DSpace at VNU: New wedelolides, (9R)-eudesman-9,12-olide d-lactones, from Wedelia trilobata

G Model PHYTOL 1175 No of Pages Phytochemistry Letters xxx (2016) xxx–xxx Contents lists available at ScienceDirect Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol New wedelolides, (9R)-eudesman-9,12-olide d-lactones, from Wedelia trilobata Toan Phan Duca , Truong Van Nguyen Thiena , Akino Jossangb , Phi Phung Nguyen Kima , Philippe Grellierc , Ginette Jaureguiberryc , Quang Ton Thata,b,c,* a Faculty of Chemistry, University of Science, Vietnam National University Ho Chi Minh City, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Vietnam Laboratoire de Chimie et Biochimie des Substances Naturelles, UMR 5154 CNRS, USM 502, Muséum National d'Histoire Naturelle, 63 rue Buffon 75005 Paris, France c Biologie Fonctionnelle des Protozoaires, USM 504, Muséum National d'Histoire Naturelle, 61 rue Buffon 75005 Paris, France b A R T I C L E I N F O Article history: Received December 2015 Received in revised form 24 May 2016 Accepted June 2016 Available online xxx Keywords: Wedelolide Sesquiterpene d-lactones Wedelia trilobata Asteraceae Antimalarial Plasmodium falciparum A B S T R A C T From the crude ethanol extract of Wedelia trilobata leaves, hexane and dichloromethane fractions exhibited in vitro antimalarial activity against the Plasmodium falciparum parasite (strain PFB), with IC50 values of 27.0 and 13.0 mg/mL, respectively Specifically, two new (9R)-eudesman-9,12-olide d-lactones, wedelolide G (1) and wedelolide H (2), were isolated from the dichloromethane extract and showed IC50 values of 3.42 and 5.96 mM, respectively Six known compounds are also present in the extract The structures of 1–8 were elucidated through spectroscopic studies ã 2016 Phytochemical Society of Europe Published by Elsevier B.V All rights reserved Contents Introduction Results and discussion Experimental General experimental procedures 3.1 Cell line and cell culture 3.2 Plant material 3.3 Extraction and isolation 3.4 Bioassays 3.5 Antimalarial activity against strain PFB 3.5.1 Sulforhodamine B (SRB) assay 3.6 Acknowledgment References Introduction As of 2015, an estimated 3.2 billion people in 97 countries and territories are at risk of being infected with malaria (compared to * Corresponding author at: Faculty of Chemistry, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam E-mail address: ttquang@hcmus.edu.vn (Q Ton That) 00 00 00 00 00 00 00 00 00 00 00 00 2.4 billion in 106 countries in 2000) (WHO, 2015) There are more cases of malaria diagnosed each year, and the risk of drug resistance is a continual threat Indeed, resistance to quinine was reported in Brazil (da Silva and Benchimol, 2014) and in southeast Asia (Giboda and Denis, 1988; Pukrittayakamee et al., 1994) The combination of sulfa drugs and pyrimethamine was successful in fighting chloroquine-resistant malaria, yet resistance again rapidly appeared in the Asian Pacific region in the late 1970s and in South America (Ferraroni and Hayes, 1979) (Hurwitz et al., 1981) In 2014, http://dx.doi.org/10.1016/j.phytol.2016.06.001 1874-3900/ã 2016 Phytochemical Society of Europe Published by Elsevier B.V All rights reserved Please cite this article in press as: T Phan Duc, et al., New wedelolides, (9R)-eudesman-9,12-olide Phytochem Lett (2016), http://dx.doi.org/10.1016/j.phytol.2016.06.001 d-lactones, from Wedelia