Antibacterial and Antiplasmodial Constituents of Beilschmiedia

Article pubs.acs.org/jnp

Antibacterial and Antiplasmodial Constituents of Beilschmiedia cryptocaryoides Ferdinand Mouafo Talontsi,† Marc Lamshöft,† Jonathan O. Bauer,‡ Andrianambinina A. Razakarivony,§ Bakoli Andriamihaja,§ Carsten Strohmann,‡ and Michael Spiteller*,† †

Institute of Environmental Research (INFU) of the Faculty of Chemistry, Chair of Environmental Chemistry and Analytical Chemistry, TU Dortmund, Otto-Hahn-Straße 6, D-44221 Dortmund, Germany ‡ Faculty of Chemistry, TU Dortmund, Otto-Hahn-Straße 6, 44227 Dortmund, Germany § Laboratory of Applied Chemistry for Natural Products, Department of Organic Chemistry, Faculty of Sciences, University of Antananarivo, Madagascar S Supporting Information *

ABSTRACT: Four new beilschmiedic acid derivatives, cryptobeilic acids A−D (1−4), and tsangibeilin B (5) have been isolated from the bark of Beilschmiedia cryptocaryoides collected from Madagascar. Their structures were elucidated using detailed spectroscopic and spectrometric methods. Cryptobeilic acid A (1) and tsangibeilin B (5) structures were confirmed by single-crystal X-ray diffraction analysis. Compounds 1−5 displayed moderate antibacterial activity against Escherichia coli 6r3, Acinetobacter calcoaceticus DSM 586, and Pseudonamas stutzeri A1501, with the minimum inhibitory concentrations ranging from 10 to 50 μM, respectively. In addition, the compounds exhibited antiplasmodial activity against erythrocytic stages of chloroquine-resistant Plasmodium falciparum strain NF54 and weak cytotoxicity against L6 cell lines.

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RESULTS AND DISCUSSION Chemical screening of the dichloromethane extract from B. cryptocaryoides using high-resolution mass spectrometry (HRMS) exhibited several peaks that were typical of beilschmiedic/endiandric acid derivatives.3 The diverse composition of the extract was revealed by detailed evaluation of the HRMS data. Besides the numerous signals in the total ion chromatogram (TIC) (m/z 200−800), the high-resolution masses extracted from the ion chromatograms (±2 ppm) of selected quasi-molecular ions (C20H29O2 → 301.21621; C20H27O3 → 315.19547; C22H21O5 → 365.13835; C22H23O4 → 351.15909) represented possible beilschmiedic/endiandric acid derivatives and indicated the presence of previously unknown derivatives (Figure 1). Further MS/MS experiments of the observed ion peaks confirmed this prediction. The fragmentation of ions with m/z 301 and 315 resulted in a product ion m/z 171, which was formed after decarboxylation and cleavage of the aliphatic chain. On the basis of these screening results four new beilschmiedic acid derivatives, namely, cryptobeilic acids A−D (1−4), were isolated using a combination of analytical and chromatographic techniques (LC-MS, preparative HPLC, silica gel, and Sephadex LH-20). Cryptobeilic acid A (1) was obtained as an optically inactive pale yellow powder. The molecular formula C20H28O3 was assigned to 1 by HRMS, suggesting seven double-bond

he genus Beilschmiedia belongs to the Lauraceae family and comprises about 250 species widely distributed in tropical and subtropical regions of the world.1 This genus is represented in tropical Africa and Madagascar by 80 species, and most of them are used locally in traditional medicine to treat uterine tumors, rheumatism, pulmonary disorders, dysentery, and headache, and as food.1,2 Beilschmiedia cryptocaryoides Kosterm. (Lauraceae) is an evergreen tree endemic to Madagascar, whose fruit, bark, and leaves are used by the local population to treat infectious diseases and malaria.2 Beilschmiedic/endiandric acids are mainly isolated as racemic mixtures, possess antibacterial and anti-inflammatory potentials, and are the most characteristic type of natural products reported from Beilschmiedia species.3−8 However, other than some traditional knowledge, no report regarding the chemical constituents of B. cryptocaryoides has been published so far. As part of our research program for identifying new bioactive secondary metabolites from natural sources,9 bioassay-guided study of the active constituents of B. cryptocaryoides bark using antibacterial and antiplasmodial activity against erythrocytic stages of chloroquine-resistant Plasmodium falciparum strain NF54 was carried out. As a result of the chemical studies, four new beilschmiedic acid derivatives, cryptobeilic acids A−D (1−4), and tsangibeilin B (5)10 were isolated and characterized. Herein we describe the isolation and structural elucidation of compounds 1−4 as well as their biological potentials. © 2013 American Chemical Society and American Society of Pharmacognosy

