Fitoterapia 103 (2015) 122–128
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Bioactive carbazole alkaloids from the stems of Clausena lansium Yi-Qian Du a, Hang Liu a,b, Chuang-Jun Li a, Jie Ma a, Dan Zhang a, Li Li a, Hua Sun a, Xiu-Qi Bao a, Dong-Ming Zhang a,⁎ a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People's Republic of China b Department of Pharmacy, Affiliated Drum Tower Hospital of Nanjing University, Zhongshan Road 321, Nanjing 210008, Jiangsu Province, People's Republic of China
a r t i c l e
i n f o
Article history: Received 25 December 2014 Accepted in revised form 16 March 2015 Keywords: Clausena lansium Claulansine L-R Anti-inflammatory Hepatoprotective
a b s t r a c t Seven new carbazole alkaloids, claulansines L-R (1–7), and six known analogues (8–13) were isolated from the stems of Clausena lansium. Their structures were elucidated on the basis of spectroscopic analyses, including UV, IR, and NMR experiments (HSQC, HMBC, and NOE experiment). Compound 7 showed moderate anti-inflammatory activities. Compounds 3, 5, 6, 8, and 12 exhibited moderate hepatoprotective activities. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental
Clausena lansium Skeels (Wampee) is a tropical species of the Rutaceae family, which is widely distributed in southern China. In traditional Chinese medicine, the leaves and roots of C. lansium are used for cough, asthma, dermatological disease, viral hepatitis, and gastro-intestinal diseases; the seeds are used to treat acute and chronic gastro-intestinal inflammation, ulcers, etc [1]. Previous phytochemical investigations have demonstrated that this plant contains coumarins, amides, carbazole alkaloids, and several other components [2–4]. Our group has also reported 17 carbazole alkaloids [5] and 17 furanocoumarins [6] from the stems of C. lansium, and several of these compounds showed selective neuroprotective effects. In a continuing search for microscale bioactive metabolites from the same parts of this plant, 200 kg stems of C. lansium were collected in Guangxi province, China. In the present investigation, 7 new (1–7) and six known carbazole alkaloids (8–13) were isolated (Fig. 1). In this paper, the isolation and structure elucidation of the new compounds as well as the biological evaluation of these compounds are reported.
2.1. General
⁎ Corresponding author. Tel./fax: +86 10 63165227. E-mail address: [email protected] (D.-M. Zhang).
http://dx.doi.org/10.1016/j.fitote.2015.03.018 0367-326X/© 2015 Elsevier B.V. All rights reserved.
Optical rotations were measured on a JASCO P2000 automatic digital polarimeter. UV spectra were recorded on a JASCO V-650 spectrophotometer, CD spectra were measured on a JASCO J-815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 spectrometer using an FT-IR microscope transmission method. NMR spectra were acquired with Bruker AVIIIHD 600, VNS-600, and Mercury-400 spectrometers in DMSO-d6. HRESIMS spectra were collected on an Agilent 1100 series LC/ MSD ion trap mass spectrometer. MPLC system was composed of two C-605 pumps (Büchi), a C-635 UV detector (Büchi), a C660 fraction collector (Büchi), and an ODS column (450mm × 60 mm, 50 μm, 400 g; YMC). Semipreparative HPLC was conducted using a Shimadzu LC-6AD instrument with an SPD-20A detector and a Daicel Chiralpak AD-H column (250 ×10 mm, 5 μm). Preparative HPLC was also performed on a Shimadzu LC-6AD instrument with a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Column chromatography (CC) was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China) and ODS (50 μm, YMC, Japan). TLC was carried out on glass precoated silica gel GF254 plates. Spots
Y.-Q. Du et al. / Fitoterapia 103 (2015) 122–128
123
Fig. 1. Structures of compounds 1–13 isolated from Clausena lansium.
were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating. 2.2. Plant materials The stems of C. lansium were collected in Liuzhou, Guangxi, China, in March 2013 and identified by Engineer Guangri Long, Forestry of Liuzhou. A voucher specimen has been deposited at the Herbarium of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (ID-S2320). 2.3. Extraction and isolation Air-dried, powdered stems of C. lansium (200 kg) were extracted with 95% ethanol (1000L × 2h × 3). The residue was suspended in water and then partitioned with EtOAc (3 × 40 L), and n-BuOH (3 × 40 L), successively. After removing the solvent, the EtOAc-soluble portion (1150 g) was fractionated via silica gel CC using a soxhlet extraction apparatus with solvents of varying polarity: petroleum ether, CHCl3, EtOAc, acetone, and methanol. Each extract was concentrated using a rotavapor prior to being separated. The CHCl3 extract (350 g) was fractionated via silica gel column chromatography, eluting with petroleum ether-EtOAc (4:1) to afford eleven fractions A0-A10 on the basis of TLC analysis. Fraction A2-A4 (78 g) was chromatographed over silica gel eluted with petroleum ether-acetone gradients (20:1, 15:1, 10:1, 5:1, 2:1) to give ten subfractions, A2-4a–A2-4j. Fraction A2-4c (3.1 g) was subject to MPLC (67% MeOH-H2O), successively using preparative HPLC (detection at 210 nm, 8 mL/min) to yield 2 (1.6 mg), 6 (4.0 mg), and 7 (5.4 mg). Fraction A2-4f (3.2 g) was chromatographed over silica gel eluted with petroleum ether-
EtOAc gradients (10:1, 5:1, 4:1, 3:1) to give nine subfractions and using preparative HPLC (detection at 210 nm, 8 mL/min) to yield 3 (2.1 mg) and 9 (7.3 mg). Fraction A2-4h (10.2 g) was subjected to MPLC (60% MeOH-H2O) successively using preparative HPLC (detection at 210 nm, 8 mL/min) to yield 4 (1.9 mg) and 5 (1.2 mg). Fraction A5-A7 (50 g) was chromatographed over silica gel eluted with petroleum ether-EtOAc gradients (20:1, 15:1, 10:1, 5:1, 2:1) to give nine subfractions, A5-7a–A5-7i. Fraction A5-7d (1.8 g) was subject to MPLC (60–100% MeOHH2O), successively using preparative HPLC (detection at 210 nm, 8 mL/min) to yield 12 (1.6 mg). Fraction A5-7f (2.5 g) was chromatographed over silica gel eluted with petroleum etherCH2Cl2 gradients (3:1, 2:1, 1:1, 1:2) to give nine subfractions and using preparative HPLC (detection at 210 nm, 8 mL/min) to yield 1 (1.3 mg), 10 (0.9 mg), 11 (1.2 mg), 8 (2.3 mg), and 13 (3.5 mg). Compound 1 was isolated by using a chiral semipreparative column (38% n-hexane/2-propanol, 2 mL/min) to yield compound 1a (0.44 mg) and 1b (0.42 mg). Claulansine L (1): light yellow powder; UV (CH3OH) λmax (log ε) 208 (6.03), 311 (6.24) nm; IR (microscope) νmax 3388, 2919, 1626, 1518, 1468, 1431, 1365, 1194, 1115 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz), see Tables 1 and 2; HRESIMS m/z 488.1320 [M + Na]+ (calcd for C25H23NO8 488.1316). Claulansine La (1a): light yellow powder (CH3OH); [α]20D + 6.8 (c 0.04, MeOH); ECD (CH3OH) λmax (Δε) 226 (−4.16), 255 (+0.41), 279.5 (−0.82) nm; claulansine Lb (1b): light yellow powder (CH3OH); [α]20D −6.9 (c 0.04, MeOH); ECD (CH3OH) λmax (Δε) 225 (+3.28), 255 (−0.07), 279.5 (+0.85) nm. Claulansine M (2): yellow powder; UV (CH3OH) λmax (log ε) 212 (6.22), 238 (6.08), 274 (6.10), 291 (6.00), 324 (5.67) nm; IR (microscope) νmax 3282, 2927, 1679, 1387, 1204, 747 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz),
