Article pubs.acs.org/jnp
Laxitextines A and B, Cyathane Xylosides from the Tropical Fungus Laxitextum incrustatum Cynthia M. Mudalungu,† Christian Richter,‡,§ Kathrin Wittstein,‡,§ Muna Ali Abdalla,† Josphat C. Matasyoh,⊥ Marc Stadler,*,‡,§ and Roderich D. Süssmuth*,† †
Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany Department of Microbial Drugs, Braunschweig, Helmholtz Centre for Infection Research GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany § German Centre for Infection Research, partner site Hannover-Braunschweig, Inhoffenstraße 7, 38124 Braunschweig, Germany ⊥ Department of Chemistry, Egerton University, P.O. Box 536, Egerton, Kenya ‡
S Supporting Information *
ABSTRACT: Bioassay-guided fractionation of the mycelial extract of a basidiomycete culture collected in Kenya led to the isolation of two new cyathane diterpenoids named laxitextines A (1) and B (2). The producer strain was characterized by detailed taxonomic studies based on rDNA using the 5.8S gene region, the internal transcribed spacer 2 (ITS2), and part of the large subunit that identified the fungus as Laxitextum incrustatum. The structures of 1 and 2 were elucidated by NMR spectroscopic and mass spectrometric analyses. Both compounds exhibited moderate activities against Grampositive bacteria Bacillus subtilis (DSM 10), Staphylococcus aureus (DSM 346), and methicillin-resistant Staph. aureus (DSM 1182). The two compounds also showed variable antiproliferative activities against mouse fibroblast (L929) and selected human cell lines (breast cancer MCF-7, epidermoid carcinoma A431, and umbilical vein endothelial HUVEC). The IC50 values with respect to the MCF-7 cell line for compounds 1 and 2 were 2.3 and 2.0 μM, respectively.
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and Sarcodon.9−11 Their biological effects have been extensively studied and include antimicrobial, agonistic effects toward the kappa-opioid receptor, anti-inflammatory effects, antiproliferative activity, and enhancement of nerve growth factor (NGF) activity.7,12,13 In previous studies, investigations on cyathane biosynthesis through feeding experiments with 13C-labeled acetate to Cyathus earlei producing cyathrathriol and derivatives indicated the positions of the labeled carbons in the cyathane skeleton by utilization of 13C NMR spectroscopy. The study concluded that this skeleton was formed via a mevalonic acid pathway and geranylgeranyl pyrophosphate followed by cationic cyclization accounting for the unusual C-6 and C-9 positions of the exocyclic methyl groups in trans configurations.14−16 Herein, we report on the isolation and structure elucidation of two new compounds, laxitextines A (1) and B (2), of the cyathane family. The two compounds were extracted from Laxitextum incrustatum collected from Kakamega forest in the western part of Kenya. Their antimicrobial and cytotoxic activities against selected test organisms are also investigated.
n recent years, screenings of microorganisms that focus on the diversity of species from untapped niches have gained considerable interest. Next to the discovery of novel strains, they are expected to deliver unprecedented chemical structures and bioactivities. The discovery and exploration of lead compounds with new mechanisms of action against Grampositive and Gram-negative bacteria is urgently needed particularly with regard to recent developments of resistance against currently used drugs.1 In this context, fungi such as basidiomycetes offer a potentially prolific source of unique bioactive secondary metabolites due to their immense biodiversity in unexplored niches.2 Wood-inhabiting fungi are well known to play an important role in the ecosystem by decomposing dead wood and their ability to degrade lignocellulose-containing material. In this regard, they are not only sources for new bioactive molecules but also of interest for various biotechnological applications.3,4 Numerous cyathane-type compounds from the higher fungi and sponges have been previously described.5,6 The cyathanes are terpenoids that have a characteristic tricyclic core structure consisting of annelated 5−6−7-membered rings. Among these are the cyathins, allocyathins, erinacines, sarcodonins, scabronines, striatals, striatoids, cyanthiwigins, and cyafrins,7,8 which were isolated from the basidiomycete genera Cyathus, Hericium, © XXXX American Chemical Society and American Society of Pharmacognosy
