Phytochemistry Letters 12 (2015) 328–331 Contents lists available at ScienceDirect Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol Terpenoids from the stem bark of Neoboutonia macrocalyx (Euphorbiaceae) Timoleon Maffo a , Pascal Wafo b, ** , Ramsay Soup Teoua Kamdem a,e, * , Raduis Melong a , Philip F. Uzor d, Pierre Mkounga a , Zulfiqar Ali c , Bonaventure Tchaleu Ngadjui a a Department of Organic Chemistry, Faculty of Science, University of Yaoundé I P.O. Box 812, Yaoundé, Cameroon Higher Teachers’ Training College, University of Yaoundé I P.O. Box 47, Yaoundé, Cameroon National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA d Department of Pharmaceutical and Medicinal Chemistry, University of Nigeria, Nsukka 410001, Nigeria e Institute für Pharmaceutische Biologie und Biotechnologie HEINRICH HEINE Universität Düsseldorf Universitat StraBe 1, Gebäude 26.23, 40225 Düsseldorf, Germany b c A R T I C L E I N F O A B S T R A C T Article history: Received 8 February 2015 Received in revised form 23 April 2015 Accepted 29 April 2015 Available online 11 May 2015 Neoboutomannin A (1), a new degraded diterpenoid monomer, and 3a-acetyl-14a-hydroxytaraxan19,28-olide (2), a new triterpenoid derivative, have been isolated from the stem bark of Neoboutonia macrocalyx Benth (Euphorbiaceae), together with the known compounds, 3-acetyl aleuritolic acid (3), 3b-acetoxy oleanolic acid (4), oleanolic acid (5), 3,3,4-tri-O-methylellagic acid (6), sitosterol 3-O-b-Dglucopyranoside (7) and sitosterol (8). Their structures were elucidated on the basis of spectral data and comparison with those present in the literature. The methanol extract of the stem bark of N. macrocalyx showed high toxicity to brine shrimp nauplii (LC50 = 0.6  0.05 mg/mL) and low antifungal activity on Candida albicans and Mucus miehei. Compound 3 exhibited moderate toxicity to brine shrimp (LC50 = 10.0  0.9 mg/mL) and compound 2 showed low antifungal activity. ã2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. Keywords: Euphorbiaceae Neoboutonia macrocalyx Terpenoids 1. Introduction Euphorbiaceae is a large family of flowering plants with 300 genera and around 7500 species. Most are herbs, but some, especially in the tropics, are also shrubs or trees (Troupin,1982). This family occurs mainly in the tropics, with the majority of the species in the Indo-Malayan region and tropical America region. There is a large variety in tropical Africa, but it is not as abundant or varied as in these two other tropical regions. Euphorbiaceae also has many species in non-tropical areas such as the Mediterranean Basin, the Middle East, South Africa, and southern USA (Troupin, 1982). The genus Neoboutonia is widely distributed in tropical West Africa and Central Africa and is represented by the species, glabrescens, melleriprain, manii and macrocalyx. The phytochemistry of the genus Neoboutonia has not been extensively studied. However, diterpenes and sterols have been reported in Neoboutonia macrocalyx; tigliane derivatives and triterpenoids were also reported from the leaves of Neoboutonia melleri (Kirira et al., 2007; Tchinda et al., 2003; Zhao * Corresponding author at: Institute für Pharmaceutische Biologie und Biotechnologie HEINRICH HEINE Universität Düsseldorf Universitat StraBe 1, Gebäude 26.23, 40225 Düsseldorf, Germany. Tel.: +491 5213040618. ** Corresponding author. E-mail addresses: wafopascal@yahoo.fr (P. Wafo), ramsay_kamdem@yahoo.fr (R.S.T. Kamdem). et al., 1998). N. macrocalyx is a plant used by traditional healers among the Meru community in Kenya. The stem bark of N. macrocalyx is used traditionally to treat headache and fever. Previous investigations indicate that the methanol extract of the stem bark of N. macrocalyx shows a high toxicity to brine shrimp nauplii (LC50 = 21.04  1.8 mg/mL). The aqueous extract of N. macrocalyx also exhibits a mild brine shrimp toxicity (LC50 = 41.69  0.9 mg/mL) (Kirira et al., 2006). In the course of our on-going research on Cameroonian medicinal plants used traditionally to treat human microbial infections, the methylene chloride–methanol (1:1) extract of the stem bark of N. macrocalyx was examined for its antifungal and cytotoxicity properties and also for its chemical constituents. This study led to the isolation and structural characterization of neoboutomannin A (1), a new degraded diterpenoid monomer and 3a-acetyl-14a-hydroxytaraxan-19,28-olide (2), a new triterpenoid derivative, along with the six known compounds (3–8). The cytotoxity and antimicrobial activities of the crude extract and compounds 2 and 3 were evaluated. 2. Results and discussion The extract of the stem bark of N. macrocalyx was subjected to repeated column chromatography to give several fractions which http://dx.doi.org/10.1016/j.phytol.2015.04.026 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. T. Maffo et al. / Phytochemistry Letters 12 (2015) 328–331 329 CH3-29, and CH3-30 respectively), two carbinolic protons at dH/dC 4.55 (1H, m, Hb-3)/81.7 and at dH/dC 4.56 (1H, d, J = 12.2 Hz, Hb-19) /79.7 attributed to C-3 and C-19 respectively, one methyl at d 2.13 (3H, s) corresponding to the methyl of an acetyl group. Its position at C-3 was determined through an HMBC experiment in which the oxymethine proton signal at d 4.55 (Hb-3) showed 3JC H correlations with C-10 (d 171.0). The configuration of the acetyl group at C-3 was assigned as a on the basis of the interactions of H-3 with CH3-23 and CH3-25 in the NOESY experiment (Fig. 2). The relative configurations of C-14, C-19 and C-17 were determined in a similar manner. The hydroxyl at C-14 is probably from hydroxylation of the olefinic carbons of the D14–15 functionality of taraxerane skeleton. From the HMBC spectrum, the proton at H-19 showed correlations to carbons at d 43.0 (C-17), 45.4 (C-18), 31.2 (C-21), 181.6 (C-28); these correlations infer the presence of a lactone group. The HMBC spectrum also showed that the lactone system is in the ring E, between the 19-hydroxyl and 28-carbonyl group. The lactone ring was deduced as b based on NOESY interactions of H-19 (d 4.56) with CH3-30. The 1H and 13C NMR chemical shifts were assigned using 1H–1H COSY, HSQC, HMBC, and TOCSY spectra (Fig. 2). The following key unambiguous HMBC correlations were observed: H-19 with C-28 and C-17; H-26 with C-14 and C-9; H-16 with C-28 and C-17. The relative configuration of 2 was deduced from the NOESY correlations (Fig. 2). The EI-MS of 2 corroborated the above structure with important fragments at m/ z 43.3 [CH3CO]; 439.3[M-CH3COOH-CH3], 454.3 [M-CH3COOH] and 411.3 [M-CH3COOH-CH3-CO]. Based on the above mentioned data, the structure of compound 2 was deduced as 3a-acetyl-14ahydroxytaraxan-19,28-olide (2). The known compounds were identified as 3-acetyl aleuritolic acid (3) (Woo and Hildebert, 1977), 3b-acetoxyoleanolic acid (4) (Maillard and Adewunmi, 1992), oleanolic acid (5) (Maillard and