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Table 1 1 H NMR data of compounds 1–7.a Position
1
1 2
6.88 s
4
8.32 s
5
7.64 s
6 7 8 1′ 2′
6.66 s
2
8.29 s 8.16 d (7.8) 7.24 t (7.2) 7.45 t (7.2) 7.55 d (7.8) 7.57 d (10.2) 5.92 d (10.2)
3
4
5
8.29 s
8.32 s
7.65 s
8.08 d (7.8) 7.20 td (7.2, 0.6) 7.39 td (7.2, 0.6) 7.47 d (7.8)
8.18 d (8.4) 7.21 td (7.8,1.2) 7.43 td (7.8,1.2) 7.52 d (8.4)
8.06 d (7.8) 7.16 td (7.8,0.6) 7.40 td (7.8,0.6) 7.51 d (7.8) 4.51 s 4.52 s
3′ 4′ 5′ 6′ 1-OCH3 1′-OCH2CH3 1′-OCH2CH3 2-OH 2-OCH3 3-CHO 3-CH2OCH2CH3 3′ and 5′-OCH3 6-OCH3 NH 1″ 2″ 3″ 1″-OH 4″-OH
1.51 s 6.66 s 3.96 s
10.86 brs 10.10 s 3.71 s 3.86 s 11.02 brs 3.83 m 3.77 m 5.59 d (6.0) 5.28 brs 8.41 brs
4.03 s
6
7
7.44 d (8.4) 7.35 dd (8.4, 1.2) 8.05 d (1.2) 8.10 d (7.8) 7.14 td (7.8,1.2) 7.38 td (7.8,1.2) 7.47 d (7.8) 4.57 s
8.05 d (7.8) 7.13 td (7.8,1.2) 7.35 td (7.8,1.2) 7.46 d (7.8) 4.57 s
3.51 q (7.2) 1.16 t (7.2)
3.52 q (7.2) 1.17 t (7.2)
1.30 s 1.04 s 6.12 s 4.00 s 3.80 q 1.20 t
6.94 s 7.64 s
3.98 s
11.03 s 10.14 s
10.08 s
4.02 s 10.29 s
11.76 brs
11.76 brs
11.83 brs
11.40 brs
11.23 brs
11.28 brs
a 1
H NMR data (δ) were measured in DMSO-d6 at 600 MHz for 2–7, and at 500 MHz for 1.
see Tables 1 and 2; HRESIMS m/z 278.1175 [M +H]+ (calcd for C18H15NO2, 278.1176). Claulansine N (3): yellow powder; UV (CH3OH) λmax (log ε) 206 (5.37), 235 (5.39), 276 (5.43), 298 (5.5), 340 (4.99) nm; IR (microscope) νmax 3306, 2943, 1687, 1456, 1388, 1333, 1289, 1207, 746 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 242.0811 [M + H]+ (calcd for C14H12NO3, 242.0812). Claulansine O (4): white powder; UV (CH3OH) λmax (log ε) 202 (5.81), 235 (5.91), 273 (5.91), 330 (5.40), 345 (5.36) nm; IR (microscope) νmax 3240, 2941, 1664, 1598, 1405, 1239,738 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 256.0970 [M + H]+ (calcd for C15H14NO3, 256.0968). Claulansine P (5): white powder; [α]25 D −38.8 (c 0.10, CH3OH); UV (CH3OH) λmax (log ε) 235 (5.85), 259 (5.57), 294 (5.41) nm; IR (microscope) νmax 3275, 2975, 1614, 1362, 1249, 1077, 939, 752 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 354.1707 [M + H]+ (calcd for C21H23NO4, 354.1700). Claulansine Q (6): yellow powder; UV (CH3OH) λmax (log ε) 235 (5.46), 259 (5.18), 294 (5.02) nm; IR (microscope) νmax 3342, 2922, 1607, 1368, 1244, 1097, 747cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see
Tables 1 and 2; HRESIMS m/z 248.1043 [M + Na]+ (calcd for C15H15NONa, 248.1046). Claulansine R (7): yellow powder; UV (CH3OH) λmax (log ε) 196 (5.66), 225 (5.86), 241 (5.93), 251 (5.85), 289 (5.35), 323 (5.09), 335 (5.05) nm; IR (microscope) νmax 3338, 2927, 1588, 1377, 1139, 600cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 255.1249 [M]+ (calcd for C16H17NO2, 255.1259). 2.4. Inhibition of nitric oxide production assay The murine microglial BV2 cells, purchased from the Cell Culture Centre at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), at 37 °C atmosphere, 5% CO2, and 100% relative humidity. The microglia cells were placed in 96-well cell culture plates (2 × 104 cell/mL) and preincubated for 24 h. Then the cells were treated with various concentrations of isolated compounds in triplicate for 1 h and continuously incubated with LPS (Sigma-Aldrich) (0.3 μg/mL) for 24 h. Curcumin was used as the positive control. After incubation, the supernatants (100 μL) were added to a solution of 100 μL of Griess reagent (a 1:1 mixture
Y.-Q. Du et al. / Fitoterapia 103 (2015) 122–128 Table 2 13 C NMR spectroscopic data of compounds 1–7a. Position
1
2
3
4
5
6
7
1 1a 2 3 4 4a 5 5a 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 1-OCH3 1′-OCH2CH3 1′-OCH2CH3 3′ and 5′-OCH3 2-OCH3 3-CHO 3-CH2OCH2CH3 3-CH2OCH2CH3 3-CH2OCH2CH3 6-OCH3 1″ 2″ 3″
97.2 146.6 159.7 117.4 123.6 118.5 104.3 116.2 140.5 148.6 111.0 132.3 132.2 104.3 148.7 136.1 148.7 104.3
138.2 132.3 116.0 122.9 121.8 122.9 120.4 123.5 120.0 126.5 111.9 140.7 119.9 131.2 76.0 27.3 27.3
130.9 138.9 151.0 121.2 116.6 117.7 120.0 123.2 120.1 125.8 111.4 140.8
137.1 138.4 152.6 122.1 116.1 120.0 120.6 123.1 120.4 126.3 111.6 140.9
111.0 139.3 126.0 128.8 120.0 122.4 120.2 122.3 118.6 125.6 110.7 140.1 72.6 64.7 15.3
145.4 129.6 106.4 129.1 112.2 129.0 120.2 122.6 118.5 125.3 111.4 140.0 72.7 64.6 15.2
60.3
60.9
144.4 132.3 121.0 129.3 111.2 124.0 120.2 122.6 118.9 126.0 111.4 140.2 69.2 81.3 77..0 23.3 29.5 100.2 60.5 64.2 15.6
194.4
62.8 189.0
a 13
55.4
56.7 193.1
192.9
125
of 0.1% naphthyl ethylene diamine and 1% sulfanilamide in 5% H3PO4) at room temperature for 20 min. NO concentration was quantified by a microplate reader at 540 nm for the amount of stable nitrite produced in the cell culture supernatants using the Griess assay [7]. 2.5. Hepatoprotective activity assay Human HepG2 hepatoma cells were cultured in DMEM medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2 + 95% air. The cells were then passaged by treatment with 0.25% trypsin in 0.02% EDTA. The MTT assay was used to assess the cytotoxicity of test samples. The cells were seeded in 96-well multiplates. After an overnight incubation at 37 °C with 5% CO2, 10 μm test samples and APAP (final concentration of 8 mM) were added into the wells and incubated for another 48 h. Then 100 μL of 0.5 mg/mL MTT was added to each well after the withdrawal of the culture medium and incubated for an additional 4 h. The resulting formazan was dissolved in 150 μL of DMSO after aspiration of the culture medium. The plates were placed on a plate shaker for 30 min and read immediately at 570 nm using a microplate reader [8]. 3. Results and discussion
57.0 62.4 53.4 87.9
C NMR data (δ) were measured in DMSO-d6 at 125 MHz for 1–7.
