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New amide alkaloid from the aerial part of Piper capense L.f. (Piperaceae)
Fitoterapia 81 (2010) 632–635
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
New amide alkaloid from the aerial part of Piper capense L.f. (Piperaceae) Ali Mohamed Kaou a, Valérie Mahiou-Leddet a,⁎, Cécile Canlet b, Laurent Debrauwer b, Sébastien Hutter c, Nadine Azas c, Evelyne Ollivier a a Laboratoire de Pharmacognosie et Ethnopharmacologie, UMR-MD3, Faculté de Pharmacie, Université de la Méditerranée (Aix-Marseille II), 27 Bd Jean Moulin, 13385 Marseille, Cedex 5, France b Laboratoire des Xénobiotiques, INRA, UMR1089, 180 Chemin de Tournefeuille BP 93173, 31027 Toulouse Cedex 3, France c Laboratoire de Parasitologie, UMR-MD3, Faculté de Pharmacie, Université de la Méditerranée (Aix-Marseille II), 27 Bd Jean Moulin, 13385 Marseille, Cedex 5, France
a r t i c l e
i n f o
Article history: Received 1 September 2009 Accepted 6 March 2010 Available online 20 March 2010
a b s t r a c t Together with apigenine dimethylether and piperchabamide A, a new amide alkaloid, Kaousine and the Z form of antiepilepsirine were isolated from the aerial part of Piper capense L.f (Piperaceae). Their structures were elucidated by spectrometric methods and their in vitro antiparasitic activities were evaluated on Plasmodium falciparum. © 2010 Elsevier B.V. All rights reserved.
Keywords: Piper capense Piperaceae Amide alkaloid Comoro Antiplasmodial activity Plasmodium falciparum
1. Introduction In a search for novel antiparasitic natural products, we have studied the aerial part of Piper capense L.f (Piperaceae). This plant is traditionally used in Comoro Islands for diarrhoea and cough, as shown by our previous ethnobotanical survey [1]. The antiplasmodial activity of different extracts was evaluated in screening tests in vitro against the chloroquine-resistant strain W2 of Plasmodium falciparum and showed that the chloromethylenic extract of P. capense had moderate in vitro activity (IC50 =7 μg/ml). Previous phytochemical studies carried out on P. capense have resulted in the identification of lignans [2,3], sesquiterpenes [4], and essential oil with high percentage of monoterpene hydrocarbons [5]. In the present paper, we describe from the chloromethylenic extract of P. capense the isolation of a new amide alkaloid, Kaousine, 1 together with the Z form of antiepilepsirine 2 [6], the known apigenine dimethylether [7], and piperchabamide A [8], isolated for the first time
from this plant material. The structures were established using NMR and mass spectrometry and spectroscopic data of known compounds were compared with reported values. 2. Experimental 2.1. General UV spectra were recorded on a Beckman DU 520 spectrophotometer and IR spectrum was taken on a Brücker Vertex 70 spectrometer. NMR spectra were recorded in CDCl3 (δ ppm) on Brücker DRX 600 spectrometer operating at 600.13 MHz and 150.91 MHz. EIMS spectra were obtained on an Ion Trap Finigan (Thermo Quest) spectrometer. HREIMS spectra were obtained on a QStar Elite (Applied Biosystems SCIEX) spectrometer. Optical rotations were taken on a Perkin Elmer 341 OROT 589 nm Polarimeter. 2.2. Plant material
⁎ Corresponding author. Tel./fax: +33 4 9183 5593. E-mail address: [email protected] (V. Mahiou-Leddet). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.03.006
The aerial part of P. capense was collected by Ali Mohamed Kaou and Ibrahim Yahaya, in the island Ngazidja (Grande
A.M. Kaou et al. / Fitoterapia 81 (2010) 632–635