Received: October 23, 2015
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DOI: 10.1021/acs.jnatprod.5b00950 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. 1H (700 MHz) and 13C (175 MHz) NMR Data for 1 and 2a 1 (in CDCl3)
a1
position
δC
1
38.4, CH2
2 3 4 5 6 7
28.2, CH2 138.6, Cq 138.5, Cq 47.3, CH 40.4, Cq 28.3, CH2
8
36.7, CH2
9 10
49.6, Cq 25.7, CH2
11
34.2, CH2
12 13 14 15
40.4, 47.8, 94.3, 65.9,
16 17 18 19 20 1′ 2′ 3′ 4′ 5′
19.1, CH3 25.1, CH3 27.2, CH 21.4, CH3 21.8, CH3 106.0, CH 78.1, Cq 95.7, Cq 70.4, CH 65.0, CH2
CH CH CH CH2
2 (in CD3OD) δH (J in Hz)
δC
1.55, m 1.60, m 2.26, m
39.6, CH2 29.2, CH2 140.6, CH 138.7, CH 43.8, CH 41.8, Cq 28.1, CH2
2.29, m 1.48, m 1.48, m 1.53, m 1.91, 1.68, 0.91, 1.80, 1.81, 2.04, 4.02, 3.54, 3.80, 0.97, 1.06, 2.72, 0.94, 0.95, 5.06,
37.6, CH2
dt (14.4, 3.5) ddd (14.3, 10.5, 9.7) m m m dd (10.9, 8.3) d (8.2) t (11.1) dd (10.9, 4.5) s s heptet (13.6, 6.7) d (6.7) d (6.7) s
3.89, t (5.1) 3.63, dd (11.7, 5.6) 4.00, dd (11.7, 4.9)
49.6, Cq 30.7, CH2
δH (J in Hz) 1.61, m 1.69, m 2.36, m
2.75, t (12.2) 1.45, 1.78, 1.55, 1.60,
ddd (13.8, 9.5, 2.2) dt (13.9, 9.2, 5.1) ddd (10.3, 8.6, 2.2) m
126.8, CH
2.56, dd (17.6, 6.5) 2.66, m 5.57, m
138.0, Cq 44.6, CH 93.8, CH 101.8, CH
3.07, m 4.20, d (8.8) 5.70, br s
17.8, CH3 25.0, CH3 28.3, CH3 22.2, CH3 21.9, CH3 106.0, CH 78.4, Cq 101.6, Cq 94.5, Cq 63.6, CH2
1.04, 1.12, 2.92, 1.03, 1.03, 4.90,
s s heptet (13.6, 6.7) d (6.8) d (6.8) s
3.51, d (12.7) 3.72, d (12.7)
H (700 MHz) and 13C (175 MHz) NMR. Chemical shifts in ppm from TMS as the internal standard.
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RESULTS AND DISCUSSION The screening of fungal strains from our sampling site revealed antibacterial activities against the Gram-positive indicator Bacillus subtilis (DSM 10) in agar diffusion assays. These strains (among them strain STMA 14285) were selected for taxonomic characterization and identification of active principles as described in the Experimental Section. Cultivation followed by bioassay-guided procedures enabled the detection of the new compounds by dereplication through HPLC-MS profiling with UV/vis detection, semipreparative chromatographic fractionation, and a subsequent database search (Dictionary of Natural Products).17 Compound 1 was obtained as a brown powder in a total yield of 7.3 mg. From the HRESIMS data evaluations, a molecular formula of C25H38O6 with seven degrees of unsaturation was established. The 13C chemical shifts in combination with HSQC data of compound 1 (see Table 1) revealed the presence of 25 carbon atoms: four methyl groups, eight methylenes, seven methines (including one ketal group C-1′ at δC 106.0 ppm), two quaternary carbons (C-6 at δC 40.4 ppm and C-9 at δC 49.6 ppm), two olefinic carbons (C-3, C-4 at δC 138.6 ppm and δC 138.6 ppm, respectively), and the rest as oxygenated carbon atoms. Its 1H NMR spectrum revealed two methyl doublets at δH 0.94 and 0.95 and two singlets at δH 0.97