Claulansine L (1) was obtained as a light yellow powder, [α]20D 0 (c 0.07, MeOH). The molecular formula, C25H23NO8, was established by HRESIMS (488.1320 [M + Na]+, calcd for 488.1316), implying 15° of unsaturation. The IR spectrum
Fig. 2. Selected HMBCs of compounds 1–7.
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displayed characteristic absorptions of amino (3388 cm−1) and aromatic ring (1626, 1518, and 1468 cm−1) groups, and the UV spectrum showed absorbances at λmax 208 and 311 nm. Five aromatic protons [δH 8.32 (1H, s), 6.88 (1H, s), 7.64 (1H, s), and 6.66 (2H, s)] and three methoxyl protons at δH 3.86 (3H, s) and 3.71 (6H, s) were observed in the 1H NMR spectrum of 1. It also displayed exchangeable resonances assignable to an amide proton at δH 11.02 (NH, brs), two phenolic hydroxyl protons [δH 10.86 (1H, brs) and 8.41 (1H, brs)], an aldehyde proton at δH 10.10 (1H, s), and an alcohol hydroxyl proton at δH 5.28 (1H, brs). The 13C NMR spectra of 1 showed 25 carbon resonances, corresponding to eighteen aromatic carbons, one oxymethine, one methine, one oxymethylene, three methoxyls, and an aldehyde. The above information coupled with biogenetic considerations and literature references indicated a carbazole skeleton and a symmetric tetrasubstituted aromatic ring in 1. In the HMBC experiment (Fig. 2), the correlation of δH 3.71/δC 148.7 placed two methoxyls at C-3′ and C-5′ of the aromatic ring; correlations of δH 3.86/C-6 and of H-4/δC 193.1 located the remaining methoxyl group at C-6 and the aldehyde at C-3 of the carbazole skeleton, respectively. Although no HMBC correlation for H-2″/C-3″ was observed, the shifts for H-3″ (δH 5.59), C-3″ (δC 87.9), H-2″ (δH 3.77), and C-2″(δC 53.4) and correlations of H-2″/C-8 and C-1′ and of H-3″/C-2′, taking the degree of unsaturation into consideration, suggested that C-7, C-8, C-2″, C-3″, and an oxygen atom at C-7 established a fivemembered ring. Additional HMBC correlation of δH 5.59/62.4 placed the CH2OH unit at C-2″. Thus, the planar structure of 1 was established. Its overall structure was the same as that of claulansine K [9]. However, the main difference was observed at the specific rotation values. The lack of distinct Cotton effects in the CD spectrum also indicated that 1 was a mixture of two enantiomers in equal amounts. This was supported by HPLC analysis of 1 on an analytical chiral column, showing two peaks with an integration of about 1:1 ratio (S8). Subsequent separation of 1 yielded 1a {[α]20D + 6.8 (c 0.04, MeOH)} and 1b {[α]20D + −6.9 (c 0.04, MeOH)}, which had opposite specific rotations and ECD data. The relative configurations of C-2″ and C-3″ were determined to be trans on the basis of the coupling constant (J = 6.0 Hz) of H-2″ with H-3″. The absolute configurations at C-2″ and C-3″ were established by applying the exciton chirality method. The CD spectrum of 1a exhibited positive chirality resulting from the exciton coupling between the two different chromophores of the long conjugated benzene ring and the carbazole alkaloid skeleton. The positive chirality indicated that the transition dipole moments of the two chromophores are in a clockwise-oriented manner (Fig. 3) and, hence, established the configuration of C-2″ and C-3″ as (2″R,3″S). This indicated that 1a and 1b had the (2″R,3″S) and (2″S,3″R) configurations, respectively. Therefore, compounds 1a and 1b were determined as claulansine La and claulansine Lb, respectively. Claulansine M (2) was obtained as a yellow powder. Its molecular formula was established as C18H15NO2 by HRESIMS (278.1175 [M + H]+, calcd for 278.1176), implying 12° of unsaturation. The 1H-NMR spectra of 2 in DMSO-d6 displayed a 1,2,3-trisubstituted carbazole alkaloid skeleton having an aromatic proton at δH 8.29 (H-4), an unsubstituted ring A [δH 8.16 (1H, d, J = 7.8 Hz, H-5), 7.24 (1-H, d, J = 7.2 Hz, H-6), 7.45 (1H, t, J = 7.2 Hz, H-7), and 7.55 (1H, t, J = 7.8 Hz, H-8)], and a
Fig. 3. CD and UV spectra of compound 1a in MeOH. Stereoview of 1a: arrows denote the electric transition dipole of the chromophores.
pyran moiety [δH 7.57 (1H, d, J = 10.2 Hz, H-1′), 5.92 (1H, d, J = 10.2 Hz, H-2′), and 1.51 (6H, s, CH3-4′, and CH3-5′)]. In the HMBC experiment, correlation of H-4/δC 192.9 located the aldehyde group at C-3; correlations of H-1′/C-1 and H-2′/C-2 placed the pyran moiety at C-1/C-2. Therefore, claulansine M was characterized as 2.
Fig. 4. CD spectrum of compound 5 in MeOH.
Y.-Q. Du et al. / Fitoterapia 103 (2015) 122–128 Table 3 Inhibitory effects of compounds against LPS-induced NO production in microglia BV2 cells. Compounds
IC50 (μM)
Compounds
IC50 (μM)
6
23.09 6.13 20.36
13 Curcumina
17.7 0.5
7 12 a
Positive control.