Comore Island) in the Grille forest in January 2004. Botanical determination of P. capense L.f. (Piperaceae) was performed by I. Yahaya and Pr J. N. Labat. Voucher specimens were deposited in the National Herbarium of the Museum Nationnal d'Histoire Naturelle (Paris, France, Piperaceae, no. P00440614). 2.3. Extraction and isolation Extraction method: Aerial part of P. capense (1.1 kg) was dried, powdered and submitted successively to a maceration process with 5 l CH2Cl2 for 14–15 h at room temperature. The extract was evaporated to dryness under vacuum affording the first CH2Cl2 extract (residue 21.5 g). The CH2Cl2 extract was fractionated by successive chromatographies on Kieselgel 60 Merck® (40–63 µm), sephadex LH 20 Pharmacia Biotech and preparative TLC 20 × 20 cm Kieselgel 60 F254 Merck® 0.5 mm: The CH2Cl2 extract (18 g) was subjected to CC (silica gel) eluting with CH2Cl2/MeOH (100/0, 98/2, 95/5, 90/10, 85/15, 80/20), in order of increasing polarity, to give 10 main fractions (G1–G10). Fraction G4 (225 mg) was separately fractionated into 4 subfractions (G4.1–G4.4) on silica gel column using toluene-AcOEt (100/0, 90/10, 80-20), whereas fraction G4.3 yielded Kaousine, 1 (13 mg). Fraction G6 (220 mg) was separated by silica gel CC using tolueneAcOEt (80/20) to obtain 3 subfractions (G6.1–G6.3). Fraction G6.2 (95 mg) was further purified by silica gel CC using toluene-AcOEt (80/20) as eluent to give apigenine dimethylether (11 mg). A part of CH2Cl2 extract (3.5 g) was subjected to CC (silica gel) eluting with CH2Cl2/MeOH (98/2, 95/5, 90/ 10, 85/15, 80/20), to give 9 main fractions (F1–F9). Fraction F5 (26 mg) was further purified by preparative TLC using CH2Cl2/MeOH (50/50) as eluent to give piperchabamide A (3.5 mg). Fraction F8 (2.15 g) was separately fractionated by silica gel CC using CH2Cl2-AcOEt (100/0, 97/3, 95/5, 90/10, 80/ 20) into 7 subfractions (F8.1–F8.7) whereas fraction F8.5 yielded: Z-antiepilepsirine, 2 (11 mg). Silica gel GF254 Merck® was used for TLC. 2.4. Kaousine (1) 3-(3-phenylpropanoyl)-7-oxa-3-aza-bicyclo[4.1.0]heptan-2-one, C14H15NO3, amorphous, [α]20 D + 17 (MeOH, c 0.115); UV λmax (MeOH) nm (Log ε): 211 (4.06).; IRfilm max ν cm−1: 3065; 3030, 2962, 2928, 2857, 2257, 1699, 1604, 1496, 1471, 1454, 1438, 1417, 1392, 1368, 1340, 1310, 1266, 1223, 1174, 1129, 1095, 1074, 1065, 1030; HREIMS m/z (%): 245.1052 (M+, 100) [245.1052calcd], EIMS 228 (18), 176 (100), 131 (44), 101 (65), 91 (39). For 1H and 13C NMR (600.13 and 150.91 MHz, CDCl3) data, see Table 1. 2.5. Z-antiepilepsirine (2) C15H17NO3, amorphous, [α]20 D + 10 (CHCl3, c 0.15); UV: λmax (MeOH) nm (Log ε) 210 (4.17), 273 (3.76), 306 (3.68). EIMS 259 (M+, 100), 175 (66), 145 (100), 117 (39), 89 (64). 1 H NMR (600.13 MHz, CDCl3) δ 6.92 (d, J = 1.5 Hz, H-5), 6.84 (dd, J = 8.2, 1.5 Hz, H-9), 6.75 (d, J = 8.2 Hz, H-8), 6.52 (d, J = 12.4 Hz, H-3), 5.95 (s, H-10), 5.92 (d, J = 12.4 Hz, H-2), 3.60 (m, H-5′), 3.35 (m, H-1′), 1.54 (m, H-2′ and H-4′), 1.27 (m, H-3′); 13C NMR (150.91 MHz, CDCl3) δ 167.3 (C-1), 147.8
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Table 1 1 H and 13C NMR data for compound 1 (600.13 and 150.91 MHz, CDCl3, J in Hz, δ in ppm). Position δΗ 2 3 4 5 6 7 8 9 10 11 12 13 14 15
– 3.56 (d, 4.2) 3.66 (brs) 2.40 (brd, 15.0) 1.98 (td, 15.0, 5.6) 4.33 (dd, 13.5, 5.6) 3.18 (td, 13.2, 3.9) – 3.24 m 2.96 (brt, 6.9) – 7.23 (brd, 7.2) 7.28 (brt, 7.2) 7.19 (brt, 7.2) 7.18 (brt, 7.2) 7.23 (brt, 7.2)
δC
H–H COSY
HMBC (H to C)
169.6 52.3 53.4 23.8
– 4 3, 5 (2.40) 5 (1.98), 6 5 (2.40), 6 5 (1.98), 6(3.18) 5, 6 (4.33) – 9 8 – – – – – –
– 2 5, 6 3 6 2, 4, 5, 7 4, 5 – 7, 9, 10 7, 8, 10, 11 – 9, 13, 14 10, 11 11/15, 12/14 10, 15 9, 11, 13
35.6 174.7 41.1 30.8 140.9 128.4 128.5 126.1 128.5 128.4
