A new ent-clerodane diterpenoid from Crassocephalum bauchiense Huch. (Asteraceae) Alembert T. Tchinda*a, Simplice R. Mouokeub, Rosalie A.N. Ngonoc, Madeleine R.E Ebellec, Aristide L.M. Kognoua,b, Diane K. Nonod, Joe D. Connollye and Michel Frédérichf a Laboratory of Phytochemistry, Institute of Medical Research and Medicinal Plant Studies, (IMPM), P.O. Box 6163, Yaounde, Cameroon b Institute of Fisheries and Aquatic Sciences, University of Douala, PO Box 2701, Douala, Cameroon c Department of Biochemistry, Faculty of Sciences, University of Douala, PO Box 24157, Douala, Cameroon. d Department of Organic Chemistry, Faculty of Science, The University of Yaounde 1, P.O. Box 812 Yaounde, Cameroon e Department of Chemistry, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, Scotland. f Laboratoire de Pharmacognosie, Université de Liège, Centre Interdisciplinaire de Recherche sur le Médicament (CIRM), Département de Pharmacie, Université de Liège, B36, B-4000, Liège, Belgium                                                                                                                         *  Corresponding author. Email :  alembertt2002@yahoo.fr Tel: 00(237)76925929   Abstract A phytochemical investigation of the whole plant of Crassocephalum bauchiense Huch. resulted in the isolation of a new clerodane diterpenoid, ent-2β,18,19trihydroxycleroda-3,13-dien-16,15-olide (1) 3’,5-dihydroxy-4’,5’,6,7,8-pentamethoxyflavone together (2) with two known flavonoids and 4’,5-dihydroxy-3’,5’,6,7,8- pentamethoxyflavone (3). The compounds were tested against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum. Compound 2 showed weak activity (IC50 = 10.1 µg/mL) whilst compounds 1 and 3 were inactive. The structures of the compounds were defined by detailed spectral analyses, especially 1H- and 13C- NMR, 1H-1H COSY, NOESY, HMBC and HR-ESIMS. Key words: Crassocephalum bauchiense; isolation; clerodane diterpenoid; flavonoids; antiplasmodial activity. 1. Introduction Crassocephalum bauchiense Hutch. is an erect bushy annual herb about 1ft. high with bright blue florets in numerous heads growing in rough open ground in Nigeria and Cameroon (Hutchison & Dalziel 1958). The plant is used in folk medicine in Cameroon to treat gastrointestinal infections, liver disorders, epilepsy, pain, arthritis, colics, behavioural disturbances in mentally-retarded children, inflammatory disorders and neuropathic pain (Arbonnier 2000; Moukeu et al. 2011). In experimental studies, the aqueous extract of the leaves showed antinociceptive activity (Taiwe et al 2012) and the fractions antibacterial and immunomodulary activities (Mouokeu et al., 2011; 2013). To the best of our knowledge, no previous phytochemical study has been done on this plant. In our efforts to investigate Cameroonian medicinal plants for the biological activity of their constituents, we carried out a phytochemical study on the dichloromethane-methanol (1:1) extract of the titled plant and report herein the isolation and structure elucidation of a new clerodane diterpenoid ent-2β,18,19-trihydroxycleroda-3,13-dien-16,15-olide (1), from the whole plant of C. bauchiense together with two known flavonoids, 3’,5-dihydroxy-4’,5’,6,7,8pentamethoxyflavone (2) and 4’,5-dihydroxy-3’,5’,6,7,8-pentamethoxyflavone (3), identified for the first time in the Crassocephalum genus. The isolated compounds were tested against the 3D7 strain of Plasmodium falciparum. Compound 2 showed weak activity. 2. Results and discussion The crude CH2Cl2-MeOH (1:1) extract of the whole plant of C. bauchiense was subjected to a series of silica gel and sephadex LH-20 chromatographic columns to give the new diterpenoid (1) and the known flavonoids 3’,5-dihydroxy-4’,5’,6,7,8-pentamethoxyflavone (or Gardenin C) (2, Sujata et al. 2013) and 4’,5-dihydroxy-3’,5’,6,7,8-pentamethoxyflavone (3, Fushiya et al. 1999) (Fig. 1). Compound 1 was isolated as an amorphous solid. Its molecular formula C20H30O5 was deduced from the HRESIMS which showed the pseudo-molecular ion peak [M+Na]+ at m/z 373.1970. Its IR spectrum exhibited characteristic absorptions for hydroxyl groups (3238 cm1 ), α,β–unsaturated-γ-lactone (1745 cm-1) and double bond (1649 cm-1) (Esquivel et al. 1989). The structure of the compound was established from its 1H- and 13C-NMR spectroscopic data (Table1), which suggested a clerodane