trilobata, G Model PHYTOL 1175 No of Pages T Phan Duc et al / Phytochemistry Letters xxx (2016) xxx–xxx resistance to the artemisinin family of drugs has developed in parts of Cambodia, Thailand, Myanmar, and Vietnam (Ashley et al., 2014) (Bosman et al., 2014) Malarone resistance was first observed in Kenya and has subsequently spread throughout many African countries (Wichmann et al., 2004) With the discovery of DDD107498, which has broad therapeutic potential and novel modes of action, the scope of treatment has been widened with the aim of eliminating emerging drug resistance This compound fights against multiple life-cycle stages of the Plasmodium parasite and features single-dose treatment, transmission blocking, and chemoprotection Furthermore, the molecular target has been identified as translation elongation factor (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA and is essential for protein synthesis (Beatriz et al., 2015) The genus Wedelia belongs to the large family Asteraceae, from which many sesquiterpene lactones have been isolated (Bohlmann et al., 1981; Salwa et al., 1996; Ragasa et al., 1993; Ferreira et al., 1994) In a survey of bioactive substances from Vietnamese plants (Jossang et al., 2003), Wedelia trilobata was investigated and used traditionally for the treatment of fever and malaria in Vietnam Two new compounds, wedelolide G (1) and wedelolide H (2), possess a new type of sesquiterpene framework, the (9R)eudesman-9,12-olide d-lactone (Quang et al., 2007) Additionally, six known compounds were isolated from this Wedelia genus for the first time: 5-hydroxymethylfurfuran (3), vanillic acid (4), 5,40 dihydroxy-7-methoxyflavone (5), 5,30 ,40 -trihydroxy-7-methoxyflavone (6), methyl caffeate (7), and trilobolide-6-O-isobutyrate (8) Herein, we describe the bioassay-guided isolation and structure elucidation of the new antimalarial products, wedelolide G (1) and wedelolide H (2) (Fig 1) Results and discussion Wedelolide G (1) was obtained as a white amorphous powder with an optical activity of [a]20D À7 (c 0.1, MeOH) The high resolution electrospray ionization mass spectrum (HR-ESI–MS), acquired in the positive mode, revealed a quasi-molecular ion at m/ z = 461.2146 [M+Na]+ (calcd for C23H34O8Na+, 461.2151), which corresponded to seven degrees of unsaturation The 13C NMR spectrum (CDCl3) showed three ester carbonyl signals at d = 163.4, 176.2, and 176.5 and two vinyl carbon signals at d 132.1 (>C¼) and 133.4 (¼CH2), accounting for four degrees of unsaturation The three remaining degrees of unsaturation are attributed to three saturated rings The 13C distortionless enhancement of polarization transfer spectrum (DEPT) (Table 1) and heteronuclear single quantum correlation spectrum (HSQC) NMR analyses indicate the presence of 23 carbon atoms, including six methyl, three methylene, eight methine, and six quaternary carbon atoms The H-1H COSY spectrum of indicates the presence of two structural fragments (Fig 2) The determination of fragment (b) was troublesome, as there is no direct COSY correlation evidence between the H-8 and H-9 protons Finally, the HMBC spectrum allows for the clear assignment of the H-9 position, vide infra Fig Structure of compound and Detailed 2D NMR data reveals the presence of two isobutyrate groups and two hydroxy groups, both of which are consistent with the molecular formula determined for The locations