Received: November 6, 2012 Published: January 15, 2013 97

dx.doi.org/10.1021/np300773x | J. Nat. Prod. 2013, 76, 97−102

Journal of Natural Products

Article

HMBC correlations (Figure 2) from H-4 (δH 4.17) and H-5 (δH 6.92) to C-1′ (δC 169.0), C-3, C-6, C-7, and C-13; from H8 (δH 5.39) to C-6, C-7, C-9, and C-13; from H-9 (δH 5.57) to C-8, C-10, C-11, C-12, and C-13; and from H-1 (δH 2.51) to C2, C-3, C-11, and C-12 established the tetracyclic partial structure of 1 with one oxygen group attached to C-4 (δC 64.1) as for beilschmiedic acid A.3 The chemical shift of H-4 (δH 4.17, d, J = 3.8 Hz) and H-5 (δH 6.92, dd, J = 3.8, 5.5 Hz) suggested a β-orientation of the OH group.3,8 Further analysis of the 1 H−1H COSY (Figure 2) and HMBC data indicated that the alkyl side chain was connected at C-11 and that the side chain had two methylene groups less than beilschmiedric acid A.3 Detailed NMR assignments are shown in Tables 1 and 2. The 1 H−1H coupling constants and NOESY data of 1 suggested the same relative configuration as reported for beilschmiedric acid A.3,8 The proposed structure and the relative configuration were later confirmed by X-ray diffraction analysis of a crystal obtained from methanol (Figure 5a). Thus, the structure of 1 was elucidated as shown, and it was named cryptobeilic acid A. High-resolution mass analysis exhibited for 2, the second optically inactive isolate, revealed the same molecular formula (C20H28O3) as 1, with seven double-bond equivalents. Analysis of its NMR data showed resonances nearly identical to those of 1 (Tables 1 and 2), except that the chemical shifts for the C-4 oxymethine in 1 (δH/δC 4.17/64.1) differed from those of 2 (δH/δC 4.25/73.1), as well as the coupling constants observed between H-4 and H-5 and between H-4 and H-3. These data implied that 2 was a stereoisomer of 1 at C-4. The 1H−1H coupling constants and the relatively high coupling constant (J4,5 = 9.6 Hz) between H-4 and H-5 suggested an αorientation of the OH group at C-4, the same as that of beilschmiedic acid H.3,8 Thus, the structure of 2 was elucidated to be as shown, and it was named cryptobeilic acid B.

equivalents. Its IR spectrum had absorption bands for OH groups at 3360 cm−1 and indications of an α,β-unsaturated carbonyl at 1686 cm−1. The NMR data (Tables 1 and 2) for 1 displayed OH protons at δH 4.84 (d, J = 5.5 Hz, OH-4) and 12.41 (br, OH-1″), seven methylenes, one methyl triplet, eight methines [one of which was oxygenated at δH 4.17 (d, J = 3.8 Hz, H-4)], four olefinic carbons (three of which were protonated), and one α,β-unsaturated carbonyl at δC 169.0.

Figure 1. UV chromatogram, TIC (total ion chromatogram), and high-resolution EICs (extracted ion chromatogram) of four selected masses (±2 ppm) of dichloromethane extract from B. cryptocaryoides. 98



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Table 2. 13C NMR Data of Compounds 1−4a

Table 1. 1H NMR Data of Compounds 1−4 (600 MHz, in CDCl3, δ in ppm, J in Hz)a no.