(C-6), 147.6 (C-7), 132.3 (CH-3), 129.8 (C-4), 123.1 (CH-9), 121.9 (CH-2), 108.2 (CH-5 and CH-8), 101.1 (CH2-10), 47.3 (CH2-1′), 42.0 (CH2-5′), 26.1 (CH2-2′), 25.2 (CH2-4′), 24.4 (CH2-3′); HMBC (1H irradiation ☛ 13C observed) δ 6.92 ☛ (C3, C-6 or C-7, C-9), 6.84 ☛ (C-3, C-7, C-8), 6.75 ☛ (C-4, C-6 or C-7), 6.52 ☛ (C-1, C-2, C-4, C-5), 5.95 ☛ (C-6 or C-7), 5.92 (C1, C-3, C-4), 3.60 (C-1, C-3), 3.35 (C-1, C-2′, C-3′, C-5′), 1.54 (C-1′, C-2′, C-5′). 2.6. Antiplasmodial bioassay 2.6.1. Plasmodium falciparum culture Assays used culture-adapted reference strain of Plasmodium falciparum type W2 from Vietnam, resistant to chloroquine, pyrimethamin and proguanil. Maintenance in continuous culture was done according to the methodology previously described [9]. Parasites were cultivated in 75 cm2 flasks containing RPMI 1640 medium (20 ml) supplemented with HEPES (25 mM; Gibco-BRL, Paisley, Scotland), NaHCO3 (25 mM), 10% of A + human serum and 1 ml of washed erythrocytes (final haematocrit 2.5%). Parasitaemia was maintained between 1 and 6%. Dilutions used non-infected A + erythrocytes. Cultures were incubated at 37 °C, 10% O2, 6% CO2, 84% N2, with 90% humidity. Medium renewal and microscopic observation (x100) by blood smears fixed with methanol and stained with 10% Giemsa's stain were performed on a daily basis. 2.6.2. Parasitaemia determination Parasite growth was assessed by flow cytometry according to a methodology previously described using hydroethidine (HE, Interchim, Montluçon, France) that is converted by metabolically active cells into ethidium [10]. After incubation with hydroethidine, parasitized and uninfected erythrocytes were all identified on the basis of fluorescence intensity and size. Triplicate assays were performed in 96-well tissue culture plates (Nunc Brand products, Fisher, Paris, France) containing 200 µl of asynchronous parasite cultures at 2% of parasitaemia and 2% haematocrit, and 5 µl of the appropriate extract dissolved in DMSO or water. Negative control treated by
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solvent (DMSO or H2O) and positive controls (Chloroquine) were added to each set of experiments. After 48 h incubation without medium change, plates were centrifuged and the upper liquids were replaced with 200 µl hydroethidine solution (0.05 mg/ml in PBS). After a 20 min incubation in the dark at 37 °C and 3 washes in PBS (Sigma, St Louis, MO, USA) a final dilution in 1 ml of PBS allowed determination by flow cytometry of the number of cell events (around 300 per second). Parasitaemia reading used a flow cytometer FACSort (Beckton Dickinson, Paris, France), equipped with an argon laser (power of 15 mW, and wavelength of 488 nm). Settings were: Forward Scatter (FSC-H), size: Voltage E-1, gain 1, mode Log; Side Scatter (SSC-H), granulosity: Voltage 250, gain1, mode Log; Fluorescence 2 (FL2), red fluorescence: Voltage 459, gain 1, mode Log.
2.6.3. Determination of the inhibitory concentration The IC values represented the mean value calculated from three independent experiments. The concentration of plant extract required to induce a 50% decrease of infected erythrocytes (IC50 Plasmodium) was calculated by non-linear regression analysis processed on dose–response curves, using the Table Curve Program.
2.6.4. Antiproliferative activity on human monocytic THP1 cells In vitro toxicity was assessed on human monocytic THP1 cells maintained at 37 °C in 6% CO2, in RPMI 1640 medium (Eurobio, Paris, France) supplemented with 10% foetal calf serum (Eurobio, Paris, France), 25 mM HEPES, 25 mM NaHCO3, and 1% Mix of 200 mM L-glutamine, 10,000 UI/ml Penicillin, 10 mg/ml Streptomycin (Sigma). Cells were subcultured every 7 days. THP1 cells were incubated with different concentrations of plant extracts previously dissolved in DMSO or distilled water. After a 72 h incubation at 37 °C and 6% CO2, in complete RPMI medium, cell growth was measured by flow cytometry after staining monocytes with 5 μl of propidium iodide (1 mg/ml, Sigma) according to a methodology previously described [10]. Antiproliferative activity was evaluated in triplicate assays by counting the number of viable cells on a 100 μl sample. Inhibitory concentration 50 (IC50) was defined as the concentration of plant extract required to induce a 50% decrease of cell growth compared to control cultures.