of these substituents were determined on the basis of HMBC correlations (Fig 3) Two isobutyrate groups are located at C-6 and C-8, based on the correlations of H-6 and H-8 to the corresponding ester carbonyl carbon atoms The secondary hydroxyl group of the partial structure (a) has an equatorial configuration, as shown by the coupling constants (J = 11.5, 5.0 Hz) The remaining hydroxy group can only be placed at C-4 because the quaternary carbon at d = 71.2 ppm shows correlations with the CH3-15 protons at d = 1.32 ppm, as well as with the two protons H-2a at d = 1.73 ppm and H-5 at d = 1.44 ppm The carbonyl carbon atom at d = 163.4 ppm shows correlations with H-7, H-9, and the exocyclic vinylidene protons at d = 5.79 and 6.64 ppm, indicating the presence of the d-lactone ring, which includes C-7, C-8, C-9, C-11, carbonyl C-12, and an oxygen atom These data suggest that compound is an eudesmanolide sesquiterpene d-lactone The relative stereochemistry of wedelolide G (1) was assigned by the NOESY spectrum (Fig 5) The CH3-15 protons show interactions with the H-8 and the H-9 protons The H-8 and H-9 protons also show a correlation, indicating that the d-lactone ring exists in the axial conformation All of these protons are situated on the same side of the molecule (Fig 5), whereas the axial proton H-5 interacts with the axial protons H-1 and H-6, situated on another side The equatorial proton H-7 exhibits a cross-peak with the vinylidene proton H-13a, which are close, spatially, to each other The stereochemistry between protons H-5 and H-15 is in accordance with that observed for trans-eudesmalonides, described in the literature (Quang et al., 2007; Yating et al., 2013; Salwa et al., 1996) The structure of wedelolide G (1) is thus established as 1a,4b-dihydroxy-6a,8b-diisobutyryloxyeudesman-9,12-olide The absolute stereochemistry of the eudesmanolide sesquiterpene d-lactone was demonstrated by mean of auxiliary chiral anisotropic reagent MTPA (Quang et al., 2007; Ohtani et al., 1991) and X-ray diffraction structure determination using Cu Ka radiation (Yating et al., 2013) Using these methods, the absolute configuration of wedelolide G (1) was determined as 1S,4S,5S,6R,7S,8S,9R,10S Wedelolide H (2) was obtained as a white amorphous powder, displaying an optical activity of [a]20D À9 (c 0.1 MeOH) The HRESI–MS spectrum, acquired in the positive mode, revealed a quasimolecular ion at m/z = 459.1989 [M + Na]+ (calcd for C23H32O8Na+, 459.1994), which corresponds to eight degrees of unsaturation The 13C NMR spectrum (CDCl3) (Table 1) exhibits similar signals to that of 1, specifically, two methine and four oxymethine carbon atoms in the d = 40À85 ppm region Detailed comparison of the NMR data of with those of suggested that both are structurally analogous, with the exception of bearing a methacrylate group instead of an isobutyrate group at the C-8 position The structure of d-lactone eudesman-9,12-olide for was determined from the HMBC correlations of the carbonyl carbon atom at d = 163.5 ppm with proton H-9 at d = 4.66 ppm, the vinylidene protons H-13a and H-13b, as well as correlations between C-9 at d = 81.9 ppm and the H-1 and methyl-15 protons The structure of wedelolide H (2) was thus determined to be 1a,4b-dihydroxy-6a-isobutyryloxy-8b-methacryloxyeudesman9,12-olide, with an absolute configuration of 1S,4S,5S,6R,7S,8S,9R,10S From the leaves of