2

3

2.51 (m) 1.21 (m) 1.54 (m)

2.35 (m) 1.32 (m) 1.50 (m)

2.48 (m) 1.55 (m) 1.65 (m)

3

1.91 (m)

1.84 (m)

3.03 (ddd, 5.7, 12.9)

4

4.17 (brd, 3.8)

4.25 (d,9.6)

5

6.92 (brdd, 3.8, 5.5)

7.06 (brs)

6.86 (brs)

3.07 (brs) 5.39 (d, 10.2)

3.20 (brs) 5.57 (d, 10.4)

3.47 (brd, 4.7) 5.46 (brd, 10.7) 5.60 (dt, 3.4, 10.7) 2.29 (m) 1.70 (m) 2.83 (m) 2.46 (m) 2.60 (m) 2.70 (m)

6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ OH-4 OH-1″ a

1

1 2

5.57 (dt, 3.4, 10.2) 1.94 (m) 1.31 (m) 2.65 (m) 2.25 (m) 1.44 (m) 1.23 (m) 1.34−1.23 (m)

5.64 (dt, 3.4, 10.2) 1.88 (m) 1.30 (m) 2.79 (m) 2.30 (m) 1.48 (m) 1.29 (m) 1.32−1.29 (m) 1.34−1.23 (m) 1.32−1.29 (m) 1.34−1.23 (m) 1.32−1.29 (m) 1.34−1.23 (m) 1.30−1.29 (m) 0.85 (t, 7.1) 0.88 (t, 6.9)

6.68 (d, 7.9) 6.57 (dd, 7.9, 1.2)

4 2.25 (m) 1.34 (m) 1.60 (dd, 11.7, 5.4) 2.58 (m) 6.22 (dt, 9.7, 2.8) 5.75 (dt, 9.7, 2.8) 3.03 (m) 2.99 (m) 5.47 (brd, 10.0) 5.66 (dt, 10.0, 3.4) 2.29 (m) 1.48 (m) 2.66 (m) 1.72 (m) 1.50 (m) 1.28 (m) 1.26 (m)

a

no.

1b

2c

3b

4b

1 2 3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″

41.0 32.4 42.5 64.1 142.0 136.1 34.1 125.8 128.7 35.9 46.7 33.1 34.6 37.5 29.7 32.5 32.2 23.0 14.8

41.6 34.1 44.4 73.1 145.6 134.7 35.6 127.4 127.7 41.1 46.1 34.1 34.7 37.5 28.9 32.2 33.6 23.0 14.0

35.4 35.2 37.4 134.9 124.3 49.7 33.2 129.8 130.6 41.6 46.4 33.5 42.5 37.6 27.4 29.7 32.3 23.1 14.5

169.0

167.8

34.0 30.4 49.6 202.0 134.3 149.8 35.0 121.7 130.6 43.7 48.1 34.1 40.2 42.6 134.3 108.5 121.9 146.2 148.0 109.3 101.2 170.7

181.3 b

Assigned by COSY, HSQC, and HMBC experiments. Recorded in CDCl3 (150 MHz, δ in ppm). cRecorded in acetone-d6 (150 MHz, δ in ppm).

1.30−1.26 (m) 1.30−1.26 (m) 1.29−1.26 (m) 0.89 (t, 6.8)

6.60 (brd, 1.2) 5.87 (s) 4.84 (d, 5.5) 12.41 (br)

8.84 (brs)

Figure 2. Selected HMBC (arrow) and 1H−1H COSY (bold) correlations for 1.

9.50 (brs)

Assigned by COSY, HSQC, and HMBC experiments.