A selectivity index (SI), corresponding to the ratio between antiparasitic and cytotoxic activities, was calculated according to the following formula: SIPlasmodium = IC50
Human = IC50 Plasmodium :
3. Results and discussion
Compound 1 was isolated as an amorphous white powder. Its molecular formula of C14H15NO3 was determined by HREIMS (m/z 245.1052, calcd 245.1052). The 1H NMR spectrum showed signals due to five aromatic protons indicating the presence of a monosubstituted benzene ring at δ 7.23 (2 CH, br d, J = 7.2 Hz, H-11 and H-15), δ 7.28 (2 CH, br t, J = 7.2 Hz, H-12 and H-14) and δ 7.19 (CH, br t, J = 7.2 Hz, H-13) (Table 1). Further, it also showed signals due to two coupled methylene, as showed by COSY analysis, at δ 2.96 (CH2, br t, J = 6.9 Hz, H-9) and δ 3.24 (CH2, m, H-8). Furthermore, the 1H NMR spectrum coupled with a detailed analysis of 1H–1H COSY data defined a –CH2CH2CHCH– systeme with diasterotopic methylene protons at δ 4.33 (1 H, d, J = 13.5; 5.6 Hz, H-6)/3.18 (1 H, td, J = 13.2; 3.9 Hz, H-6); δ 2.40 (1 H, brd, J = 15.0 Hz, H-5)/1.98 (1 H, td, J = 15.0; 5.6 Hz, H-5); δ 3.66 (1 H, br s, H-4) and δ 3.56 (1H, d, J = 4.2 Hz, H-3). The 13C NMR spectrum indicated the presence of two carbonyl groups (δ 169.6 and 174.7). Furthermore, 1H and 13C NMR spectroscopic data indicated an amidic carbonyl (δ 169.6) and epoxidation at 3,4 position, δ 52.3 and 53.4. HMBC cross peak between the carbonyl carbon at δ 169.6 and the epoxymethine proton at δ 3.56 (H-3) demonstrated that the epoxy and carbonyl functionalities are vicinal. The full structural characterisation of 1 was then accomplished by examination of the 2D homo and heteronuclear correlated NMR spectra (Table 1) leading to the structure assignment of 1. The syn relationship for H-3 and H-4 was established by a 4.2 Hz coupling constant
Table 2 In vitro antiplasmodial activity of Piper capense compounds on W2 strain of Plasmodium falciparum, in vitro cytotoxic activity against human cell line (THP1) and selectivity index. Compounds
Kaousine 1 Z-antiepilepsirine 2 Apigenine dimethylether Chloroquine b a b c
Plasmodium falciparum, W2 (IC50)
THP1 cell line c (IC50)
µg/ml
µM
µg/ml
µM
20 7 12.5 –
82 27 42 0.7
34 N 50 17 –
139 N 193 57 40
Selectivity index was calculated according to the following formula: SIPlasmodium = IC50 Human/IC50 Plasmodium. Chloroquine was used as the antiplasmodial drug compound of reference. Doxorubicine was used as a drug compound of reference for human cell toxicity (IC50 = 0.06 µM).
SI a
1.7 N7 1.4 57
A.M. Kaou et al. / Fitoterapia 81 (2010) 632–635
observed between the two vicinal protons [11]. Thus the structure of 1 was determined as 3-(3-phenylpropanoyl)-7oxa-3-aza-bicyclo[4.1.0]heptan-2-one and named Kaousine. The molecular formula of Z-antiepilepsirine 2, C15H17 NO3, was deduced from EIMS (M+, 259) and the 1H and 13C NMR spectra. When comparing the 1H NMR spectrum of 2 with that of 1-[1-oxo-3(3,4-methylenedioxy-5-methoxyphenyl)2Z-propenyl]piperidine [4], the signals were superimposable except the downfield signals due to H-5, and H-9, the lack of signal corresponding to the methoxy group and the signal at δ 6.75 (1 H, d, J = 8.2 Hz) corresponding to H-8. COSY, HSQC and HMBC correlations confirmed the similarity between 2 and antiepilepsirine and led to the complete assignment of all the remaining protons of 2. The geometry of the double bonds (Δ2) was determined to be Z from the coupling constant of 12.4 Hz. This was corroborated by the downfield signals of H2 and H-3 in the Z isomer when compared with the E isomer [12]. The Z form of antiepilepsirine is isolated here for the first time. The antiplasmodial activity of the purified compounds was evaluated in vitro against the chloroquine-resistant strain W2 of Plasmodium falciparum, according to Azas, 2002 (Table 2). Lower activity was observed for Kaousine and Apigenine dimethylether (IC50 = 20 and 12.5 μg/ml), whereas Antiepilepsirine demonstrated the same activity that the chloromethylenic extract of P. capense (IC50 = 7 μg/ml). Due to the small amount available, Piperchabamide A could not be evaluated. Acknowledgements The authors wish to thank Professor Jean-Noël Labat and I. Yahaya for identification of plant material, and Fathi Mabrouki for technical assistance.