Wedelia trilobata, the crude ethanol, hexane, and dichloromethane extracts showed in vitro antimalarial activity against Plasmodium falciparum parasite (strain PFB) with IC50 values of 25.0, 27.0, and 13.0 mg/mL, respectively Two new compounds, wedelolide G (1) and wedelolide H (2), were isolated from the dichloromethane extract, and they are examples of the Please cite this article in press as: T Phan Duc, et al., New wedelolides, (9R)-eudesman-9,12-olide Phytochem Lett (2016), http://dx.doi.org/10.1016/j.phytol.2016.06.001 d-lactones, from Wedelia trilobata, G Model PHYTOL 1175 No of Pages T Phan Duc et al / Phytochemistry Letters xxx (2016) xxx–xxx Table 1 H NMR and 13 C NMR Spectroscopic Data of Compounds and (CDCl3).a Position dC, type dH (J in Hz) dC, type dH (J in Hz) 10 11 12 13 14 15 70.5, CH 28.2, CH2 41.6, CH2 71.2, C 43.7, CH 74.2, CH 44.1, CH 64.7, CH 81.9, CH 44.6, C 132.1, C 163.4, C 133.4, CH2 25.3, CH3 14.5, CH3 3.95 (1H, dd, 11.5, 5.0) 1.73 (1H, m), 1.65 (1H, m) 1.61 (1H, m), 1.42 (1H, m) 70.5, CH 28.1, CH2 41.6, CH2 71.2, C 43.7, CH 74.2, CH 44.1, CH 65.2, CH 81.9, CH 44.6, C 132.0 163.5 133.5 25.3, CH3 14.5, CH3 3.95 (1H, dd, 11.5, 5.0) 1.80 (1H, m), 1.72 (1H, m) 1.69 (1H, m), 1.51 (1H, m) 6-ester 18.9, CH3 19.2, CH3 34.0, CH 176.5, C 1.28 (3H, d, 7.0) 1.24 (3H, t, 7.0) 2.47 (1H, quint, 7.0) 18.7, CH3 19.2, CH3 34.7, CH 176.4, C 1.28 (3H, s) 1.25 (3H, s) 2.64 (1H, quint, 7.0) 8-ester 18.7, CH3 18.8, CH3 34.7, CH 176.2, C 1.13 (3H, d, 7.0) 1.11 (3H, d, 7.0) 2.64 (1H, quint, 7.0) 18.7, CH3 127.3, CH2 135.6, C 166.3, C 1.13 (3H, d, 7.0) 6.07 (1H, t, 1.0), 5.59 (3H, t, 1.5) a The 1H and 13 1.44 (1H, d, 3.0) 5.49 (1H, dd, 4.0, 3.0) 3.23 (1H, dd, 6.5, 4.0) 5.39 (1H, dd, 3.5, 2.0) 4.60 (1H, t, 2.5) 6.66 (1H, s), 5.82 (1H, s) 1.30 (3H, s) 1.32 (3H, s) 1.45 (1H, d, 3.0) 5.50 (1H, dd, 4.0, 3.0) 3.27 (1H, dd, 6.5, 3.5) 5.48 (1H, dd, 3.5, 2.0) 4.66 (1H, t, 2.5) 6.66 (1H, s), 5.84 (1H, s) 1.31 (3H, s) 1.33 (3H, s) C NMR spectra were measured at 500 and 125 MHz, respectively Fig Structural fragments of compound rare sesquiterpene (9R)-eudesman-9,12-olide d-lactone, wedelolides A (10) and B (11) (Quang et al., 2007) were isolated from the extract, also Other commonly known eudesmanolide sesquiterpene lactones generally contain 6,12- (Spring et al., 2000) or 8,12-g-lactones, e.g., trilobolides (9) and (10) (Bohlmann et al., 1981; Salwa et al., 1996; Ragasa et al., 1993), with various configurations of the A/B and B/C ring junctions and various configurations of C-1, C-4, C-5, C-6, C-7, C-8, C-9, and C-10 The in vitro antimalarial activity of wedelolide G (1) and wedelolide H (2), with IC50 values of 3.42, 5.96 mM (Table 2), were more active than Fig Significant HMBC (!) correlations of compounds and Table In vitro antimalarial activity of isolated compounds from Wedelia trilobata and positive control against Plasmodium falciparum parasite (strain PFB) Compound Antimalarial activity against strain PFB; IC50 (mM) Wedelolide A (10) Wedelolide B (11) Wedelolide G (1) Wedelolide H (2) Trilobolide (8) Trilobolide (9) Chloroquine 4.22 9.15 3.42 5.96 32.67 19.70 0.08 Please cite this article in press as: T Phan Duc, et al., New wedelolides, (9R)-eudesman-9,12-olide Phytochem Lett (2016), http://dx.doi.org/10.1016/j.phytol.2016.06.001 Cytotoxicity; IC50 (mM) 16.05 17.78 d-lactones, from Wedelia trilobata, G Model PHYTOL 1175 No of Pages T Phan Duc et al / Phytochemistry Letters xxx (2016) xxx–xxx Fig Structures of the previously reported trilobolide (8), trilobolide (9), wedelolide A (10), and wedelolide B (11) Organic Chemistry, University of Science, Vietnam National University, Ho Chi Minh City 3.4 Extraction and isolation Fig Selected NOE interactions of wedelolide G (1) the previously reported trilobolides (8) and (9) (32.67 and 19.70 mM), as well as those of wedelolide A (10) and wedelolide B (11) (4.22 and 9.15 mM) (Fig 4) Chloroquine was used as a positive control, with an IC50 = 0.08 mM (Quang et al., 2007) It must be noted that wedelolide G (1) and wedelolide H (2) are toxic to Hep G2 cells (IC50 = 16.05 and 17.78 mM, respectively, Table 2) Experimental 3.1 General experimental procedures The NMR spectra were acquired on a Bruker Advance III 500 MHz spectrometer (Bruker Biospin) with tetramethylsilane (TMS) as an internal standard, with chemical shifts expressed in d (ppm) values The HR-ESI–MS were determined with a MicrOQIITOF mass spectrometer (Bruker Daltonics) The optical rotation values were measured on a Kruss digital polarimeter Analytical and preparative TLC were performed on precoated Merck Kieselgel 60F254 or RP-18F254 plates (0.25 mm or 0.5 mm thickness) 3.2 Cell line and cell culture Hep G2 cells (HB-8065) were purchased from the American Type Culture Collection (Manassas, VA, USA) Cells were cultured at 37  C and 5% CO2, in Eagle’s Minimal Essential Medium (E’MEM) supplemented with 10% (v/v) FBS (Sigma), mM L-glutamine (Sigma), 20 mM HEPES (Sigma), 0.025 mg/mL amphotericine B (Sigma), 100 IU/mL penicillin G (Sigma), and 100 mg/mL streptomycin (Sigma) 3.3 Plant material Leaves of W trilobata were collected in Ho Chi Minh City, Vietnam in November 2011 The plant identification was by pharmacist Phan Duc Binh, Associate Editor–in–Chief of the Vietnam Bimonthly Magazine of Drug and Health A voucher specimen was deposited in the herbarium of the Department of The crude EtOH residue of dried leaf powder of W trilobata (1.8 kg), was successively extracted with hexane and CH2Cl2 The CH2Cl2 extract was subjected to silica gel column chromatography (CC) eluted with hexane/CHCl3 (9.5:0.5 to 0:10, v/v, L), followed by CHCl3/MeOH (9.5:0.5 to 2:8, v/v, L), affording sixteen fractions (1–16) Bioactive fraction (12.6 g), obtained from the elution of CHCl3/MeOH of 9.5:0.5, was further purified by preparative TLC (CH2Cl2/MeOH, 9:1) followed by RP-C18 HPLC eluted with CH3CN/ H2O (65:35), to afford new compounds, wedelolide G (1) (10.0 mg) and wedelolide H (2) (5.0 mg), respectively Fraction (16.43 g), obtained from the elution of CHCl3/MeOH of 9:1, was subjected to silica gel CC, eluted with hexane/AcOEt (from 9:1 to 5:5, v/v), to yield sub-fractions 8A–8 K Sub-fraction 8C was extensively chromatographed on columns of silica gel and Sephadex LH-20 (CHCl3ÀMeOH, 6:4, 3.2 Â 140 cm) to afford compounds (7.6 mg), (12.8 mg), and (6.3 mg) Sub-fraction 8E was submitted to a silica gel CC, eluted with increasing concentrations of AcOEt in petroleum ether (from 8:2 to 5:5, v/v), as well as preparative TLC eluted with hexane/acetone (9:1, v/v) yielding (7.1 mg), (34.8 mg), and (9.1 mg) The structures of these eight compounds (Fig 1) are identified by spectroscopic methods and optical activity Wedelolide G (1) [(1S,4S,5S,6R,7S,8S,9R,10S)-1a,4b-dihydroxy6a,8b-diisobutyryloxyeudesman-9,12-olide]: white, amorphous solid, [a]20D À7 (c 0.1, MeOH); HR-ESI–MS m/z = 461.2146 [M+Na]+ (calcd for C23H34O8, 461.2151); 1H NMR (CDCl3) and 13C NMR (CDCl3), see Table Wedelolide H (2) [(1S,4S,5S,6R,7S,8S,9R,10S)-1a,4b-dihydroxy6a-isobutyryloxy-8b-methacryloxyeudesman-9,12-olide]: white, amorphous solid, [a]20D À9 (c 0.1, MeOH); HR-ESI–MS m/ z = 459.1989 [M + Na]+ (calcd for C23H32O8, 459.1995); 1H NMR (CDCl3) and 13C NMR (CDCl3), see Table 5-Hydroxymethylfurfuran (3): yellow, amorphous solid 1H NMR (CDCl3): 9.55 (1H, s, H), 7.21 (1H, d, J = 3.5 Hz, H-3), 6.50 (1H, d, J = 3.5 Hz, H-4), 4.69 (2H, s) 13C NMR (CDCl3): 152.4 (C-2), 123.0 (C3), 110.1 (C-4), 160.9 (C-5), 57.6 (À ÀCH2OH), 177.8 (À ÀCHO) 4-Hydroxy-3-methoxybenzoic acid (vanillic acid) (4): white, amorphous solid 1H NMR (DMSO-d6): 7.44-7.42 (2H, m, H-2, H-6), 6.83 (1H, d, J = 9.0 Hz, H-5), 3.55 (3H, s, ÀÀOCH3) 13C NMR (DMSOd6): 167.4 (À ÀCOOH), 150.9 (C-4), 147.1 (C-3), 123.4 (C-6), 122.0 (C1), 115.0 (C-5), 112.8 (C-2), 55.5 (3-OCH3) 5,40 -Dihydroxy-7-methoxyflavone (5): yellow, amorphous sol1 id H NMR (DMSO-d6): 12.97 (1H, brs, 5-OH), 7.97 (2H, d, 9.0 Hz, H20 ,60 ), 6.95 (2H, d, 9.0 Hz, H-30 ,50 ), 6.86 (1H, s, H-3), 6.79 (1H, d, 2.0 Hz, H-8), 6.39 (1H, d, 2.0 Hz, H-6), 3.88 (3H, s, ÀOCH3) 13C NMR (DMSO-d6): 56.0 (7-OCH3), 164.1 (C-2), 103.0 (C-3), 181.9 (C-4), 161.3 (C-5), 97.9 (C-6), 165.1 (C-7), 92.7 (C-8), 157.4 (C-9), 104.6 (C-10), 121.0 (C-10 ), 128.5 (C-20 ,60 ), 115.9 (C-30 ,50 ), 161.2 (C-40 ) Please cite this article in press as: T Phan Duc, et al., New wedelolides, (9R)-eudesman-9,12-olide Phytochem Lett (2016), http://dx.doi.org/10.1016/j.phytol.2016.06.001 d-lactones, from Wedelia trilobata, G Model PHYTOL 1175 No of Pages T Phan Duc et al / Phytochemistry Letters xxx (2016) xxx–xxx 5,30 ,40 -Trihydroxy-7-methoxyflavone (6): yellow, amorphous solid 1H NMR (DMSO-d6): 13.05 (1H, brs, 5-OH), 7.42 (1H, dd, 8.5, 2.0 Hz, H-60 ), 7.37 (1H, d, 2.0 Hz, H-20 ), 6.89 (1H, d, 8.5 Hz, H-50 ), 6.71 (1H, d, 2.0 Hz, H-8), 6.33 (1H, d, 2.0 Hz, H-6), 6.66 (1H, s, H-3), 3.98 (3H, s, ÀOCH3) 13C NMR (DMSO-d6): 55.9 (7-OCH3), 164.8 (C-2), 101.5 (C-3), 181.4 (C-4), 161.2 (C-5), 97.7 (C-6), 164.8 (C-7), 92.7 (C-8), 157.0 (C-9), 104.5 (C-10), 119.7 (C-10 ), 111.6 (C-20 ), 146.6 (C-30 ), 154.0 (C-40 ), 115.5 (C-50 ), 119.7 (C-60 ) Methyl caffeate (7): white needles 1H NMR (DMSO-d6): 7.48 (1H, d, J = 15.5 Hz, H-3), 7.05 (1H, d, J = 2.0 Hz, H-20 ), 7.00 (1H, dd, J = 8.5 Hz; 2.0 Hz, H-60 ), 6.76 (1H, d, J = 8.0 Hz, H-50 ), 6.26 (1H, d, J = 16.0 Hz, H-2) 13C NMR (DMSO-d6): 51.2 (1-OCH3), 167.0 (C-1), 113.7 (C-2), 145.2 (C-3), 125.5 (C-10 ), 114.8 (C-20 ), 145.6 (C-30 ), 148.3 (C-40 ), 115.7 (C-50 ), 121.4 (C-60 ) Trilobolide-6-O-isobutyrate (8): white, amorphous solid 1H NMR (CDCl3): 6.26 (1H, d, J = 4.0 Hz, H-13a), 5.98 (1H, dd, 3.0, 1.5 Hz, H-7), 5.67 (1H, dd, J = 3.0; 1.5 Hz, H-6), 5.24 (1H, d, J = 4.5 Hz, H-9), 4.89 (1H, dd, J = 8.0; 4.5 Hz, H-8), 2.60 (1H, sept, J = 7.0 Hz, H-19), 2.01 (3H, s, H-23), 1.95 (3H, s, H-17), 1.36 (3H, s, H-15), 1.24 (3H, d, J = 7.0 Hz, H-20), 1.22 (3H, d, J = 7.0 Hz, H-21).13C NMR (CDCl3): 73.2 (1), 24.2 (C-2), 41.8 (C-3), 71.3 (C-4), 45.1 (C-5), 68.2 (C-6), 43.7 (C7), 72.2 (C-8), 70.8 (C-9), 41.5 (C-10), 134.3 (C-11), 169.5 (C-12), 119.3 (C-13), 26.6 (C-14), 14.5 (C-15), 170.7 (C-16), 21.2 (C-17), 176.4 (C-18), 34.6 (C-19), 18.6 (C-20), 19.2 (C-21), 169.3 (C-22), 20.4 (C23) 3.5 Bioassays 