Compound 3 was assigned the molecular formula C22H20O5 on the basis of HRMS, accounting for 12 double-bond equivalents. Its IR spectrum had absorption bands for OH groups at 3345 cm−1, α,β-unsaturated carbonyls (1700 and 1644 cm−1), and indications of a substituted aromatic ring (1590, 756 cm−1). The 1H and 13C NMR data indicated one exchangeable proton at δH 8.84 (br, OH-1″), two α,βunsaturated carbonyl atoms (δC 170.7, C-1″ and 202.0, C-4), two methylene groups, 10 olefinic/aromatic carbons (six of which were protonated), seven methines, and one methylenedioxy group (δH 5.87/δC 101.2). These data suggested that the tetracyclic part of 3 was nearly identical to that of 1, except that the C-4 oxymethine was oxidized to a ketone as in beilschmiedic acid F.11 Interpretation of 1H−1H COSY and HMBC data (Figure 3) confirmed the tetracyclic moiety, and correlations from H-5 (δH 6.86, brs) to C-4 (δC 202.0), C-6 (δC 149.8), and C-1″ (δC 170.7) and from H-7 (δH 3.47, brd) to C1″ (δC 170.7), C-6 (δC 149.8), and C-8 (δC 121.7) placed the carboxylic acid group at C-1″ as in 1. However, correlations from H-11 (δH 1.70, m) to C-1 (δC 34.0), C-10 (δC 43.7), and C-1′ (δC 42.6) and from H2-1′ (δH 2.60) to C-11 and C-2′ supported the presence of one 11-substituted methylenedioxyaromatic moiety as in tsagibeilin A.10 Further analysis of 2D NMR data (Figure 3) confirmed the structure of 3 as shown.



Compound 4 had the molecular formula C20H28O2 with seven double-bond equivalents and with five methylene groups less than erythrophloin B.12 Analysis of its 1H and 13C NMR data (Tables 1 and 2) confirmed the same tetracyclic moiety and also the alkyl side chain. Interpretation of the 1H−1H COSY and HMBC data (Figure 4) confirmed the proposed structure of 4. Its congener 5, which was obtained as a major product during this study, featured the same tetracyclic 99



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Figure 5. (a) ORTEP plot of cryptobeilic acid (1) with the displacement ellipsoids drawn at the 50% probability level. (b) ORTEP plot of tsangibeilin B (5) with the displacement ellipsoids drawn at the 50% probability level.

Table 3. Antibacterial, Antiplasmodial, and Cytotoxicity (L6) Potentials of Compounds 1−5 antimicrobial activitya (MIC, μg/mL) compound

E.cc

A.cd

P.se

S.pf

antiplasmodial activityb (IC50, μM)

1 2 3 4 5 difloxacin ampicilin chloroquine podophyllotoxin

10 20 >50 50 50 5 5

>50 20 >50 >50 >50 5 5

>50 10 >50 >50 >50 20 20

>50 >50 >50 >50 >50 20 20

17.7 5.35 14 10.8 8.2

cytotoxicityb (IC50, μM) 59.5 20.4 59.3 61 21.5

0.002 0.008

Each MIC value was assayed in triplicate. bIC50 values are the means of two independent assays; the individual values varied less than ±50%. c Escherichia coli 6r3. dAcinetobacter calcoaceticus DSM 586. ePseudonamas stutzeri A1501. fSerratia plymuthica C48. a

for antiplasmodial activity against chloroquine-resistant Plasmodium falciparum strain NF54 (Table 3). All of the tested compounds exhibited antiplasmodial activity against the NF54 strain, with cryptobeilic acid B (2) showing the best potency, with an IC50 value of 5.325 μM, followed by compounds 5 [8.2 μM] and 4 [10.8 μM], compared to chloroquine [0.002 μM] (Table 3). However, the IC50 values against the L6 cell lines indicated higher cytotoxicity of compound 2 (20.4 μM) (Table 3) compared to compounds 4, 1, and 3. Using high-resolution mass spectrometry as chemical screening of the crude extract enabled the isolation and structural elucidation of four new beilschmiedic acid derivatives, namely, cryptobeilic acids A−D (1−4), from B. cryptocaryoides collected at Ranomafana-Ifanadiana, Madagascar. The new compounds 1 and 2 showed moderate antibacterial activity in one or more of four bacterial strains (Escherichia coli 6r3, Acinetobacter calcoaceticus DSM 586, Pseudonamas stutzeri A1501, and Serratia plymuthica C48) and in vitro antiplasmodial potential. Compounds 1−5 were all weakly cytotoxic on L6 cells. These