diterpenoid (Esquivel et al., 1989). The 13C NMR spectrum of 1 revealed 20 C-atoms, including a lactone carbonyl and two trisubstituted double bonds, three methines (one oxygenated), eight methylenes (three oxygenated), a secondary methyl group and a tertiary methyl group and two fully substituted carbons. The presence of an α,β-unsaturated γ-lactone moiety was evident from the lowfield signal of the olefinic proton H-14 (δH 7.35, brs), showing HMBC correlations with the lactone carbonyl C-16 (δC 175.3), C-12 (δC 128.1), C-13 (δC 133.2) and C-15 (δC 70.6). This was further supported by the other long range correlations between the oxymethylene protons H2-14 ( δH 4.81, brs,) and C-13 and C-16. The two methyls signals at δH 0.88 (d, J = 7.8 Hz) and δH 0.82 (s) in the 1H NMR spectrum of compound 1 were assigned to Me-17 and Me-20 respectively. The characteristic Me-18 and Me-19 of clerodane diterpenoids (Merrit & Ley 1992) were oxidized to oxymethylenes which were observed in the 13 C NMR spectrum at δC 62.5 [δH 4.19 (H-18a) and 3.89 (H-18b), both d, J = 12.8 Hz] and δC 64.5 5 [(δH 4.02 (H-19a) and 3.71 (H-19b), both d, J = 10.8 Hz] respectively. The positioning of these methylene groups at C-4 and C-5 was based on the observation of HMBC correlations between H-18b and C-3 (δC 128.6) and C-4 (δC 147.5) and between H-19a and C-6 and C-10 (Table 1). Compound 1 is isomeric with a previously reported clerodane, ent-7β,18,19-trihydroxycleroda-3,13-dien-16,15-olide, isolated from another source (Esquivel et al., 1989). In addition, their NMR data are very close, the difference being the position of the secondaryhydroxyl group which was fixed at C-2 (δC 68.1) in 1 by the HMBC correlations observed between H-2 (δH 4.28,) and C-1, C-3 and C-4. The HMBC correlation between H2-1 and C-2 as well as the COSY connectivity between H2-1 and H-2 further confirmed the attachment of the hydroxyl group at C-2. The relative configuration of compound 1 was determined from the NOESY spectrum (Table 1) and by comparison of NMR data with those of reported analogues (Das et al. 2005; Esquivel et al. 1989; Gu et al. 2014). The important NOESY cross peaks included those between H-10 and H-2 and H-8 one one hand, Me-17 and Me-20 and H-19a on the other hand. These results revealed the trans AB-ring junction and the relative configurations at C-2, C-8 and C-9. In vitro antiplasmodial activity of compounds 1, 2 and 3 was evaluated against the chloroquino-sensitive 3D7 strain of P. falciparum. Compound 2 was moderately active (IC50 = 10.1 µg/mL) whilst compounds 1 and 3 were inactive (IC50 > 40 µg/mL). However, at higher concentrations, compound 3 inhibited 42% of the parasites. Compounds 2 and 3 have the same A and B rings. The two flavonoids differ in the positions of the hydroxy and methoxy groups in ring B. The presence of these groups at C-3’ and C-4’ in compound 1 may be responsible for his activity while the positions of these groups in compound 3 may account for the reduction in activity. 3. Experimental 4.1 General experimental procedures The optical activity was determined with an AA-10 Automatic (ANALIS) polarimeter The UV spectra were recorded with an U-2910 spectrometer (λmax in nm). The IR spectrum was recorded on a Perkin-Elmer 1750 FTIR spectrometer. The HRESI mass spectra were recorded with a Bruker APEX-Qe 9.4T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a hybrid quadrupole analyzer and using an electrospray source.   NMR spectra were recorded in MeOH-d4 on a Bruker 500 MHz NMR AV II spectrometer equipped with a cryoprobe, with TMS as an internal reference. Chemical shifts were recorded in δ (ppm) and the coupling constants (J) are in hertz (Hz). Column chromatography (CC) was carried out on silica gel (70-230 mesh, Merck). TLC was performed on Merck precoated silica gel 60 F254 aluminum foil and compounds were detected using 10% sulfuric acid in ethanol as spray reagent. 