3.5.1 Antimalarial activity against strain PFB Plasmodium falciparum was grown by the technique of Frappier F (Frappier et al., 1996), to a hematocrit of 4% of human red blood cells of O+ blood group The culture was maintained in RPMI 1640 containing 25 mM HEPES, pH 7.3; 0.3 g/L-glutamine; g/L of NaHCO3; g/L of glucose, supplemented with 10% decomplemented human serum; and 5000 U streptomycin-penicillin The culture was maintained at 37  C under an atmosphere of 91% nitrogen, 6% oxygen, and 3% carbon dioxide The culture medium was changed daily to remove lactic acid due to carbohydrate catabolism parasites and thus keep the medium pH within the physiological range (pH = 7.2–7.4) An intake of healthy red blood cells twice per week is required to maintain the P falciparum cycle Cultivation is monitored via a blood smear stained 20 Giemsa diluted 1/20 in distilled water Parasitaemia is determined by counting the number of parasitized red blood cells out of 1000 with a microscope at a magnification of 100Â The P falciparum strain used in this work was the PFB Strain (strain cloned by limiting dilution, from Brazil, known for its chloroquine resistant, with an IC50 of 0.089 Ỉ 0.017 mM A strain is considered resistant to chloroquine from an IC50 of 70–100 nM.) 3.6 Sulforhodamine B (SRB) assay The assay was performed as previously described, with some modifications (Skehan et al., 1990) Cells seeded at a density of 10,000 cells/well in 96-well plates were cultured for 24 h before being incubated with compounds at different concentrations for 48 h Treated cells were fixed with cold 50% (w/v) trichloroacetic acid (Merck) solution for 1–3 h, washed, and stained with 0.2% (w/ v) SRB (Sigma) for 20 After five washes with 1% acetic acid (Merck), protein-bound dye was solubilized in 10 mM Tris base (Promega) solution Optical density values were determined using a 96-well micro-titer plate reader (Synergy HT, Biotek Instruments) at wavelengths of 510 nm and 620 nm Percentage of growth inhibition (I%) was calculated according to the formula: I % = (1 À [ODt/ODc] Â 100%, where ODt and ODc are the optical density value of test sample and control sample, respectively Dose-response curves were plotted to determine IC50 values using the GraphPad Prism software (GraphPad, San Diego, CA) Acknowledgment This work was supported by grant # 104.01-2011.46 from Vietnam’s National Foundation for Science and Technology Development (NAFOSTED) References Ashley, E.A., Dhorda, 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systemic acquired resistance J Agric Food Chem 61, 3884–3890 Please cite this article in press as: T Phan Duc, et al., New wedelolides, (9R)-eudesman-9,12-olide Phytochem Lett (2016), http://dx.doi.org/10.1016/j.phytol.2016.06.001 d-lactones, from Wedelia trilobata, ... accounting for four degrees of unsaturation The three remaining degrees of unsaturation are attributed to three saturated rings The 13C distortionless enhancement of polarization transfer spectrum (DEPT)... et al., New wedelolides, (9R)-eudesman-9,12-olide Phytochem Lett (2016), http://dx.doi.org/10.1016/j.phytol.2016.06.001 Cytotoxicity; IC50 (mM) 16.05 17.78 d-lactones, from Wedelia trilobata, G... as: T Phan Duc, et al., New wedelolides, (9R)-eudesman-9,12-olide Phytochem Lett (2016), http://dx.doi.org/10.1016/j.phytol.2016.06.001 d-lactones, from Wedelia trilobata, G Model PHYTOL 1175