structure and differed from 4 only in the side chain. Complete assignment of the NMR data of 4 was achieved through 2D NMR experiments. All of the physicochemical data are in full agreement with the proposed structure of 4 as shown, which was named cryptobeilic acid D. In addition, the single-crystal Xray structure of tsangibeilin B (5) is reported herein for the first time in Figure 5b. Beilschmiedic/endiandric acids have been isolated frequently as racemates from species of Beilschmiedia and Endiandra and reported to exhibit a number of biological properties, including antibacterial,3,5 anti-inflammatory,10 and cytotoxic8b activities. Given the traditional use of B. cryptocaryoides to treat infectious diseases and the fact that some Beilschmiedia species are used for malaria treatment, compounds 1−5 were assayed for their antimicrobial, antiplasmodial, and cytotoxic activities (Table 3). Compounds 1 and 2 showed higher antimicrobial activity against Escherichia coli 6r3, Acinetobacter calcoaceticus DSM 586, and Pseudonamas stutzeri A1501 compared to 5, as shown in Table 3. Furthermore, compounds 1−5 were assayed in vitro 100



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nine subfractions (Fr.3A1 to Fr.3A9) after TLC and LC-MS analyses. The active subfraction Fr.A3A4 was subjected to preparative HPLC (Nucleodur column C18 Isis) using MeOH−H2O as eluent to yield tsangibeilin B (5, 17.2 mg, tR 13.5 min) and cryptobeilic acid A (4, 3.6 mg, tR 14.2 min), while fraction Fr.3A3 purified by preparative HPLC (Nucleodur C18 Isis column) with MeOH−H2O as eluent afforded cryptobeilic acid A (1, 3.1 mg; tR 11.4 min), cryptobeilic acid B (2, 2.4 mg; tR 11.6 min), and cryptobeilic acid C (3, 5.2 mg; tR 12.7 min). The other fractions and subfractions were analyzed by high-resolution LCMS and found to contain only known compounds. Cryptobeilic acid A (1): pale yellow powder; [α]20D ±0 (c 0.20, CHCl3); IR (film) νmax 3360, 2923, 1686, 1238, 763 cm−1; LC-UV [(MeOH(aq) in H2O/0.05% FA)] λmax 205 and 225 nm; 1H NMR and 13C NMR (CDCl3) see Tables 1 and 2; HRMS m/z 315.19543 [M + H]+ (calcd for C20H27O3, 315.19547). X-ray crystallographic analysis of cryptobeilic acid A (1): pale yellow blocks, 0.20 × 0.10 × 0.10 mm3, C20H28O3, Mr = 316.42, triclinic, space group P1,̅ a = 8.8869(17) Å, b = 12.351(2) Å, c = 16.241(4) Å, α = 77.84(2)°, β = 86.67(2)°, γ = 89.966(17)°, V = 1739.7(6) Å3, Z = 4, ρ = 1.208 Mg·m−3; 30 449 reflections measured with 2θ in the range 4.60−50.00°, 6138 unique reflections (Rint = 0.1643); R1 = 0.1116, wR2 = 0.2986 (all data). Cryptobeilic acid B (2): pale yellow powder; [α]20D ±0 (c 0.10, CHCl3); IR (film) νmax 3342, 2923, 1679, 1231, 756 cm−1; LC-UV [(MeOH(aq) in H2O/0.05% FA)] λmax 205 and 