 19 carbon signals including two H H H H H H H H 13 651 7 108.90 145.74 147.73 110.10 32.01 57.22 50.06 146.94 151.22 110.34 127.37 45.87 190.98 132.40 128.09 129.04 135.40 41.03 60.57 55.44 100.96 - 8 106.44 149.73 151.99 109.29 28.21 57.17 145.56 146.24 151.84 127.88 124.40 121.72 139.47 131.74 123.17 134.97 121.35 62.53 57.61 103.58 - 9 121.25 147.62 148.62 105.21 27.01 56.04 144.03 108.15 144.47 147.93 112.38 121.85 150.81 130.57 120.70 121.05 133.11 104.58 102.45 methoxy carbon signals (δ 55.83, δ 55.76). In the low magnet field, four oxygenated-olefin quaternary (δ 145.24, δ 145.11, δ 144.98, δ 143.21), three olefin quaternary (δ 132.84, δ 129.84, δ 124.85), and five olefin methine (δ 120.74, δ 115.58, δ 113.73, δ 110.51, δ 110.39) carbon signals were observed. Except for them, one nitrogenated-methine (δ 64.43), one nitrogenated-methylene (δ 46.53), one nitrogenatedmethyl (δ 42.25), and two methylene (δ 40.90, δ 24.82) carbon signals were assigned. The position of two C C C C C C C C C C C C C C C C C C C 652 Jong-Ki Lee et al. Table 2. Inhibition activity on LDL oxidation of the alkaloids from the tuber of Compounds 1 2 3 4 5 6 (5 µg/mL) inhibition rate (%)* .82±0.3 .72±0.6 .74±0.3 43±0.2 29±0.5 25±0.3 IC50 (µM)* 2.1±0.2 2.4±0.1 2.0±0.2 *The data are presented as the mean±standard deviation of three replications. **Probucol was used as a positive control. methoxy groups were determined at C-3' and C-4' by HMBC experiments as well as with comparison of NMR data with those in the literature [Debourges et al., 1987; Cui et al., 2007]. The characteristics of being optically inactive, a specific rotation value of zero, indicated that compound 6 was a racemic mixture of the enantiomers. Finally, compound 6 was identified as tetrahydroprotopapaverine. Compound 7, colorless prisms, showed absorbance bands due to the conjugated carbonyl (1655 cm−1) and the double bond (1504 cm−1) in the IR spectrum. The molecular weight was determined to be 369 from the molecular ion [M]+ at m/z 369 in the EI-MS spectrum. In the 1H-NMR (400 MHz, CDCl3) spectrum, two singlet olefin methine (δH 6.88. δH 6.56), two doublet olefin methine showing vicinal coupling with each other (δH 6.84, δH 6.73, d, J=8.4 Hz), one dioxygenated-methylene (δH 5.85, 2H, s), and two methoxy (δH 3.78, δH 3.71) protons signals were observed. In the high magnet field, two nitrogenated-methylene [δH 3.93, 2H, br. s; δH 2.54, 2H, br. s), one nitrogenated-methyl (δH 1.79), and two allyl methylene (δH 3.68, 2H, br. s; δH 2.68, 2H, br. s) proton signals were identified. The above described 1HNMR data allowed the assumption that compound 7 was a protopine-type isoquinoline alkaloid. The 13C-NMR (100 MHz, CDCl3) spectrum showed 21 carbon signals including one dioxygenated-methylene (δC 100.96) and two methoxy (δC 60.57, 55.44) carbon signals. The other 18 carbon signals included one ketone (δC 190.98), four oxygenated-olefin quaternary (δC 151.22, δC 147.73, δC 146.94, δC 145.74], four olefin quaternary (δC 135.40, δC 132.40, δC 129.04, δC 128.09), and four olefin methine (δC 127.37, δC 110.34, δC 110.10, δC 108.90) carbon signals in the low magnet field, and two nitrogenated-methylene (δC 57.22, δC 50.06), one nitrogenated-methyl (δC 41.03), and two methylene (δC 45.87, δC 32.01) carbon signals in the high magnet field. The position of a dioxygenatedmethylene and two methoxy groups were determined to be between C-2 and C-3, and at C-9 and C-10, respectively, using HMBC experiments. The proton signal of dioxymethylene (δH 5.85) showed a correlation with C-2 (δC 145.74) and C-3 (δC 147.73), and two methoxy proton signals (δH 3.78, δH 3.71), respectively, correlated with C- C. turtschaninovii 7 8 9 Probucol** 34±0.2 - 11±0.1 - 38±0.3 - .85±0.4 6.8±0.1 10 (δC 151.22) and C-9 (δC 146.94). Consequently, compound 7 was identified through comparison of spectroscopic data with those in the literature [Isawa et al., 1982; Takahashi et al., 1985] as allocryptopine, a protopine-type alkaloid, which has been previously isolated from Argemone mexicana [Chang et al., 2003]. The nine alkaloids isolated from the tuber of C. turtschaninovii were evaluated for inhibition of LDLoxidation activity. The oxidation of LDL cholesterol is an important step in the formation of atherosclerotic lesions [Diaz et al., 1997]. This hypothesis is supported by some observational studies that demonstrate associations between oxidized LDL cholesterol and both the presence of atherosclerotic lesions [Regnstrom et al., 1992] as well the progression of carotid artery atherosclerosis [Salonen et al., 1992]. Compounds 1-3 showed high inhibition activities with inhibitions of 82±0.3, 72±0.6, and 74±0.3 % at 5 µg/mL, respectively; compounds 4 and 9 had moderate activities with inhibitions of 43±0.2 and 38± 0.3%, respectively; and compounds 5-8 had inhibitions of less than 29±0.5% (Table 2). Compounds 1-3 demonstrated LDL antioxidant activities with IC50 values of 2.1±0.2, 2.4±0.1, and 2.0±0.2 µM, respectively, which were about three times higher than that of the positive control, probucol, which had an IC50 value of 6.8±0.1 µM in this experiment. And their activity were also higher than those of quinoline alkaloids, 3,8-dihydroxyquinoline and 2,8dihydroxy-3,4-dimethoxyquinoline, from Scolopendra subspinipes [Yoon et al., 2006]. Therefore, the alcohol extracts or some alkaloids from Corydalis tuber could be developed as functional food or raw materials for medicine to prevent or remedy atherosclerosis. Acknowledgment. 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