Claulansine N (3) was isolated as a yellow powder. The molecular formula, C14H11NO3, was determined on the basis of its HRESIMS. The UV and IR spectra were similar to those of 2, indicating compound 3 is also a 1,2,3-trisubstituted carbazole alkaloid. The 1D NMR spectra of 3 revealed the presence of an aldehyde group (δH 10.08), a hydroxy group (δH 11.03), and a methoxy group (δH 3.96). In the HMBC spectrum, correlation of H-4 (δH 8.29)/3-CHO (δc 194.4) indicated that the aldehyde group was attached to C-3; correlation of δH 10.08/C-2 placed the OH at C-2. The location of the methoxy group assigned to C1 was further confirmed by an NOE difference experiment showing strong enhancements of NH (δH 11.76) on irradiation of OCH3 (δH 3.96). Hence, claulansine N was characterized as 3. The elemental composition of claulansine O (4) was established as C15H13NO3 by HRESIMS. The NMR data of 4 revealed a 1,2,3-trisubstituted carbazole alkaloid skeleton with an aldehyde group and two methoxy groups. Comparison of the 1H and 13C NMR data of 4 and 3 indicated them to be very closely related analogues, differing only in the presence of a C-2 methoxy group [δH 4.03 (3H, s, 2-OCH3)/δc 62.8] in 4, instead of the hydroxy group in 3. Thus, structure 4 was assigned as claulansine O. Claulansine P (5) showed the molecular formula C21H23NO4 by the HRESIMS. Analyses of the 1D and 2D NMR data of 5 indicated that it possessed the same skeleton and substitution mode as claulansine B [5], except for the ethyoxyl group [δH 3.80 (2H, q, 1′-OCH2CH3)/δc 64.2 and δH 1.20 (3H, t, 1′OCH2CH3/δc 15.6]. In the HMBC experiment, the correlation of H-1′/δc 64.2 placed the OCH2CH3 at C-1′. Thus, the planar structure of 5 was established and its possible biogenetic pathway was also speculated (S36). The relative configurations of 5 were assigned from the NOESY spectrum (in CDCl3), in which a correlation of H-6′/H-1′ indicated that these two protons are in a cis configuration. A negative Cotton effect at 249 nm in the CD spectrum (Fig. 4)
Table 4 Hepatoprotective effects of compounds 3, 5, 6, 8, and 12 (1 × 10−5 mol/L) against APAP-induced toxicity in HepG2 cells. Compounds
OD (mean ± SD)
Cell survival rate (% of normal)
Control APAP 8 mM 3 5 6 8 12 Bicyclola
2.376 1.451 1.625 1.631 1.620 1.658 1.650 1.650
100.00 61.09 66.63 66.87 65.87 67.99 67.64 69.43
a
± ± ± ± ± ± ± ±
0.280 0.114⁎⁎⁎ 0.055# 0.057# 0.029# 0.056# 0.056# 0.190#
Positive control substance. ⁎⁎⁎ P b 0.001, compared with control. # P b 0.05, compared with model (APAP).
127
suggested that the absolute configurations at C-1′, C-2′, and C6′ of 5 were the same as those of claulansine B. Thus, the structure of claulansine P (5) was proposed as shown. Claulansine Q (6) was found to possess the molecular formula C15H15NO according to the HRESIMS peak at m/z 248.1043 [M + Na]+. Analysis of the 1D NMR data revealed that 6 was a monosubstituted carbazole. In addition, an ABX system [δH 7.44 (1H, d, J = 8.4 Hz, H-1), 7.35 (1H, dd, J = 8.4, 1.2 Hz, H-2), and 8.05 (1H, d, J = 1.2 Hz, H-4)] also confirmed the presence of one substituent on the B ring. In the HMBC spectrum of 6, correlations of δH H-3′/C-2′ (64.6), δH H-2′/C-1′ (72.5), and C-3′ (15.3), δH 4.57/C-2, C-3, C-4, C-2′, and C-4a determined the attachments of ethyoxymethyl group at C-3. Then 6 could be determined as a 3- ethyoxymethylcarbazole. Claulansine R (7) gave a molecular formula of C16H17NO2. The NMR data revealed that 7 possessed the same skeleton and substitution mode as 6, except for the C-1 methoxy group in 7. The HMBC correlations from H-4 (δH 7.64), and H-2 (δH 6.94) to δc 72.7 (3-CH2OCH2CH3), and from H-2 (δH 6.94) to δc 112.2 (C-4) and 129.0 (C-4a) supported the location of the ethyoxymethyl and methoxyl group at C-3 and C-1, respectively. Hence, claulansine Q was characterized as 7. Seven known carbazole alkaloids, glycozoline (8) [4], mukonal (9) [10], 3-nethoxymethylcarbazole (10) [11], indizoline (11) [4], methyl carbazole-3- carboxylate (12) [10], and murrayacine (13) [12], were also identified on the basis of their spectroscopic profiles (NMR, UV, and MS) and comparison to published data. In an anti-inflammatory assay, compound 7 exhibited an inhibitory effect on LPS-stimulated NO production in murine microglial BV2 cells with curcumin as a positive control as shown in Table 3, whereas the other compounds were inactive in this assay (IC50 value N10 μm). All isolates (1–13) were also bioassayed for their hepatoprotective activities against N-acetyl-p-aminophenol (APAP)-induced toxicity in HepG2 (human hepatocellular liver carcinoma cell line) cells, using the hepatoprotective activity drug bicyclol as the positive control. As shown in Table 4, compounds 3, 5, 6, 8, and 12 exhibited moderate hepatoprotective activities. Acknowledgments We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, for the measurement of the UV, IR, CD, NMR, and HRESIMS spectra. This research program is financially supported by the National Natural Science Foundation of China (No.21272278) and the National Megaproject for Innovative Drugs (No.2012ZX09301002-002). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.03.018. References [1] Adebajo AC, Iwalewa EO, Obuotor EM, Ibikunle GF, Omisore NO, Adewunmi CO, et al. Pharmacological properties of the extract and some isolated compounds of Clausena lansium stem bark: anti-trichomonal, antidiabetic, anti-inflammatory, hepatoprotective and antioxidant effects. J Ethnopharmacol 2009;122:10–9.
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[2] Maneerat W, Prawat U, Saewan N, Laphookhieo S. New coumarins from Clausena lansium twigs. J Braz Chem Soc 2010;21:665–8. [3] Yang MH, Chen YY, Huang L. Three novel cyclic amides from Clausena lansium. Phytochemistry 1988;27:445–50. [4] Li WS, McChesney JD, El-Feraly FS. Carbazole alkaloids from Clausena lansium. Phytochemistry 1991;30:343–6. [5] Liu H, Li CJ, Yang JZ, Ning N, Si YK, Li L, et al. Carbazole alkaloids from the stems of Clausena lansium. J Nat Prod 2012;75:677–82. [6] Liu H, Li F, Li CJ, Yang JZ, Li L, Chen NH, et al. Bioactive furanocoumarins from stems of Clausena lansium. Phytochemistry 2014;107:141–7. [7] Zhang D, Liu R, Sun L, Huang C, Wang C, Zhang DM, et al. Anti-inflammatory activity of methyl salicylate glycosides isolated from Gaultheria yunnanensis (Franch.) Rehder. Molecules 2011;16:3875–84.