Adewunmi, 1992), 3,30 ,4-tri-O-methylellagic acid (6) (Khac et al., 1990), sitosterol 3-O-b-D-glucopyranoside (7) and sitosterol (8) (Lawson et al., 1988). The known compounds were identified as 3-acetyl aleuritolic acid (3) (Woo and Hildebert, 1977), 3b-acetoxyoleanolic acid (4) (Maillard and Adewunmi, 1992), oleanolic acid (5) (Maillard and Adewunmi, 1992), 3,30 ,4-tri-O-methylellagic acid (6) (Khac et al., 1990), sitosterol 3-O-b-D-glucopyranoside (7) and sitosterol (8) (Lawson et al., 1988). Results of the antimicrobial test showed that the extract was active against the microbes used but was more active against the fungi, Candida albicans and Mucor miehei (inhibition zone of 10 mm each). Compounds 2 and 3 also showed moderate antifungal were further purified to yield two new compounds 1 and 2, together with six known compounds. Compound 1 was obtained as a yellow powder from hexane/ EtOAc (60:40) fraction. The 1H and 13C NMR data (Table 1) indicated the presence of 14 non-exchangeable protons and 16 carbon atoms corresponding to the molecular formula C16H14O3 (10 of unsaturation) which was deduced from its mass, 1 H and 13C NMR spectra and comparison with literature data of neoboutomannin (1b). The 1H NMR data of 1 revealed the presence of three methyl singlet resonances at d 1.20, 1.20 and 2.22; two vinylic proton singlet resonances at d 7.08 and 6.49; two aromatic proton singlet resonances at d 7.40 and 7.82, and a hydroxyl group resonance at d 10.62. The HMBC spectroscopic data were used to construct the skeleton of 1. Cross-peaks were observed in HMBC spectra between: H-1 (d 7.08) and C-3 (d 207.9), C-9 (d 129.5), C-5 (d 161.3), C-4 (d 45.3); H-6 (d 6.49) and C-10 (d 154.3), C-4 (d 45.3), and C-7 (d 183.3); H-11 (d 7.40) and C-12 (d 160.0), C-10 (d 154.3), C-9 (d 129.5) and C-8 (d 123.3); H-14 (d 7.82) and C-12 (d 160.0), C-15 (d 16.4) and C-7 (d 183.3); H-15 (d 2.22) and C-12 (d 160.0), C-13 (d 130.0), C-14 (d 129.3). The numbering system used for 1 (Figs. 1 and 2) is the same as neoboutomannin (1b) and reflects its putative diterpenoid biogenetic origin (Tene et al., 2008). Thus compound 1 is a new degraded diterpenoid monomer to which a trivial name, neoboutomannin A, is given. Compound 2 was an optically active white amorphous powder from n-hexane/EtOAc (37:3) fraction. It was assigned the molecular formula C32H50O5 from the 1H and broad band-decoupled 13C NMR spectra and ESI-HRMS [M + Na]+ at m/z 537.3564 (calcd. 537.3550). The IR spectrum showed absorptions for a hydroxyl group (3430.32 and 3300.92 cm 1), a lactone group (1732.00 cm 1), an ester group (1684.00 cm 1), and a gem-dimethyl (1386.00 and 1375.00 cm 1) (Lontsi et al., 1998). It gave a positive Liebermann–Burchard test for triterpenes. The 13C NMR spectrum of compound 2 revealed 32 carbon signals which were sorted by DEPT 13C NMR as eight methyls, ten methylenes, three methines, seven quaternary carbons, two oxymethine and one quaternary alcohol (Table 2). Further analysis of these spectra revealed resonances for two carboxyl groups at d 171.0 and 181.6; two oxymethine groups at d 79.7 and 80.7, and eight methyl groups at d 16.6, 17.3, 21.3, 21.7, 22.9, 24.1, 27.9, and 33.5. A detailed analysis of the 1H NMR spectrum of 2 (Table 2) confirmed the characteristic features for a triterpenic taraxerane parent structure (Mahato and Kundu, 1994). It was characterized by signals of seven tertiary methyls at dH/dC 0.84/16.6, 0.84/27.9, 0.95/17.3, 