225 nm; 1H NMR and 13C NMR see Tables 1 and 2; HMBC see Figure 3; HRMS m/z 315.19546 [M + H]+ (calcd for C20H27O3, 315.19547). Cryptobeilic acid C (3): yellow oil; [α]20D ±0 (c 0.30, CHCl3); IR (film) νmax 3360, 2923, 1693, 1483, 1434, 1029, 924, 756 cm−1; LCUV [(MeOH(aq) in H2O/0.05% FA)] λmax 254 and 290 sh nm; 1H NMR and 13C NMR see Tables 1and 2; HRMS m/z 365.1384 [M + H]+ (calcd for C22H21O5, 365.13835). Cryptobeilic acid D (4): yellow oil; [α]20D ±0 (c 0.30, CHCl3); IR (film) νmax 3398, 2916, 1693, 1231 cm−1; LC-UV [(MeOH(aq) in H2O/0.05% FA)] λmax 232 sh nm; 1H NMR and 13C NMR see Tables 1and 2; HRMS m/z 301.2161 [M + H]+ (calcd for C20H29O2, 301.2162). X-ray crystallographic analysis of tsangibeilin B (5): colorless needles, 0.20 × 0.10 × 0.10 mm3, C22H22O4, Mr = 350.40, orthorhombic, space group Pbca, a = 15.791(3) Å, b = 7.964(4) Å, c = 26.713(7) Å, V = 3359.5(19) Å3, Z = 8, ρ = 1.386 Mg·m−3; 65 151 reflections measured with 2θ in the range 5.16−50.00°, 2952 unique reflections (Rint = 0.1580); R1 = 0.0683, wR2 = 0.1782 (all data). Antimicrobial Assay. Four bacterial species (Escherichia coli 6r3, Acinetobacter calcoaceticus DSM 586, Pseudonamas stutzeri A1501, Serratia plymuthica C48) from our culture collection were used for the antimicrobial tests. The tests of compounds 1−5 were carried out by the paper disk diffusion method according to our earlier descriptions.9c,13 Antiplasmodial Activity. In vitro activity against erythrocytic stages of P. falciparum was assayed using a 3H-hypoxanthine incorporation assay,14,15 the chloroquine- and pyrimethamine-resistant K1 strain that originated from Thailand,16 and the standard drug chloroquine (Sigma C6628). Compounds 1−5 were dissolved in DMSO at 10 μg/mL and added to parasite cultures incubated in RPMI 1640 medium without hypoxanthine, supplemented with HEPES (5.94 g/L), NaHCO3 (2.1 g/L), neomycin (100 U/mL), Albumax (5 gL), and washed human red cells A+ at 2.5% hematocrit (0.3% parasitemia). Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg/mL were prepared. The 96-well plates were incubated in a humidified atmosphere at 37 °C; 4% CO2, 3% O2, 93% N2. After 48 h 50 μL of 3H-hypoxanthine (=0.5 μCi) was added to each well of the plate. The plates were incubated for a further 24 h under the same conditions. The plates were then harvested with a Betaplate cell harvester (Wallac, Zurich, Switzerland), and the red blood cells transferred onto a glass fiber filter, then washed with distilled water. The dried filters were inserted into a plastic foil with 10 mL of scintillation fluid and counted in a Betaplate liquid scintillation counter (Wallac, Zurich, Switzerland). IC50 values were calculated from sigmoidal inhibition curves by linear regression.