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References [1] Mohamed Kaou A, Mahiou-Leddet V, Hutter S, Aïnouddine S, Hassani S, Yahaya I, et al. Antimalarial activity of crude extracts from nine African medicinal plants. J Ethnopharmacol 2008;116:74–83. [2] Green TP, Galinis DL, Wiemer DF. Three neolignans from the roots of Piper capense. Phytochem 1991;30:1649–52. [3] Green TP, Wiemer DF. Four neolignan ketones from Piper capense. Phytochem 1991;30:3759–62. [4] Chen TB, Green TP, Wiemer DF. Capentin: a novel sesquiterpene from the roots of Piper capense. Tetrahedron Lett 1992;33:5673–6. [5] Martins AP, Salgueiro L, Vila R, Tomi F, Canigueral S, Casanova J, et al. Essential oils from four Piper species. Phytochem 1998;49:2019–23. [6] Wei K, Li W, Koike K, Pei Y, Chen Y, Nikaido T. New amide alkaloids from the roots of Piper nigrum. J Nat Prod 2004;67:1005–9. [7] Kartnig T, Bucar F, Neuhold S. Flavonoids from the aboveground part of Lycopus virginicus. Planta Med 1993;59:563–5364. [8] Morikawa T, Matsuda H, Yamaguchi I, Pongpiriyadacha Y, Yoshikawa M. New amides and gastroprotective constituents from the fruit of Piper Chaba. Planta Med 2004;70:152–9. [9] Trager W, Jensen J. Human malaria parasites in continuous culture. Science 1976;193:673–5. [10] Azas N, Laurencin N, Delmas F, Di Giorgio C, Gasquet M, Laget M, et al. Synergistic in vitro antimalarial activity of plant extracts used as traditional herbal remedies in Mali. Parasitol Res 2002;88:165–71. [11] Seeram NP, Lewis PA, Jacobs H. 3, 4-epoxy-8, 9-dihydropiplartine. A new amide from Piper verrucosum. J Nat Prod 1996;59:436–7. [12] Venkatasamy R, Faas L, Young AR, Raman A, Hider RC. Effects of piperine analogues on stimulation of melanocyte proliferation and melanocyte differentiation. Bioorg Med Chem 2004;12:1905–20.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
New amide alkaloid from the aerial part of Piper capense L.f. (Piperaceae) Ali Mohamed Kaou a, Valérie Mahiou-Leddet a,⁎, Cécile Canlet b, Laurent Debrauwer b, Sébastien Hutter c, Nadine Azas c, Evelyne Ollivier a a Laboratoire de Pharmacognosie et Ethnopharmacologie, UMR-MD3, Faculté de Pharmacie, Université de la Méditerranée (Aix-Marseille II), 27 Bd Jean Moulin, 13385 Marseille, Cedex 5, France b Laboratoire des Xénobiotiques, INRA, UMR1089, 180 Chemin de Tournefeuille BP 93173, 31027 Toulouse Cedex 3, France c Laboratoire de Parasitologie, UMR-MD3, Faculté de Pharmacie, Université de la Méditerranée (Aix-Marseille II), 27 Bd Jean Moulin, 13385 Marseille, Cedex 5, France
a r t i c l e
i n f o
Article history: Received 1 September 2009 Accepted 6 March 2010 Available online 20 March 2010
a b s t r a c t Together with apigenine dimethylether and piperchabamide A, a new amide alkaloid, Kaousine and the Z form of antiepilepsirine were isolated from the aerial part of Piper capense L.f (Piperaceae). Their structures were elucidated by spectrometric methods and their in vitro antiparasitic activities were evaluated on Plasmodium falciparum. © 2010 Elsevier B.V. All rights reserved.
Keywords: Piper capense Piperaceae Amide alkaloid Comoro Antiplasmodial activity Plasmodium falciparum
1. Introduction In a search for novel antiparasitic natural products, we have studied the aerial part of Piper capense L.f (Piperaceae). This plant is traditionally used in Comoro Islands for diarrhoea and cough, as shown by our previous ethnobotanical survey [1]. The antiplasmodial activity of different extracts was evaluated in screening tests in vitro against the chloroquine-resistant strain W2 of Plasmodium falciparum and showed that the chloromethylenic extract of P. capense had moderate in vitro activity (IC50 =7 μg/ml). Previous phytochemical studies carried out on P. capense have resulted in the identification of lignans [2,3], sesquiterpenes [4], and essential oil with high percentage of monoterpene hydrocarbons [5]. In the present paper, we describe from the chloromethylenic extract of P. capense the isolation of a new amide alkaloid, Kaousine, 1 together with the Z form of antiepilepsirine 2 [6], the known apigenine dimethylether [7], and piperchabamide A [8], isolated for the first time