[8] Hao ZY, Liang D, Luo H, Liu YF, Ni G, Zhang Q J, et al. Bioactive sesquiterpenoids from the rhizomes of Acorus calamus. J Nat Prod 2012; 75:1083–9. [9] Deng HD, Mei WL, Wang H, Guo ZK, Dong WH, Wang H, et al. Carbazole alkaloids from the peels of Clausena lansium. J Asian Nat Prod Res 2014;16: 1024–8. [10] Wu TS, Huang SC, Wu PL, Teng CM. Carbazole alkaloids from Clausena excavata and their biological activity. Phytochemistry 1996;43:133–40. [11] Yan SY, Cui CB, Cai B, Qu GX, Yao XS. New carbazole alkaloid from Clausena dunniana Levl. Chin J Med Chem 2001;11:345–6. [12] Khan NU, Naqvi SW. Constituents of the genus Clausena. J Sci Ind Res 1988; 47:543–6.
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Bioactive carbazole alkaloids from the stems of Clausena lansium Yi-Qian Du a, Hang Liu a,b, Chuang-Jun Li a, Jie Ma a, Dan Zhang a, Li Li a, Hua Sun a, Xiu-Qi Bao a, Dong-Ming Zhang a,⁎ a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People's Republic of China b Department of Pharmacy, Affiliated Drum Tower Hospital of Nanjing University, Zhongshan Road 321, Nanjing 210008, Jiangsu Province, People's Republic of China
a r t i c l e
i n f o
Article history: Received 25 December 2014 Accepted in revised form 16 March 2015 Keywords: Clausena lansium Claulansine L-R Anti-inflammatory Hepatoprotective
a b s t r a c t Seven new carbazole alkaloids, claulansines L-R (1–7), and six known analogues (8–13) were isolated from the stems of Clausena lansium. Their structures were elucidated on the basis of spectroscopic analyses, including UV, IR, and NMR experiments (HSQC, HMBC, and NOE experiment). Compound 7 showed moderate anti-inflammatory activities. Compounds 3, 5, 6, 8, and 12 exhibited moderate hepatoprotective activities. © 2015 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental
Clausena lansium Skeels (Wampee) is a tropical species of the Rutaceae family, which is widely distributed in southern China. In traditional Chinese medicine, the leaves and roots of C. lansium are used for cough, asthma, dermatological disease, viral hepatitis, and gastro-intestinal diseases; the seeds are used to treat acute and chronic gastro-intestinal inflammation, ulcers, etc [1]. Previous phytochemical investigations have demonstrated that this plant contains coumarins, amides, carbazole alkaloids, and several other components [2–4]. Our group has also reported 17 carbazole alkaloids [5] and 17 furanocoumarins [6] from the stems of C. lansium, and several of these compounds showed selective neuroprotective effects. In a continuing search for microscale bioactive metabolites from the same parts of this plant, 200 kg stems of C. lansium were collected in Guangxi province, China. In the present investigation, 7 new (1–7) and six known carbazole alkaloids (8–13) were isolated (Fig. 1). In this paper, the isolation and structure elucidation of the new compounds as well as the biological evaluation of these compounds are reported.
2.1. General
⁎ Corresponding author. Tel./fax: +86 10 63165227. E-mail address: [email protected] (D.-M. Zhang).
http://dx.doi.org/10.1016/j.fitote.2015.03.018 0367-326X/© 2015 Elsevier B.V. All rights reserved.
Optical rotations were measured on a JASCO P2000 automatic digital polarimeter. UV spectra were recorded on a JASCO V-650 spectrophotometer, CD spectra were measured on a JASCO J-815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 spectrometer using an FT-IR microscope transmission method. NMR spectra were acquired with Bruker AVIIIHD 600, VNS-600, and Mercury-400 spectrometers in DMSO-d6. HRESIMS spectra were collected on an Agilent 1100 series LC/ MSD ion trap mass spectrometer. MPLC system was composed of two C-605 pumps (Büchi), a C-635 UV detector (Büchi), a C660 fraction collector (Büchi), and an ODS column (450mm × 60 mm, 50 μm, 400 g; YMC). Semipreparative HPLC was conducted using a Shimadzu LC-6AD instrument with an SPD-20A detector and a Daicel Chiralpak AD-H column (250 ×10 mm, 5 μm). Preparative HPLC was also performed on a Shimadzu LC-6AD instrument with a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Column chromatography (CC) was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China) and ODS (50 μm, YMC, Japan). TLC was carried out on glass precoated silica gel GF254 plates. Spots
Y.-Q. Du et al. / Fitoterapia 103 (2015) 122–128
123
Fig. 1. Structures of compounds 1–13 isolated from Clausena lansium.
were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating. 2.2. Plant materials The stems of C. lansium were collected in Liuzhou, Guangxi, China, in March 2013 and identified by Engineer Guangri Long, Forestry of Liuzhou. A voucher specimen has been deposited at the Herbarium of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (ID-S2320). 2.3. Extraction and isolation Air-dried, powdered stems of C. lansium (200 kg) were extracted with 95% ethanol (1000L × 2h × 3). The residue was suspended in water and then partitioned with EtOAc (3 × 40 L), and n-BuOH (3 × 40 L), successively. After removing the solvent, the EtOAc-soluble portion (1150 g) was fractionated via silica gel CC using a soxhlet extraction apparatus with solvents of varying polarity: petroleum ether, CHCl3, EtOAc, acetone, and methanol. Each extract was concentrated using a rotavapor prior to being separated. The CHCl3 extract (350 g) was fractionated via silica gel column chromatography, eluting with petroleum ether-EtOAc (4:1) to afford eleven fractions A0-A10 on the basis of TLC analysis. Fraction A2-A4 (78 g) was chromatographed over silica gel eluted with petroleum ether-acetone gradients (20:1, 15:1, 10:1, 5:1, 2:1) to give ten subfractions, A2-4a–A2-4j. Fraction A2-4c (3.1 g) was subject to MPLC (67% MeOH-H2O), successively using preparative HPLC (detection at 210 nm, 8 mL/min) to yield 2 (1.6 mg), 6 (4.0 mg), and 7 (5.4 mg). Fraction A2-4f (3.2 g) was chromatographed over silica gel eluted with petroleum ether-