compounds were all isolated as racemic mixtures and represent the first report on chemical constituents of this plant.

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EXPERIMENTAL SECTION

General Experimental Procedures. The NMR spectra were recorded on a Bruker DRX-500 500 MHz and a Varian Inova 600 MHz spectrometer. Chemical shifts (δ) were quoted in parts per million (ppm) from internal standard tetramethylsilane. Optical rotation was measured on a Perkin-Elmer polarimeter, model 241. Preparative HPLC was run for 20 min on a Gilson apparatus with UV detection at 206 nm using a Nucleodur C18 Isis column (MachereyNagel, Düren, Germany), 5 μm (250 × 16 mm), with H2O (0.1% HCOOH) (A)−MeOH (0.1% HCOOH) (B) with a gradient program as follows (flow rate 8 mL min−1): 70% A linear to 75% B for 15 min, linear gradient to 20% A over 13 min, linear gradient to 0% A over 2 min, after 100% B isocratic for 7 min, the system was then returned to its initial condition (70% A) over 1 min and was equilibrated for 2 min. IR spectra were recorded on a Perkin-Elmer (model 1600) FTIR spectrometer. Column chromatography (CC) was carried out on silica gel (230−400 mesh). Size exclusion chromatography was done on Sephadex LH-20 (lipophilic Sephadex), Amersham Biosciences. Highresolution mass spectra were obtained with an LTQ-Orbitrap spectrometer (Thermo Fisher, USA) equipped with an APCI source. The spectrometer was operated in positive mode (1 spectrum s−1; mass range 200−800, with nominal mass resolving power of 60 000 at m/z 400 with a scan rate of 1 Hz) with automatic gain control to provide high-accuracy mass measurements within 2 ppm deviation using an internal standard, bis(2-ethylhexyl)phthalate, m/z = 391.284286. The spectrometer was attached to an Agilent (Santa Clara, CA, USA) 1200 HPLC system consisting of an LC pump, PDA detector (λ = 215 nm), autosampler (injection volume 10 μL), and column oven (30 °C). MS/MS experiments were performed by a collision-induced decay (35 eV) mode. The following parameters were used for experiments: spray voltage 5 kV, capillary temperature 190 °C, tube lens 70 V. Nitrogen was used as sheath gas (50 arbitrary units) and auxiliary gas (10 arbitrary units). Helium served as the collision gas. The separations were performed by using a Nucleodur RP Isis column (Macherey-Nagel, Düren, Germany), 1.8 μm (50 × 2 mm) with a H2O (+0.1% HCOOH) (A)−acetonitrile (+0.1% HCOOH) (B) gradient (flow rate 400 μL min−1). Samples were analyzed by using a gradient program as follows: 80% A isocratic for 1 min, linear gradient to 100% B over 25 min, after 100% B isocratic for 8 min, the system returned to its initial condition (80% A) within 0.5 min and was equilibrated for 4.5 min. X-ray Analysis. Single-crystal X-ray diffraction analysis was performed on an Oxford Diffraction Xcalibur S diffractometer at 173(2) K using graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The crystal structures were solved with direct methods (SHELXS97; Sheldrick, 2008) and refined against F2 with the fullmatrix least-squares method (SHELXL97; Sheldrick, 2008). A multiscan absorption correction using the CrysAlis RED program (Oxford Diffraction, 2006) was employed. The non-hydrogen atoms were refined anisotropically. All of the hydrogen atoms were placed in geometrically calculated positions, and each was assigned a fixed isotropic displacement parameter based on a riding model. Plant Material. The bark of B. cryptocaryoides was collected in October 2011 at Ranomafana-Ifanadiana, Madagascar. The plant was identified by Dr. Rabarison Harison, botanist at the Faculty of Sciences, University of Antananarivo, Madagascar. Extraction and Isolation. Powdered air-dried bark of B. cryptocaryoides (0.4 kg) was extracted three times with MeOH− CH2Cl2 mixtures at room temperature for 48 h. The extract was concentrated to dryness under reduced pressure, followed by suspension in H2O and extraction with CH2Cl2. The resulting active CH2Cl2-soluble extract (45.0 g) was subjected to LC-MS and LC-MS/ MS and further chromatographed on silica gel eluted with hexane− ethyl acetate mixtures (100:0 to 0:100) to yield six main fractions after TLC and LC-MS profiling (Fr.1 to Fr.6). The active fraction, Fr.3, was further purified on Sephadex LH-20 CC, eluted with MeOH, to afford 101



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In Vitro Cytotoxicity with L6 Cells. Assays were performed in 96-well microtiter plates, each well containing 100 μL of RPMI 1640 medium, supplemented with 1% L-glutamine (200 mM) and 10% fetal bovine serum, and 4000 L-6 cells (a primary cell line derived from rat skeletal myoblasts).17,18 Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 μg/mL were prepared. After 70 h of incubation the plates were inspected under an inverted microscope to ensure growth of the controls and sterile conditions. A 10 μL amount of Alamar Blue was then added to each well, and the plates were incubated for another 2 h. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. The IC50 values were calculated by linear regression (Huber 1993) from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Corporation, Sunnyvale, CA, USA). The IC50 values in μM are the means of two independent assays; the individual values varied less than ±50%.