from this plant material. The structures were established using NMR and mass spectrometry and spectroscopic data of known compounds were compared with reported values. 2. Experimental 2.1. General UV spectra were recorded on a Beckman DU 520 spectrophotometer and IR spectrum was taken on a Brücker Vertex 70 spectrometer. NMR spectra were recorded in CDCl3 (δ ppm) on Brücker DRX 600 spectrometer operating at 600.13 MHz and 150.91 MHz. EIMS spectra were obtained on an Ion Trap Finigan (Thermo Quest) spectrometer. HREIMS spectra were obtained on a QStar Elite (Applied Biosystems SCIEX) spectrometer. Optical rotations were taken on a Perkin Elmer 341 OROT 589 nm Polarimeter. 2.2. Plant material
⁎ Corresponding author. Tel./fax: +33 4 9183 5593. E-mail address: [email protected] (V. Mahiou-Leddet). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.03.006
The aerial part of P. capense was collected by Ali Mohamed Kaou and Ibrahim Yahaya, in the island Ngazidja (Grande
A.M. Kaou et al. / Fitoterapia 81 (2010) 632–635
Comore Island) in the Grille forest in January 2004. Botanical determination of P. capense L.f. (Piperaceae) was performed by I. Yahaya and Pr J. N. Labat. Voucher specimens were deposited in the National Herbarium of the Museum Nationnal d'Histoire Naturelle (Paris, France, Piperaceae, no. P00440614). 2.3. Extraction and isolation Extraction method: Aerial part of P. capense (1.1 kg) was dried, powdered and submitted successively to a maceration process with 5 l CH2Cl2 for 14–15 h at room temperature. The extract was evaporated to dryness under vacuum affording the first CH2Cl2 extract (residue 21.5 g). The CH2Cl2 extract was fractionated by successive chromatographies on Kieselgel 60 Merck® (40–63 µm), sephadex LH 20 Pharmacia Biotech and preparative TLC 20 × 20 cm Kieselgel 60 F254 Merck® 0.5 mm: The CH2Cl2 extract (18 g) was subjected to CC (silica gel) eluting with CH2Cl2/MeOH (100/0, 98/2, 95/5, 90/10, 85/15, 80/20), in order of increasing polarity, to give 10 main fractions (G1–G10). Fraction G4 (225 mg) was separately fractionated into 4 subfractions (G4.1–G4.4) on silica gel column using toluene-AcOEt (100/0, 90/10, 80-20), whereas fraction G4.3 yielded Kaousine, 1 (13 mg). Fraction G6 (220 mg) was separated by silica gel CC using tolueneAcOEt (80/20) to obtain 3 subfractions (G6.1–G6.3). Fraction G6.2 (95 mg) was further purified by silica gel CC using toluene-AcOEt (80/20) as eluent to give apigenine dimethylether (11 mg). A part of CH2Cl2 extract (3.5 g) was subjected to CC (silica gel) eluting with CH2Cl2/MeOH (98/2, 95/5, 90/ 10, 85/15, 80/20), to give 9 main fractions (F1–F9). Fraction F5 (26 mg) was further purified by preparative TLC using CH2Cl2/MeOH (50/50) as eluent to give piperchabamide A (3.5 mg). Fraction F8 (2.15 g) was separately fractionated by silica gel CC using CH2Cl2-AcOEt (100/0, 97/3, 95/5, 90/10, 80/ 20) into 7 subfractions (F8.1–F8.7) whereas fraction F8.5 yielded: Z-antiepilepsirine, 2 (11 mg). Silica gel GF254 Merck® was used for TLC. 2.4. Kaousine (1) 3-(3-phenylpropanoyl)-7-oxa-3-aza-bicyclo[4.1.0]heptan-2-one, C14H15NO3, amorphous, [α]20 D + 17 (MeOH, c 0.115); UV λmax (MeOH) nm (Log ε): 211 (4.06).; IRfilm max ν cm−1: 3065; 3030, 2962, 2928, 2857, 2257, 1699, 1604, 1496, 1471, 1454, 1438, 1417, 1392, 1368, 1340, 1310, 1266, 1223, 1174, 1129, 1095, 1074, 1065, 1030; HREIMS m/z (%): 245.1052 (M+, 100) [245.1052calcd], EIMS 228 (18), 176 (100), 131 (44), 101 (65), 91 (39). For 1H and 13C NMR (600.13 and 150.91 MHz, CDCl3) data, see Table 1. 2.5. Z-antiepilepsirine (2) C15H17NO3, amorphous, [α]20 D + 10 (CHCl3, c 0.15); UV: λmax (MeOH) nm (Log ε) 210 (4.17), 273 (3.76), 306 (3.68). EIMS 259 (M+, 100), 175 (66), 145 (100), 117 (39), 89 (64). 1 H NMR (600.13 MHz, CDCl3) δ 6.92 (d, J = 1.5 Hz, H-5), 6.84 (dd, J = 8.2, 1.5 Hz, H-9), 6.75 (d, J = 8.2 Hz, H-8), 6.52 (d, J = 12.4 Hz, H-3), 5.95 (s, H-10), 5.92 (d, J = 12.4 Hz, H-2), 3.60 (m, H-5′), 3.35 (m, H-1′), 1.54 (m, H-2′ and H-4′), 1.27 (m, H-3′); 13C NMR (150.91 MHz, CDCl3) δ 167.3 (C-1), 147.8
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Table 1 1 H and 13C NMR data for compound 1 (600.13 and 150.91 MHz, CDCl3, J in Hz, δ in ppm). Position δΗ 2 3 4 5 6 7 8 9 10 11 12 13 14 15
– 3.56 (d, 4.2) 3.66 (brs) 2.40 (brd, 15.0) 1.98 (td, 15.0, 5.6) 4.33 (dd, 13.5, 5.6) 3.18 (td, 13.2, 3.9) – 3.24 m 2.96 (brt, 6.9) – 7.23 (brd, 7.2) 7.28 (brt, 7.2) 7.19 (brt, 7.2) 7.18 (brt, 7.2) 7.23 (brt, 7.2)
δC
H–H COSY
HMBC (H to C)
169.6 52.3 53.4 23.8
– 4 3, 5 (2.40) 5 (1.98), 6 5 (2.40), 6 5 (1.98), 6(3.18) 5, 6 (4.33) – 9 8 – – – – – –
– 2 5, 6 3 6 2, 4, 5, 7 4, 5 – 7, 9, 10 7, 8, 10, 11 – 9, 13, 14 10, 11 11/15, 12/14 10, 15 9, 11, 13