EtOAc gradients (10:1, 5:1, 4:1, 3:1) to give nine subfractions and using preparative HPLC (detection at 210 nm, 8 mL/min) to yield 3 (2.1 mg) and 9 (7.3 mg). Fraction A2-4h (10.2 g) was subjected to MPLC (60% MeOH-H2O) successively using preparative HPLC (detection at 210 nm, 8 mL/min) to yield 4 (1.9 mg) and 5 (1.2 mg). Fraction A5-A7 (50 g) was chromatographed over silica gel eluted with petroleum ether-EtOAc gradients (20:1, 15:1, 10:1, 5:1, 2:1) to give nine subfractions, A5-7a–A5-7i. Fraction A5-7d (1.8 g) was subject to MPLC (60–100% MeOHH2O), successively using preparative HPLC (detection at 210 nm, 8 mL/min) to yield 12 (1.6 mg). Fraction A5-7f (2.5 g) was chromatographed over silica gel eluted with petroleum etherCH2Cl2 gradients (3:1, 2:1, 1:1, 1:2) to give nine subfractions and using preparative HPLC (detection at 210 nm, 8 mL/min) to yield 1 (1.3 mg), 10 (0.9 mg), 11 (1.2 mg), 8 (2.3 mg), and 13 (3.5 mg). Compound 1 was isolated by using a chiral semipreparative column (38% n-hexane/2-propanol, 2 mL/min) to yield compound 1a (0.44 mg) and 1b (0.42 mg). Claulansine L (1): light yellow powder; UV (CH3OH) λmax (log ε) 208 (6.03), 311 (6.24) nm; IR (microscope) νmax 3388, 2919, 1626, 1518, 1468, 1431, 1365, 1194, 1115 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz), see Tables 1 and 2; HRESIMS m/z 488.1320 [M + Na]+ (calcd for C25H23NO8 488.1316). Claulansine La (1a): light yellow powder (CH3OH); [α]20D + 6.8 (c 0.04, MeOH); ECD (CH3OH) λmax (Δε) 226 (−4.16), 255 (+0.41), 279.5 (−0.82) nm; claulansine Lb (1b): light yellow powder (CH3OH); [α]20D −6.9 (c 0.04, MeOH); ECD (CH3OH) λmax (Δε) 225 (+3.28), 255 (−0.07), 279.5 (+0.85) nm. Claulansine M (2): yellow powder; UV (CH3OH) λmax (log ε) 212 (6.22), 238 (6.08), 274 (6.10), 291 (6.00), 324 (5.67) nm; IR (microscope) νmax 3282, 2927, 1679, 1387, 1204, 747 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz),
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Table 1 1 H NMR data of compounds 1–7.a Position
1
1 2
6.88 s
4
8.32 s
5
7.64 s
6 7 8 1′ 2′
6.66 s
2
8.29 s 8.16 d (7.8) 7.24 t (7.2) 7.45 t (7.2) 7.55 d (7.8) 7.57 d (10.2) 5.92 d (10.2)
3
4
5
8.29 s
8.32 s
7.65 s
8.08 d (7.8) 7.20 td (7.2, 0.6) 7.39 td (7.2, 0.6) 7.47 d (7.8)
8.18 d (8.4) 7.21 td (7.8,1.2) 7.43 td (7.8,1.2) 7.52 d (8.4)
8.06 d (7.8) 7.16 td (7.8,0.6) 7.40 td (7.8,0.6) 7.51 d (7.8) 4.51 s 4.52 s
3′ 4′ 5′ 6′ 1-OCH3 1′-OCH2CH3 1′-OCH2CH3 2-OH 2-OCH3 3-CHO 3-CH2OCH2CH3 3′ and 5′-OCH3 6-OCH3 NH 1″ 2″ 3″ 1″-OH 4″-OH
1.51 s 6.66 s 3.96 s
10.86 brs 10.10 s 3.71 s 3.86 s 11.02 brs 3.83 m 3.77 m 5.59 d (6.0) 5.28 brs 8.41 brs
4.03 s
6
7
7.44 d (8.4) 7.35 dd (8.4, 1.2) 8.05 d (1.2) 8.10 d (7.8) 7.14 td (7.8,1.2) 7.38 td (7.8,1.2) 7.47 d (7.8) 4.57 s
8.05 d (7.8) 7.13 td (7.8,1.2) 7.35 td (7.8,1.2) 7.46 d (7.8) 4.57 s
3.51 q (7.2) 1.16 t (7.2)
3.52 q (7.2) 1.17 t (7.2)
1.30 s 1.04 s 6.12 s 4.00 s 3.80 q 1.20 t
6.94 s 7.64 s
3.98 s
11.03 s 10.14 s
10.08 s
4.02 s 10.29 s
11.76 brs
11.76 brs
11.83 brs
11.40 brs
11.23 brs
11.28 brs
a 1
H NMR data (δ) were measured in DMSO-d6 at 600 MHz for 2–7, and at 500 MHz for 1.
see Tables 1 and 2; HRESIMS m/z 278.1175 [M +H]+ (calcd for C18H15NO2, 278.1176). Claulansine N (3): yellow powder; UV (CH3OH) λmax (log ε) 206 (5.37), 235 (5.39), 276 (5.43), 298 (5.5), 340 (4.99) nm; IR (microscope) νmax 3306, 2943, 1687, 1456, 1388, 1333, 1289, 1207, 746 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 242.0811 [M + H]+ (calcd for C14H12NO3, 242.0812). Claulansine O (4): white powder; UV (CH3OH) λmax (log ε) 202 (5.81), 235 (5.91), 273 (5.91), 330 (5.40), 345 (5.36) nm; IR (microscope) νmax 3240, 2941, 1664, 1598, 1405, 1239,738 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 256.0970 [M + H]+ (calcd for C15H14NO3, 256.0968). Claulansine P (5): white powder; [α]25 D −38.8 (c 0.10, CH3OH); UV (CH3OH) λmax (log ε) 235 (5.85), 259 (5.57), 294 (5.41) nm; IR (microscope) νmax 3275, 2975, 1614, 1362, 1249, 1077, 939, 752 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 354.1707 [M + H]+ (calcd for C21H23NO4, 354.1700). Claulansine Q (6): yellow powder; UV (CH3OH) λmax (log ε) 235 (5.46), 259 (5.18), 294 (5.02) nm; IR (microscope) νmax 3342, 2922, 1607, 1368, 1244, 1097, 747cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see
Tables 1 and 2; HRESIMS m/z 248.1043 [M + Na]+ (calcd for C15H15NONa, 248.1046). Claulansine R (7): yellow powder; UV (CH3OH) λmax (log ε) 196 (5.66), 225 (5.86), 241 (5.93), 251 (5.85), 289 (5.35), 323 (5.09), 335 (5.05) nm; IR (microscope) νmax 3338, 2927, 1588, 1377, 1139, 600cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 255.1249 [M]+ (calcd for C16H17NO2, 255.1259). 2.4. Inhibition of nitric oxide production assay The murine microglial BV2 cells, purchased from the Cell Culture Centre at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), at 37 °C atmosphere, 5% CO2, and 100% relative humidity. The microglia cells were placed in 96-well cell culture plates (2 × 104 cell/mL) and preincubated for 24 h. Then the cells were treated with various concentrations of isolated compounds in triplicate for 1 h and continuously incubated with LPS (Sigma-Aldrich) (0.3 μg/mL) for 24 h. Curcumin was used as the positive control. After incubation, the supernatants (100 μL) were added to a solution of 100 μL of Griess reagent (a 1:1 mixture
Y.-Q. Du et al. / Fitoterapia 103 (2015) 122–128 Table 2 13 C NMR spectroscopic data of compounds 1–7a. Position
1
2
3
4
5
6
7
1 1a 2 3 4 4a 5 5a 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 1-OCH3 1′-OCH2CH3 1′-OCH2CH3 3′ and 5′-OCH3 2-OCH3 3-CHO 3-CH2OCH2CH3 3-CH2OCH2CH3 3-CH2OCH2CH3 6-OCH3 1″ 2″ 3″
97.2 146.6 159.7 117.4 123.6 118.5 104.3 116.2 140.5 148.6 111.0 132.3 132.2 104.3 148.7 136.1 148.7 104.3
138.2 132.3 116.0 122.9 121.8 122.9 120.4 123.5 120.0 126.5 111.9 140.7 119.9 131.2 76.0 27.3 27.3
130.9 138.9 151.0 121.2 116.6 117.7 120.0 123.2 120.1 125.8 111.4 140.8
137.1 138.4 152.6 122.1 116.1 120.0 120.6 123.1 120.4 126.3 111.6 140.9