and 1.06. The majority of methylene signals appeared between δH 0.91 and 2.25 with the exception of four diastereotopic highly deshielded protons exhibiting 1H chemical shifts between δH 3.54 and 4.00 ppm (C-15, C-5′). Further 2D NMR analysis by means of heteronuclear HMBC and homonuclear COSY correlations enabled determination of the basic carbon skeletal structure of the molecule. The main differences from the closely related compound CJ-14,258 (4) are the variations at position C-3′ (without hydroxylation) and C-15, bearing a hydroxy group.10,18 The other related compound to 1 is striatoid A (5), with the presence of a ketal moiety (C-15) and a hydroxy group at C-1.7 The identified carbon−carbon bond connections of the structure were further supported by COSY correlations: H-1/H-2, H-7/ H-8, H-4′/H-5′, and H-5/H-10/H-11/H-12/H-13/H-14 (as indicated by the emboldened sections on the structures of Figure 2). The molecular formula of 1 as well as the observed chemical shifts was consistent with the presence of a cyathane xyloside moiety and consequently led to its structural assignment as in Figure 1. The relative stereochemistry of compound 1 was determined by the analysis of the NOE differential effects along with cyathane-skeleton biogenetic considerations as earlier described.5,7 In the ROESY spectrum of 1, the cross-peak correlations of 17-CH3/H-5/H-13/H-1′ suggested an αB
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observable differences at C-15 and C-4′, where a characteristic oxygen bridge is located (Figure 2). From the 13C NMR spectrum of 2, two other characteristic ketal carbon signals were observed at δC = 101.6 ppm (C-3′) and δC = 101.8 ppm (C-15). With the help of the HSQC spectrum, the latter signal with a corresponding low-field 1H-shift (δH 5.70, 1H) was identified as a methine carbon and the former as a ketal carbon atom. Confirmation of the assignments to the two positions was attained by long-range couplings in the HMBC spectrum. This therefore revealed a close structural resemblance to striatoid B (6), with lack of a hydroxy moiety at C-1 as the main difference.7 The relative stereochemistry of 2 was partially in accordance with that of 1 based on the correlations derived from its ROESY spectrum. Hence, the structure of 2 was named laxitextine B (2). Aside from the two new isolated compounds 1 and 2, striatal D (3) (Figure 1) was also isolated, and its NMR correlations matched with previously reported data.14 Biological Activities of the Compounds. The purified compounds displayed moderate antibacterial activity against B. subtilis (DSM 10) with an inhibition concentration of 33.3 μg/ mL (76.7 μM) for 1 and 16.7 μg/mL (37.4 μM) for 2 (see Table 2). Further evaluation of 1 and 2 against Staphylococcus
Figure 1. Isolated compounds laxitextines A (1) and B (2), striatal D (3), and the known congeners CJ-14,258 (4) and striatoids A (5) and B (6).
Table 2. Antimicrobial MIC [in μg/mL] and IC50 [in μM] Values from Selected Cell Lines MIC (μg/mL)a test organism Bacillus subtilis DSM 10 Staphylococcus aureus DSM 346 Methicillin-resistant Staph. aureus DSM 1182
Figure 2. Homonuclear and heteronuclear NMR correlations of laxitextines A (1) and B (2).
orientation of the groups, whereas the correlations of H-12/H14/16-CH3 suggested a β-orientation (Figure 3). With all the correlations considered, the compound was determined as laxitextine A (1). The 1H and 13C NMR values are summarized in Table 1.
1
a
b
1
d
2a
2b
oxyc 1.0 1.0
33.3 7.8
n.t. 6.6
16.7 15.7
n.t. n.i.e
7.8
n.i
62.5
n.i.
vanc
1.6
IC50 (μM)f cell line
1
2
epoc
umbilical vein endothelial cells HUVEC mouse fibroblast cells L929 epidermoid carcinoma cells A431 breast cancer cells MCF-7
4.8 17.3 5.8 2.3
3.6 16.1 4.9 2.0
0.0004 0.0038 0.0006 0.0004
a a 1 and 1b represent MIC in (μg/mL) for laxitextine A (1) using 20 and 2 μL of 1 mg/mL solution, respectively. Also, 2a and 2b represent laxitextine B (2) using 20 and 2 μL of 1 mg/mL solution, respectively. c Positive controls used (oxy = oxytetracycline, van = vancomycin, epo = epothilone). The positive control values were obtained with 2 μL of solution. d n.t. = not tested. en.i. = no inhibition. f The IC 50 concentrations (μM) for 1 and 2 against cell lines were carried out using 3 μL of compound solutions.