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(8) (a) Banfield, J. E.; Black, D. S.; Collins, D. J.; Hyland, B. P. M.; Lee, J. J.; Pranowo, S. R. Aust. J. Chem. 1994, 47, 587−607. (b) Williams, R. B.; Martin, S. M.; Hu, J.-F.; Norman, V. L.; Goering, M. G.; Loss, S.; O’Neil-Johnson, M.; Eldridge, G. R.; Starks, C. M. J. Nat. Prod. 2012, 75, 1319−1325. (c) Yang, P. S.; Cheng, M. J.; Chenc, J. J.; Chen, I. S. Helv. Chim. Acta 2008, 91, 2130−2138. (9) (a) Talontsi, M. F.; Matasyoh, J. C.; Ngoumfo, R. M.; Chepkorir, R. Pestic. Biochem. Physiol. 2011, 99, 82−85. (b) Matasyoh, J. C.; Talontsi, M. F.; Ngoumfo, R. M.; Chepkorir, R. Nat. Prod. J. 2011, 1, 121−124. (c) Talontsi, M. F.; Nwemeguela, K. T. J.; Dittrich, B.; Douanla-Meli, C.; Laatsch, H. Planta Med. 2012, 78, 1020−1023. (10) Huang, Y.-T.; Chang, H.-S.; Wang, G.-J.; Cheng, M.-J.; Chen, C.-H.; Yang, Y.-J.; Chen, I.-S. J. Nat. Prod. 2011, 74, 1875−1880. (11) Chouna, J. R.; Nkeng-Efouet, P. A.; Lenta, B. N.; Wansi, J. D.; Neumann, B.; Stammler, H.-G.; Kimbu, S. F.; Sewald, N. Helv. Chim. Acta 2011, 94, 1071−1076. (12) Yang, P.-S.; Cheng, M.-J.; Peng, C.-F.; Chen, J.-J.; Chen, I.-S. J. Nat. Prod. 2009, 72, 53−58. (13) Awouafack, M. D.; Spiteller, P.; Lamshöft, M.; Kusari, S.; Ivanova, B.; Tane, P.; Spiteller, M. J. Nat. Prod. 2011, 74, 272−278. (14) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents Chemother. 1979, 16, 710−718. (15) Matile, H.; Pink, J. R. L. In Immunological Methods; Lefkovits, I., Pernis, B., Eds.; Academic Press: San Diego, CA, USA, 1990; pp 221− 234. (16) Thaithong, S.; Beale, G. H.; Chutmongkonkul, M. Trans. R. Soc. Trop. Med. Hyg. 1983, 77, 228−231. (17) Ahmed, S. A.; Gogal, R. M.; Walsh, J. E. J. Immunol. Methods 1994, 170, 211−224. (18) Page, B.; Page, M.; Noel, C. Int. J. Oncol. 1993, 3, 473−476.

ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of compounds 1−5 are available free of charge via the Internet at http://pubs.acs.org. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication nos. CCDC 898512 (1) and CCDC 898513 (5). Copies of the data can be obtained free of charge on application to Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK [fax: (+44) 1223-336-033; e-mail: [email protected]].

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 231 755 4080. Fax: +49 231 755 4085. E-mail: m. [email protected]; [email protected]. de. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the German Academic Exchange Service (DAAD) initiative “Welcome to Africa” and the Ministry of Innovation, Science, Research and Technology of the State of North Rhine-Westphalia, Germany, and the German Research Foundation (DFG) for funding a highresolution mass spectrometer. We thank J. Hardes and C. Kostrzewa for technical assistance. J.O.B. thanks the Fonds der Chemischen Industrie (FCI) for a doctoral scholarship.

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REFERENCES

(1) (a) Fouilloy, R. Flore du Cameroun (Lauracées, Myristicaées et Momimiacées); Muséum National d’Histoire Naturelle: Paris, 1974, Vol. 18, pp 7−13. (b) van der Werff, H. A Synopsis of the Genus Beilschmiedia (Lauraceae) in Madagascar; 2003, Vol. 3, pp 77−92. (2) Iwu, M. M. Handbook of African Medicinal Plants; CRC: Boca Raton, 1993; p 435. (3) Chouna, J. R.; Nkeng-Efouet, P. A.; Lenta, B. N.; Wansi, J. D.; Kimbu, S. F.; Sewald, N. Phytochemistry 2009, 70, 684−688. (4) Lenta, B. N.; Tantangmo, F.; Devkota, K. P.; Wansi, J. D.; Chouna, J. R.; Soh, R. C. F.; Neumann, B.; Stammler, H.-G.; Tsamo, E.; Sewald, N. J. Nat. Prod. 2009, 72, 2130−2134. (5) Chen, J. J.; Chou, E. T.; Duh, C. Y; Yang, S. Z.; Chen, I. S. Planta Med. 2006, 72, 351−357. (6) Clezy, P. S.; Nichol, A. W.; Gellert, E. Experientia 1963, 19, 1−2. (7) Harborne, J. B.; Mendez, J. Phytochemistry 1969, 8, 763−764. 102


Antibacterial and Antiplasmodial Constituents of Beilschmiedia

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