111.0 139.3 126.0 128.8 120.0 122.4 120.2 122.3 118.6 125.6 110.7 140.1 72.6 64.7 15.3
145.4 129.6 106.4 129.1 112.2 129.0 120.2 122.6 118.5 125.3 111.4 140.0 72.7 64.6 15.2
60.3
60.9
144.4 132.3 121.0 129.3 111.2 124.0 120.2 122.6 118.9 126.0 111.4 140.2 69.2 81.3 77..0 23.3 29.5 100.2 60.5 64.2 15.6
194.4
62.8 189.0
a 13
55.4
56.7 193.1
192.9
125
of 0.1% naphthyl ethylene diamine and 1% sulfanilamide in 5% H3PO4) at room temperature for 20 min. NO concentration was quantified by a microplate reader at 540 nm for the amount of stable nitrite produced in the cell culture supernatants using the Griess assay [7]. 2.5. Hepatoprotective activity assay Human HepG2 hepatoma cells were cultured in DMEM medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2 + 95% air. The cells were then passaged by treatment with 0.25% trypsin in 0.02% EDTA. The MTT assay was used to assess the cytotoxicity of test samples. The cells were seeded in 96-well multiplates. After an overnight incubation at 37 °C with 5% CO2, 10 μm test samples and APAP (final concentration of 8 mM) were added into the wells and incubated for another 48 h. Then 100 μL of 0.5 mg/mL MTT was added to each well after the withdrawal of the culture medium and incubated for an additional 4 h. The resulting formazan was dissolved in 150 μL of DMSO after aspiration of the culture medium. The plates were placed on a plate shaker for 30 min and read immediately at 570 nm using a microplate reader [8]. 3. Results and discussion
57.0 62.4 53.4 87.9
C NMR data (δ) were measured in DMSO-d6 at 125 MHz for 1–7.
Claulansine L (1) was obtained as a light yellow powder, [α]20D 0 (c 0.07, MeOH). The molecular formula, C25H23NO8, was established by HRESIMS (488.1320 [M + Na]+, calcd for 488.1316), implying 15° of unsaturation. The IR spectrum
Fig. 2. Selected HMBCs of compounds 1–7.
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displayed characteristic absorptions of amino (3388 cm−1) and aromatic ring (1626, 1518, and 1468 cm−1) groups, and the UV spectrum showed absorbances at λmax 208 and 311 nm. Five aromatic protons [δH 8.32 (1H, s), 6.88 (1H, s), 7.64 (1H, s), and 6.66 (2H, s)] and three methoxyl protons at δH 3.86 (3H, s) and 3.71 (6H, s) were observed in the 1H NMR spectrum of 1. It also displayed exchangeable resonances assignable to an amide proton at δH 11.02 (NH, brs), two phenolic hydroxyl protons [δH 10.86 (1H, brs) and 8.41 (1H, brs)], an aldehyde proton at δH 10.10 (1H, s), and an alcohol hydroxyl proton at δH 5.28 (1H, brs). The 13C NMR spectra of 1 showed 25 carbon resonances, corresponding to eighteen aromatic carbons, one oxymethine, one methine, one oxymethylene, three methoxyls, and an aldehyde. The above information coupled with biogenetic considerations and literature references indicated a carbazole skeleton and a symmetric tetrasubstituted aromatic ring in 1. In the HMBC experiment (Fig. 2), the correlation of δH 3.71/δC 148.7 placed two methoxyls at C-3′ and C-5′ of the aromatic ring; correlations of δH 3.86/C-6 and of H-4/δC 193.1 located the remaining methoxyl group at C-6 and the aldehyde at C-3 of the carbazole skeleton, respectively. Although no HMBC correlation for H-2″/C-3″ was observed, the shifts for H-3″ (δH 5.59), C-3″ (δC 87.9), H-2″ (δH 3.77), and C-2″(δC 53.4) and correlations of H-2″/C-8 and C-1′ and of H-3″/C-2′, taking the degree of unsaturation into consideration, suggested that C-7, C-8, C-2″, C-3″, and an oxygen atom at C-7 established a fivemembered ring. Additional HMBC correlation of δH 5.59/62.4 placed the CH2OH unit at C-2″. Thus, the planar structure of 1 was established. Its overall structure was the same as that of claulansine K [9]. However, the main difference was observed at the specific rotation values. The lack of distinct Cotton effects in the CD spectrum also indicated that 1 was a mixture of two enantiomers in equal amounts. This was supported by HPLC analysis of 1 on an analytical chiral column, showing two peaks with an integration of about 1:1 ratio (S8). Subsequent separation of 1 yielded 1a {[α]20D + 6.8 (c 0.04, MeOH)} and 1b {[α]20D + −6.9 (c 0.04, MeOH)}, which had opposite specific rotations and ECD data. The relative configurations of C-2″ and C-3″ were determined to be trans on the basis of the coupling constant (J = 6.0 Hz) of H-2″ with H-3″. The absolute configurations at C-2″ and C-3″ were established by applying the exciton chirality method. The CD spectrum of 1a exhibited positive chirality resulting from the exciton coupling between the two different chromophores of the long conjugated benzene ring and the carbazole alkaloid skeleton. The positive chirality indicated that the transition dipole moments of the two chromophores are in a clockwise-oriented manner (Fig. 3) and, hence, established the configuration of C-2″ and C-3″ as (2″R,3″S). This indicated that 1a and 1b had the (2″R,3″S) and (2″S,3″R) configurations, respectively. Therefore, compounds 1a and 1b were determined as claulansine La and claulansine Lb, respectively. Claulansine M (2) was obtained as a yellow powder. Its molecular formula was established as C18H15NO2 by HRESIMS (278.1175 [M + H]+, calcd for 278.1176), implying 12° of unsaturation. The 1H-NMR spectra of 2 in DMSO-d6 displayed a 1,2,3-trisubstituted carbazole alkaloid skeleton having an aromatic proton at δH 8.29 (H-4), an unsubstituted ring A [δH 8.16 (1H, d, J = 7.8 Hz, H-5), 7.24 (1-H, d, J = 7.2 Hz, H-6), 7.45 (1H, t, J = 7.2 Hz, H-7), and 7.55 (1H, t, J = 7.8 Hz, H-8)], and a
Fig. 3. CD and UV spectra of compound 1a in MeOH. Stereoview of 1a: arrows denote the electric transition dipole of the chromophores.
pyran moiety [δH 7.57 (1H, d, J = 10.2 Hz, H-1′), 5.92 (1H, d, J = 10.2 Hz, H-2′), and 1.51 (6H, s, CH3-4′, and CH3-5′)]. In the HMBC experiment, correlation of H-4/δC 192.9 located the aldehyde group at C-3; correlations of H-1′/C-1 and H-2′/C-2 placed the pyran moiety at C-1/C-2. Therefore, claulansine M was characterized as 2.