Figure 3. Selected ROESY correlations of laxitextine A (1) indicating selected α-oriented (green) and β-oriented (blue) groups.
Compound 2 was also obtained as a brown powder in a yield of 3.0 mg. The observed molecular mass with HRESIMS corresponded to the molecular formula of C25H34O7, from which nine degrees of unsaturation were deduced. The NMR spectroscopic data of 2 in CD3OD (Table 1) were similar to the chemical shifts of 1 in CDCl3, suggesting that 2 was also a cyathane diterpenoid having the same carbon backbone. However, the 1H NMR of compound 2 in CDCl3 gave unusually broad signals for various resonances (probably due to tautomerization), which were later resolved by solvent change, i.e., measurements in CD3OD and DMSO-d6, enabling the correct assignment of the proton signals. On the basis of the skeletal relationship of the two metabolites, compound 2 contains a carbon skeleton similar to 1 with the main
aureus (DSM 346) and methicillin-resistant (MRSA) Staph. aureus (DSM 1182) showed laxitextine A (1) as the most potent one, inhibiting Staph. aureus (DSM 346) at 7.8 μg/mL (17.9 μM). Compound 1 also exhibited anti-MRSA activity with an MIC value of 7.8 μg/mL (17.9 μM), similar to that of Staph. aureus (DSM 346) strain, as shown in Table 2. However, compound 2 was less potent in relation to the anti-MRSA activity, indicating its MIC value to be 62.5 μg/mL (140.0 μM). These findings are paralleled by previous investigations on the anti-MRSA activity of related compounds containing the tricyclic ring system of the aglycon, which conclusively showed that the core cyathane ring structure was imperative for the observed activity.10 C
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days with monitoring until complete depletion of the carbon source as previously described.23 Extraction and Isolation. The culture broth was separated by centrifugation (10 min at 1000g) and filtered. The obtained wet mycelia were extracted twice with acetone in an ultrasonic bath at 40 °C for 30 min, after which it was filtered and evaporated in vacuo. The remaining aqueous residue was diluted to 150 mL with distilled water and extracted twice with 150 mL of ethyl acetate. The combined EtOAc phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give 180 mg of crude extract. The crude extract was adsorbed on an RP solid-phase cartridge (Strata-X 33 mm, Polymeric Reversed Phase; Phenomenex, Aschaffenburg, Germany) and eluted with methanol to yield 160 mg of the crude substance as described previously.24 For fractionation and further purification of the crude extract, a preparative RP HPLC (PLC 2020, Gilson, Middleton, WI, USA) equipped with a VP Nucleodur 100-7 18 ec column (125 × 40 mm, 7 μm; Macherey-Nagel, Düren, Germany) was used. The mobile phase was composed as follows: solvent A: H2O (Milli-Q, Millipore, Schwalbach, Germany) with 0.05% trifluoroacetic acid; B: acetonitrile with 0.05% trifluoroacetic acid. The gradient used was 30% to 100% acetonitrile in 40 min, 100% for 15 min at a flow rate of 40 mL/min. UV detection was carried out at λ = 210, 254, and 350 nm, and fractions were combined according to the observed peaks [tRt = 26.9− 27.5 min (1) and 24.4−25.0 min (2)]. The identity of the compounds was confirmed by HPLC-MS (Agilent 1260 Infinity Systems) equipped with a diode array detector, attached to an ion trap MS (amaZon speed, Bruker), with an electrospray ionization source, C18 Acquity UPLC BEH column (2.1 × 50 mm, 1.7 μm), and the following gradient (same solvents as above + 0.1% formic acid): 5% B for 0.5 min, increasing to 100% B in 19.5 min, isocratic conditions 100% B for 10 min, flow rate = 0.6 mL/min, UV detection 200−600 nm; the compounds eluted at tR 12.9−13.1 min for 1 and 12.4−12.6 min for 2, respectively. The physicochemical properties of the isolated compounds are as summarized below. Laxitextine A (1): brown powder; [α]23 D −254 (c 0.68, MeOH); UV (MeOH) λmax (log ε) 218 (3.04); IR (KBr) ν 3342, 2932, 2863, 1672, 1455, 1373, 1359, 1315, 