35.6 174.7 41.1 30.8 140.9 128.4 128.5 126.1 128.5 128.4
(C-6), 147.6 (C-7), 132.3 (CH-3), 129.8 (C-4), 123.1 (CH-9), 121.9 (CH-2), 108.2 (CH-5 and CH-8), 101.1 (CH2-10), 47.3 (CH2-1′), 42.0 (CH2-5′), 26.1 (CH2-2′), 25.2 (CH2-4′), 24.4 (CH2-3′); HMBC (1H irradiation ☛ 13C observed) δ 6.92 ☛ (C3, C-6 or C-7, C-9), 6.84 ☛ (C-3, C-7, C-8), 6.75 ☛ (C-4, C-6 or C-7), 6.52 ☛ (C-1, C-2, C-4, C-5), 5.95 ☛ (C-6 or C-7), 5.92 (C1, C-3, C-4), 3.60 (C-1, C-3), 3.35 (C-1, C-2′, C-3′, C-5′), 1.54 (C-1′, C-2′, C-5′). 2.6. Antiplasmodial bioassay 2.6.1. Plasmodium falciparum culture Assays used culture-adapted reference strain of Plasmodium falciparum type W2 from Vietnam, resistant to chloroquine, pyrimethamin and proguanil. Maintenance in continuous culture was done according to the methodology previously described [9]. Parasites were cultivated in 75 cm2 flasks containing RPMI 1640 medium (20 ml) supplemented with HEPES (25 mM; Gibco-BRL, Paisley, Scotland), NaHCO3 (25 mM), 10% of A + human serum and 1 ml of washed erythrocytes (final haematocrit 2.5%). Parasitaemia was maintained between 1 and 6%. Dilutions used non-infected A + erythrocytes. Cultures were incubated at 37 °C, 10% O2, 6% CO2, 84% N2, with 90% humidity. Medium renewal and microscopic observation (x100) by blood smears fixed with methanol and stained with 10% Giemsa's stain were performed on a daily basis. 2.6.2. Parasitaemia determination Parasite growth was assessed by flow cytometry according to a methodology previously described using hydroethidine (HE, Interchim, Montluçon, France) that is converted by metabolically active cells into ethidium [10]. After incubation with hydroethidine, parasitized and uninfected erythrocytes were all identified on the basis of fluorescence intensity and size. Triplicate assays were performed in 96-well tissue culture plates (Nunc Brand products, Fisher, Paris, France) containing 200 µl of asynchronous parasite cultures at 2% of parasitaemia and 2% haematocrit, and 5 µl of the appropriate extract dissolved in DMSO or water. Negative control treated by
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solvent (DMSO or H2O) and positive controls (Chloroquine) were added to each set of experiments. After 48 h incubation without medium change, plates were centrifuged and the upper liquids were replaced with 200 µl hydroethidine solution (0.05 mg/ml in PBS). After a 20 min incubation in the dark at 37 °C and 3 washes in PBS (Sigma, St Louis, MO, USA) a final dilution in 1 ml of PBS allowed determination by flow cytometry of the number of cell events (around 300 per second). Parasitaemia reading used a flow cytometer FACSort (Beckton Dickinson, Paris, France), equipped with an argon laser (power of 15 mW, and wavelength of 488 nm). Settings were: Forward Scatter (FSC-H), size: Voltage E-1, gain 1, mode Log; Side Scatter (SSC-H), granulosity: Voltage 250, gain1, mode Log; Fluorescence 2 (FL2), red fluorescence: Voltage 459, gain 1, mode Log.
2.6.3. Determination of the inhibitory concentration The IC values represented the mean value calculated from three independent experiments. The concentration of plant extract required to induce a 50% decrease of infected erythrocytes (IC50 Plasmodium) was calculated by non-linear regression analysis processed on dose–response curves, using the Table Curve Program.
2.6.4. Antiproliferative activity on human monocytic THP1 cells In vitro toxicity was assessed on human monocytic THP1 cells maintained at 37 °C in 6% CO2, in RPMI 1640 medium (Eurobio, Paris, France) supplemented with 10% foetal calf serum (Eurobio, Paris, France), 25 mM HEPES, 25 mM NaHCO3, and 1% Mix of 200 mM L-glutamine, 10,000 UI/ml Penicillin, 10 mg/ml Streptomycin (Sigma). Cells were subcultured every 7 days. THP1 cells were incubated with different concentrations of plant extracts previously dissolved in DMSO or distilled water. After a 72 h incubation at 37 °C and 6% CO2, in complete RPMI medium, cell growth was measured by flow cytometry after staining monocytes with 5 μl of propidium iodide (1 mg/ml, Sigma) according to a methodology previously described [10]. Antiproliferative activity was evaluated in triplicate assays by counting the number of viable cells on a 100 μl sample. Inhibitory concentration 50 (IC50) was defined as the concentration of plant extract required to induce a 50% decrease of cell growth compared to control cultures.