Fig. 4. CD spectrum of compound 5 in MeOH.
Y.-Q. Du et al. / Fitoterapia 103 (2015) 122–128 Table 3 Inhibitory effects of compounds against LPS-induced NO production in microglia BV2 cells. Compounds
IC50 (μM)
Compounds
IC50 (μM)
6
23.09 6.13 20.36
13 Curcumina
17.7 0.5
7 12 a
Positive control.
Claulansine N (3) was isolated as a yellow powder. The molecular formula, C14H11NO3, was determined on the basis of its HRESIMS. The UV and IR spectra were similar to those of 2, indicating compound 3 is also a 1,2,3-trisubstituted carbazole alkaloid. The 1D NMR spectra of 3 revealed the presence of an aldehyde group (δH 10.08), a hydroxy group (δH 11.03), and a methoxy group (δH 3.96). In the HMBC spectrum, correlation of H-4 (δH 8.29)/3-CHO (δc 194.4) indicated that the aldehyde group was attached to C-3; correlation of δH 10.08/C-2 placed the OH at C-2. The location of the methoxy group assigned to C1 was further confirmed by an NOE difference experiment showing strong enhancements of NH (δH 11.76) on irradiation of OCH3 (δH 3.96). Hence, claulansine N was characterized as 3. The elemental composition of claulansine O (4) was established as C15H13NO3 by HRESIMS. The NMR data of 4 revealed a 1,2,3-trisubstituted carbazole alkaloid skeleton with an aldehyde group and two methoxy groups. Comparison of the 1H and 13C NMR data of 4 and 3 indicated them to be very closely related analogues, differing only in the presence of a C-2 methoxy group [δH 4.03 (3H, s, 2-OCH3)/δc 62.8] in 4, instead of the hydroxy group in 3. Thus, structure 4 was assigned as claulansine O. Claulansine P (5) showed the molecular formula C21H23NO4 by the HRESIMS. Analyses of the 1D and 2D NMR data of 5 indicated that it possessed the same skeleton and substitution mode as claulansine B [5], except for the ethyoxyl group [δH 3.80 (2H, q, 1′-OCH2CH3)/δc 64.2 and δH 1.20 (3H, t, 1′OCH2CH3/δc 15.6]. In the HMBC experiment, the correlation of H-1′/δc 64.2 placed the OCH2CH3 at C-1′. Thus, the planar structure of 5 was established and its possible biogenetic pathway was also speculated (S36). The relative configurations of 5 were assigned from the NOESY spectrum (in CDCl3), in which a correlation of H-6′/H-1′ indicated that these two protons are in a cis configuration. A negative Cotton effect at 249 nm in the CD spectrum (Fig. 4)
Table 4 Hepatoprotective effects of compounds 3, 5, 6, 8, and 12 (1 × 10−5 mol/L) against APAP-induced toxicity in HepG2 cells. Compounds
OD (mean ± SD)
Cell survival rate (% of normal)
Control APAP 8 mM 3 5 6 8 12 Bicyclola
2.376 1.451 1.625 1.631 1.620 1.658 1.650 1.650
100.00 61.09 66.63 66.87 65.87 67.99 67.64 69.43
a
± ± ± ± ± ± ± ±
0.280 0.114⁎⁎⁎ 0.055# 0.057# 0.029# 0.056# 0.056# 0.190#
Positive control substance. ⁎⁎⁎ P b 0.001, compared with control. # P b 0.05, compared with model (APAP).
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suggested that the absolute configurations at C-1′, C-2′, and C6′ of 5 were the same as those of claulansine B. Thus, the structure of claulansine P (5) was proposed as shown. Claulansine Q (6) was found to possess the molecular formula C15H15NO according to the HRESIMS peak at m/z 248.1043 [M + Na]+. Analysis of the 1D NMR data revealed that 6 was a monosubstituted carbazole. In addition, an ABX system [δH 7.44 (1H, d, J = 8.4 Hz, H-1), 7.35 (1H, dd, J = 8.4, 1.2 Hz, H-2), and 8.05 (1H, d, J = 1.2 Hz, H-4)] also confirmed the presence of one substituent on the B ring. In the HMBC spectrum of 6, correlations of δH H-3′/C-2′ (64.6), δH H-2′/C-1′ (72.5), and C-3′ (15.3), δH 4.57/C-2, C-3, C-4, C-2′, and C-4a determined the attachments of ethyoxymethyl group at C-3. Then 6 could be determined as a 3- ethyoxymethylcarbazole. Claulansine R (7) gave a molecular formula of C16H17NO2. The NMR data revealed that 7 possessed the same skeleton and substitution mode as 6, except for the C-1 methoxy group in 7. The HMBC correlations from H-4 (δH 7.64), and H-2 (δH 6.94) to δc 72.7 (3-CH2OCH2CH3), and from H-2 (δH 6.94) to δc 112.2 (C-4) and 129.0 (C-4a) supported the location of the ethyoxymethyl and methoxyl group at C-3 and C-1, respectively. Hence, claulansine Q was characterized as 7. Seven known carbazole alkaloids, glycozoline (8) [4], mukonal (9) [10], 3-nethoxymethylcarbazole (10) [11], indizoline (11) [4], methyl carbazole-3- carboxylate (12) [10], and murrayacine (13) [12], were also identified on the basis of their spectroscopic profiles (NMR, UV, and MS) and comparison to published data. In an anti-inflammatory assay, compound 7 exhibited an inhibitory effect on LPS-stimulated NO production in murine microglial BV2 cells with curcumin as a positive control as shown in Table 3, whereas the other compounds were inactive in this assay (IC50 value N10 μm). All isolates (1–13) were also bioassayed for their hepatoprotective activities against N-acetyl-p-aminophenol (APAP)-induced toxicity in HepG2 (human hepatocellular liver carcinoma cell line) cells, using the hepatoprotective activity drug bicyclol as the positive control. As shown in Table 4, compounds 3, 5, 6, 8, and 12 exhibited moderate hepatoprotective activities. Acknowledgments We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, for the measurement of the UV, IR, CD, NMR, and HRESIMS spectra. This research program is financially supported by the National Natural Science Foundation of China (No.21272278) and the National Megaproject for Innovative Drugs (No.2012ZX09301002-002). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.03.018. References [1] Adebajo AC, Iwalewa EO, Obuotor EM, Ibikunle GF, Omisore NO, Adewunmi CO, et al. Pharmacological properties of the extract and some isolated compounds of Clausena lansium stem bark: anti-trichomonal, antidiabetic, anti-inflammatory, hepatoprotective and antioxidant effects. J Ethnopharmacol 2009;122:10–9.
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