1201, 1065, 1024, 991, 827, 765 cm−1; 1H (700 MHz) and 13C NMR (175 MHz) data (CDCl3), see Table 1; positive ion HRMS m/z 457.2563 [M + Na]+ (calcd for C25H38O6Na+, 457.2561). Laxitextine B (2): brown powder; [α]23 D −146 (c 0.30, MeOH); UV (MeOH) λmax (log ε) 220 (3.18); IR (KBr) ν 3341, 2928, 2863, 1679, 1454, 1376, 1257, 1180, 1132, 1076, 1023, 989, 930 cm−1; 1H (700 MHz) and 13C NMR (175 MHz) data (MeOD), see Table 1; positive ion HRMS m/z 447.2386 [M + H]+ (calcd for C25H34O7H+, 447.2377). Antimicrobial Assays. Minimum inhibitory concentrations (MIC) were conducted in a serial dilution assay with the pure compounds following a previously described protocol25,26 using various test organisms for antibacterial activities. The tests were done by taking a 20 μL stock solution of each compound at 1 mg/mL in MeOH. In the case of positive controls vancomycin and oxytetracycline, 2 μL stock solutions were used. Assays were conducted in 96-well microtiter plates in EBS medium [0.5% casein peptone, 0.5% glucose, 0.1% meat extract, 0.1% yeast extract, 50 mM HEPES (11.9 g/L), pH 7.0] for bacteria.27 Cytotoxicity Assay. In vitro cytotoxic effects (IC50) were determined with 3.0 μL [from stock solutions containing 1 mg/mL] of the compounds against a number of mammalian cell lines by serial dilution (60 μL) in 96-well plates for tissue cultures (Falcon). The cell lines included the mouse fibroblast cell line L929, breast cancer cell line MCF-7, epidermoid carcinoma cell line A431, and umbilical vein endothelial cell line (HUVEC). The cell line L929 was cultured in DMEM (Lonza) and MCF-7 and A431 were cultured in RPMI (Gibco), all supplemented with 10% fetal bovine serum (Gibco) and incubated under 5% CO2 at 37 °C for 5 days. Methanol was used as the negative control and epothilone A as the positive control. The assays were conducted in accordance with literature descriptions.25
In the proliferation assays against selected cell lines, the two laxitextine compounds manifested moderate activities against the mouse fibroblast cell line (L929) and selected human cell lines (breast cancer MCF-7, epidermoid carcinoma A431, and umbilical vein endothelial HUVEC). Comparable proliferative effects were shown by 1 and 2 upon being subjected to the aforementioned cell lines, with the IC50 values of the most inhibited cell line MCF-7 occurring at 2.3 and 2.0 μM, respectively. The least inhibited cell line by compounds 1 and 2 was L929, at 17.3 and 16.3 μM, respectively (Table 2). In previous reports, some cyathane derivatives have shown mild antimicrobial activities and limited cytotoxic effects against primary tumor cells such as murine leukemia cells (P388) and the lung cancer tumor cells (A549).19 In conclusion, two new cyathane xyloside diterpenoid derivatives produced as secondary metabolites in fermented mycelial cultures of the basidiomycete L. incrustatum were isolated and their chemical structures elucidated. The compounds laxitextines A (1) and B (2) are herein documented for the first time since the wood-inhabiting fungus of the Laxitextum genus was first taxonomically characterized in 1981. This fungus belongs to the division Basidiomycota, order Russulales, and Hericiaceae family.20,21 This family comprises the genera Hericium, Dentipellis, and Laxitextum, with Hericium being the most studied.9,22 The discovery of cyathanes in Laxitextum reconfirms the phylogenetic relationships of this genus to Hericium and also points toward the possible role as chemotaxonomic markers for the family Hericiaceae. However, secondary metabolites from the third genus, Dentipellis, have to our knowledge not been studied.