A selectivity index (SI), corresponding to the ratio between antiparasitic and cytotoxic activities, was calculated according to the following formula: SIPlasmodium = IC50
Human = IC50 Plasmodium :
3. Results and discussion
Compound 1 was isolated as an amorphous white powder. Its molecular formula of C14H15NO3 was determined by HREIMS (m/z 245.1052, calcd 245.1052). The 1H NMR spectrum showed signals due to five aromatic protons indicating the presence of a monosubstituted benzene ring at δ 7.23 (2 CH, br d, J = 7.2 Hz, H-11 and H-15), δ 7.28 (2 CH, br t, J = 7.2 Hz, H-12 and H-14) and δ 7.19 (CH, br t, J = 7.2 Hz, H-13) (Table 1). Further, it also showed signals due to two coupled methylene, as showed by COSY analysis, at δ 2.96 (CH2, br t, J = 6.9 Hz, H-9) and δ 3.24 (CH2, m, H-8). Furthermore, the 1H NMR spectrum coupled with a detailed analysis of 1H–1H COSY data defined a –CH2CH2CHCH– systeme with diasterotopic methylene protons at δ 4.33 (1 H, d, J = 13.5; 5.6 Hz, H-6)/3.18 (1 H, td, J = 13.2; 3.9 Hz, H-6); δ 2.40 (1 H, brd, J = 15.0 Hz, H-5)/1.98 (1 H, td, J = 15.0; 5.6 Hz, H-5); δ 3.66 (1 H, br s, H-4) and δ 3.56 (1H, d, J = 4.2 Hz, H-3). The 13C NMR spectrum indicated the presence of two carbonyl groups (δ 169.6 and 174.7). Furthermore, 1H and 13C NMR spectroscopic data indicated an amidic carbonyl (δ 169.6) and epoxidation at 3,4 position, δ 52.3 and 53.4. HMBC cross peak between the carbonyl carbon at δ 169.6 and the epoxymethine proton at δ 3.56 (H-3) demonstrated that the epoxy and carbonyl functionalities are vicinal. The full structural characterisation of 1 was then accomplished by examination of the 2D homo and heteronuclear correlated NMR spectra (Table 1) leading to the structure assignment of 1. The syn relationship for H-3 and H-4 was established by a 4.2 Hz coupling constant
Table 2 In vitro antiplasmodial activity of Piper capense compounds on W2 strain of Plasmodium falciparum, in vitro cytotoxic activity against human cell line (THP1) and selectivity index. Compounds
Kaousine 1 Z-antiepilepsirine 2 Apigenine dimethylether Chloroquine b a b c
Plasmodium falciparum, W2 (IC50)
THP1 cell line c (IC50)
µg/ml
µM
µg/ml
µM
20 7 12.5 –
82 27 42 0.7
34 N 50 17 –
139 N 193 57 40
Selectivity index was calculated according to the following formula: SIPlasmodium = IC50 Human/IC50 Plasmodium. Chloroquine was used as the antiplasmodial drug compound of reference. Doxorubicine was used as a drug compound of reference for human cell toxicity (IC50 = 0.06 µM).
SI a
1.7 N7 1.4 57
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observed between the two vicinal protons [11]. Thus the structure of 1 was determined as 3-(3-phenylpropanoyl)-7oxa-3-aza-bicyclo[4.1.0]heptan-2-one and named Kaousine. The molecular formula of Z-antiepilepsirine 2, C15H17 NO3, was deduced from EIMS (M+, 259) and the 1H and 13C NMR spectra. When comparing the 1H NMR spectrum of 2 with that of 1-[1-oxo-3(3,4-methylenedioxy-5-methoxyphenyl)2Z-propenyl]piperidine [4], the signals were superimposable except the downfield signals due to H-5, and H-9, the lack of signal corresponding to the methoxy group and the signal at δ 6.75 (1 H, d, J = 8.2 Hz) corresponding to H-8. COSY, HSQC and HMBC correlations confirmed the similarity between 2 and antiepilepsirine and led to the complete assignment of all the remaining protons of 2. The geometry of the double bonds (Δ2) was determined to be Z from the coupling constant of 12.4 Hz. This was corroborated by the downfield signals of H2 and H-3 in the Z isomer when compared with the E isomer [12]. The Z form of antiepilepsirine is isolated here for the first time. The antiplasmodial activity of the purified compounds was evaluated in vitro against the chloroquine-resistant strain W2 of Plasmodium falciparum, according to Azas, 2002 (Table 2). Lower activity was observed for Kaousine and Apigenine dimethylether (IC50 = 20 and 12.5 μg/ml), whereas Antiepilepsirine demonstrated the same activity that the chloromethylenic extract of P. capense (IC50 = 7 μg/ml). Due to the small amount available, Piperchabamide A could not be evaluated. Acknowledgements The authors wish to thank Professor Jean-Noël Labat and I. Yahaya for identification of plant material, and Fathi Mabrouki for technical assistance.
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