4.2 Plant material The whole plant of C. bauchiense was collected at Dschang, in the West Region of Cameroon. The botanical identification was done at the National Herbarium in Yaounde (Cameroon) by referring to the sample number 7954/SRF/Cam. A voucher specimen of the plant is kept in the Herbarium of the Department of Plant Biology of the University of Dschang under the code number 0033/UDs/PB. 4.3 Extraction and isolation of compounds The whole plant of C. bauchiense was dried at room temperature for two weeks and powdered. The powder (1.5 Kg) was macerated with a mixture of CH2Cl2-MeOH (4L, 1:1) for two days. After filtration, the solvent was removed under reduced pressure using a rotary evaporator (45° C) to yield of 25 g (1.6%) of the crude extract which was further fractionated by column chromatography (CC) on 200 g of silica gel 60 with hexane-EtOAc and EtOAcMeOH mixtures of increasing polarity. Thirty-three fractions of 300 ml each were collected and pooled according to their TLC profile. Compound 2 (30 mg) crystallized from fraction 13 collected from the column with the mixture 60:40. Fractions 14-17 collected from the main column with the mixtures hexane-EtOAc 60:40 to 50:50 were further chromatographed on a silica gel CC with hex-EtOAc (90-10) to yield 29 fractions. Compound 3 (12 mg) crystallized from fraction 11. Fraction 28 obtained from the main column with the mixture hexane-EtOAc 30:70 was chromatographed on a Si gel CC eluting with CHCl3-MeOH (98:2 to 90:10) to give 13 fractions. Sub-fractions 6 and 7 were regrouped and passed through several Sephadex LH20 columns using methanol as eluent to give compound 1 (8 mg). Compound 1: Greyish amorphous powder; [α]25D: +23.1 (c 0.03, MeOH); UV (MeOH) λmax 215 nm; IR (KBr) 3238, 2952, 2912, 2869, 1745, 1720, 1649, 1448, 1386, 1350, 1209, 1083, 1039, 842 cm-1; 1H and 13 C NMR data see Table 1; HRESIMS: m/z 373.19705 ([M+Na]+, calcd. for C20H30NaO5, m/z 373.19855). 4.4 In vitro antiplasmodial assay Continuous culture of the P. falciparum chloroquine sensitive 3D7 strains was assessed following the procedure already described in Frederich et al. (2002). The parasites were obtained from Prof. Grellier (Museum d’Histoire Naturelle, Paris, France). Each compound was applied in a series of eight threefold dilutions from 0.02 to 40 µg/ml for a pure substance) on two rows of a 96-well microplate and were tested in triplicate (n=3). Parasite growth was estimated by determination of lactate dehydrogenase activity as described previously in Kenmogne et al. (2006). Artemisinin (98%, Sigma–Aldrich) and chloroquine were used as positive controls (IC50 4.12 ng/mL). Acknowledgements Authors acknowledge the financial support of Aires Sud (Appuis Intégrés pour le Renforcement des Equipes Scientifiques du Sud), Ministère Français des Affaires Etrangères et Européennes, France (grant No 7082), the Fund for Scientific Research-FNRS under grant T.0190.13 and a research fellowship to ATT at the Department of Pharmacognosy, University of Liège, Belgium. The authors are grateful to Ms. J. Widart, Laboratory of Mass Spectrometry, Giga Center, University of Liege, for the recording of the mass spectrum. References Arbonnier M. 2000. Arbres, arbustes et lianes des zones sèches d’Afrique de l’Ouest. CIRAD, Montpellier, 541 p. Das B, Ramu R, Venkateswarlu K, Rao Y K, Reddy M R, Ramakrishna K V S, Harakishore K, Murty U S. 2006. 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Revised by FN Hepper, Crown Agents, London vol. 2, pp. 244-248.   Table 1: 1H (500 MHz) and13C NMR (125 MHz) data of compound 1 (recorded in MeOH-d4) Pos. δH Mult., J (Hz) δC 1 2 3 4 5 6a b 1.95 4.28 5.70 2.20 1.17 ov brt (7.5) brs ov dt 27.1 68.1 128.6 147.5 42.9 30.6 - 7 8 9 10 11a 11b 12a b 13 14 1.45 1.59 1.58 1.66 1.54 2.17 2.02 7.35 m ov ov ov ov ov ov brs 26.4 35.8 38.1 45.0 35.8 HMBC (1H-13C) C-2, C-3, C-10 C-1, C-3, C-4 C-1, C-5, C-18 C-8 C-4, C-5, C-7, C19 C-8, C-12 C-9, C-13, C-12 18.14 C-8, C-10, C-13 15 16 17 4.81 0.88 brs d(6.6) 70.6 175.3 14.7 18a 18b 19a 4.19 3.89 4.02 d(12.8) d(12.8) d(10.8) 62.2 19b 20 3.71 0.82 d(10.8) s   133.2 145.9 64.5 17.8 1 H-1H COSY NOESY H-2, H-10 H-1, H-3 H-2 H-12b, H-6b H-7, H-6a H-2 H-1, H-3, H-10 H-2, H-18b H-6b H-6a, H-10 H-6b H-17 H-9, H-11 H-10, H-11 Me-17 Me-17 H-2, H-6b, H-8, Me-17 - H-12b H-12a C-12, C-13, C-15, H-15 C-16 C-13, C-16 H-14 C-7, C-8, C-9 H-8, H-11 C-3, C-4, C-5 H-18b C-4, C-5 H-18a C-4, C-5, C-6, C- H-19b 10 C-4, C-6, C-10 H-19a C-8, C-9, C-10 H-17 H2-15 H-14 H-7, H-8, , H11b, H3-20 H-18b H-18a, H-3 H-19b, H3-20 H-19a H-19a, H3-17      15 O 16 O 14 13 11 12 23 H 1 HO 9 8 2 17 10 5 20 7 3 4 6 19 OH 18 OH   1   OH OMe OMe MeO O OMe MeO OH     O 2   OMe OH OMe MeO O OMe MeO OH O   3   Fig. 1 : Structures of the isolated compounds