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation data were obtained on a Jasco P-2000 polarimeter, IR spectra were measured with an FTIR-4100 spectrometer (Jasco), and NMR spectra were recorded on a Bruker Ascend 700 spectrometer with a 5 mm TXI cryoprobe (1H 700 MHz, 13C 175 MHz) spectrometer. Highresolution ESIMS measurements were performed on an Exactive Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) coupled to an Agilent 1200 HPLC system (Agilent Technologies, Waldbronn, Germany). A Grom-Sil ODS-4 HR 3 μm, 50 mm × 2.0 mm, column (Grace, Grace GmbH & Co KG, Worms, Germany) was used for column chromatography. Strain Collection and Characterization. The fungal material was collected from the Kakamega tropical rain forest located in the western part of Kenya, East Africa (lat: 0.2870/0°17′13″, long: 34.8870/ 34°53′13″). A voucher specimen is deposited at Egerton University, Kenya, with the collection number STMA 14285. The culture was isolated from the fruiting body and cultivated on YMG agar plates. Combined studies of morphological characters and the sequencing of parts of the rDNA (5.8S gene region, the internal transcribed spacer 2 (ITS2), and part of the large subunit (LSU)) identified the fungus as Laxitextum incrustatum (Figure S1).20 The sequences are deposited at GenBank: KT722621 (ITS) and KT722622 (LSU). The genus Laxitextum is closely related to Hericium, which is known as a producer of several compounds of the cyathane type such as hericenones and erinacines.9 Fermentation of Laxitextum incrustatum (STMA 14285). Pieces of well-grown agar of L. incrustatum from YMG plates were inoculated in Q6/2 medium consisting of 1.0% glycerol, 0.25% glucose, and 0.5% cottonseed flour, pH 7.2, in a 500 mL Erlenmeyer flask containing 200 mL of media and incubated at 23 °C for 7 days. This 7-day-old seed culture was then used to inoculate 14 other flasks (2.8 L) of the same medium composition after maceration into a homogeneous phase. The inoculated flasks were incubated at 23 °C on a rotary shaker at 140 rpm agitation. The culture was grown for 14 D
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(18) Saito, T.; Aoki, F.; Hirai, H.; Inagaki, T.; Matsunaga, Y.; Sakakibara, T.; Sakemi, S.; Suzuki, Y.; Watanabe, S.; Suga, O.; Sujaku, T.; Smogowicz, A. A.; Truesdell, S. J.; Wong, J. W.; Nagahisa, A.; Kojima, Y.; Kojima, N. J. Antibiot. 1998, 51, 983−990. (19) Peng, J.; Walsh, K.; Weedman, V.; Bergthold, J. D.; Lynch, J.; Lieu, K. L.; Braude, I. A.; Kelly, M.; Hamann, M. T. Tetrahedron 2002, 58, 7809−7819. (20) Hjortstam, K.; Ryvarden, L. Mycotaxon 1981, 13, 35−40. (21) Zhou, L. W.; Dai, Y. C. Mycologia 2013, 105, 636−649. (22) Miller, S. L.; Larsson, E.; Larsson, K. H.; Verbeken, A.; Nuytinck, J. Mycologia 2006, 98, 960−970. (23) Stadler, M.; Hellwig, V.; Mayer-Bartschmid, A.; Denzer, D.; Wiese, B.; Burkhardt, N. J. Antibiot. 2005, 58, 775−786. (24) Surup, F.; Thongbai, B.; Kuhnert, E.; Sudarman, E.; Hyde, D. K.; Stadler, M. J. Nat. Prod. 2013, 76, 2141−2144. (25) Okanya, P. W.; Mohr, K. I.; Gerth, K.; Jansen, R.; Müller, R. J. Nat. Prod. 2011, 74, 603−608. (26) Surup, F.; Mohr, K. I.; Jansen, R.; Stadler, M. Phytochemistry 2013, 95, 252−258. (27) Halecker, S.; Surup, F.; Kuhnert, E.; Mohr, K. I.; Brock, N. L.; Dickschat, J. S.; Junker, C.; Schulz, B.; Stadler, M. Phytochemistry 2014, 100, 86−91.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00950. Spectral data from NMR, IR, and MS, description of the characterization of the producer organism, and antimicrobial activity photograph (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +49 531 6181-4240. Fax: +49 531 6181 9499. E-mail:
[email protected]. *Tel: +49 (0)3031478774. Fax: (+49) 030-314-79651. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful for the financial support by the Alexander von Humboldt Foundation, the ASAFEM Project (Grant no. IC-070) under the ERAfrica Programme, and the German Academic Exchange Service (DAAD) for offering a joint Ph.D. scholarship with the Kenya National Council of Science, Technology and Innovation (NACOSTI) to C.M.M. We thank C. Kaksochke for recording the NMR spectra, W. Collisi for the technical assistance in conducting the bioassays, and Dr. F. Surup for the valuable scientific discussions.
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REFERENCES
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