0.85/22.9, 1.12/21.7, 0.96/24.1, and 0.92/33.5 (3H each, s, CH3-24, CH3-23, CH3-25, CH3-26, CH3-27, Table 1 1 H and 13C NMR spectroscopic data of compounds 1 and 1b (DMSO-d6) C. No Neoboutomanin A (1) dC 1 3 4 5 6 7 8 9 10 11 12 13 14 15 18 19 12-OH 127.9 207.9 45.3 161.3 120.5 183.3 123.3 129.5 154.3 111.6 160.0 130.0 129.3 16.4 23.0 23.0 – C. No dH (mult.) 7.08 (s) – – – 6.49 (s) – – – – 7.40 (s) – – 7.82 (s) 2.22 (s) 1.20 (s) 1.20 (s) 10.62(s) 0 1/1 3/30 4/40 5/50 6/60 7/70 8/80 9/90 10/100 11/110 12/120 13/130 14/140 15/150 18/180 19/190 12/12-OH Neoboutomanin (1b) (Tene et al., 2008) dC dH (mult.) 133.5 204.6 45.7 160.3 121.4 183.4 124.0 130.0 151.8 112.1 160.6 130.0 130.3 16.5 22.8 23.9 – – – – – 6.76 (s) – – – – 7.14 (s) – – 7.94 (s) 2.21 (s) 1.44 (s) 1.33 (s) 10.53 (s) 330 T. Maffo et al. / Phytochemistry Letters 12 (2015) 328–331 Fig. 1. Structures of compounds 1, 1b and 2. Fig. 2. Key correlations from HMBC 1 and 2, and NOESY of 2. activity (inhibition zone of 10 mm each). The crude extract showed a higher toxicity against brine shrimp nauplii (LC50 = 0.6  0.05 mg/mL) than compounds 2 (LC50 > 100.00 mg/mL) and 3 (LC50 = 10.0  0.9 mg/mL). In the present study, the extract and compounds 2 and 3 showed LC50 of less than 1000 mg/mL indicating their potential as cytotoxic and antitumor agents. However, further studies are needed to determine their possible role in cancer. filtration, the solution was evaporated in vacuum to yield 150.3 g of crude extract. A portion (140 g) of this extract was subjected to flash chromatography over silica gel (300 g, 80  5 cm) eluting with n-hexane/EtOAc in order of increasing polarity to give four Table 2 1 H and 13C NMR data of compound 2 [CDCl3; d ppm (mult., J = Hz)] C. No. 3.1. General experimental procedure The optical rotation was measured on a PerkinElmer polarimeter 241 at the sodium D line. The NMR spectra were recorded on a Varian Inova-500 spectrophotometer. The chemical shifts are given in d values with TMS as internal reference, and coupling constants are given in Hz. The ESI and ESI-HR mass spectra were recorded on an APEX IV FTICR mass spectrometer Bruker Daltonik, at 7 T. Column chromatography was performed on silica gel (type 60, 70–230 mesh, E. Merck). TLC experiments were carried out on silica gel pre-coated plates (E. Merck, 0.25 mm), and detection was achieved by UV light (254 or 366 nm) and spraying with 10% H2SO4 followed by heating. All solvents were distilled before use. 3.2. Plant material The plant material was collected at Manjo, Littoral region of Cameroon in May 2009 and identified by Mr. Victor Nana, the botanist at the National Herbarium. The voucher specimen (Ref 50111 HNC) has been deposited in the National Herbarium, Yaounde, Cameroon. 3.3. Extraction and isolation The air-dried and powdered stem bark (3 kg) was extracted with MeOH/CH2Cl2 (1:1, 6 L  2, 48 h) at room temperature. After 2 13 3. Material and methods 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 10 20 C 33.7, CH2 23.5, CH2 80.7, CH 37.7, C 56.1, CH 18.3, CH2 44.3, CH2 40.4, C 47.9, CH 37.8, C 18.0, CH2 38.6, CH2 44.9, C 76.1, C 34.2, CH2 32.3, CH2 43.0, C 45.4, CH 79.7, CH 28.9, C 31.2, CH2 35.0, CH2 27.9, CH3 16.6, CH3 17.3, CH3 22.9, CH3 21.7, CH3 181.6, C 24.1, CH3 33.5, CH3 171.0, C¼O 21.3, CH3 1 H 1.20; 1.97 (m) 1.65; 1.61 (t, 2.3) 4.55 (m) – 1.19 (m) 1.54; 1.49 (m) 1.30; 1.56 (m) – 1.47 (m) – 1.57; 1.45 (m) 1.30; 1.07 (m) – – 1.30; 1.07 (m) 1.64; 1.71(m) – 2.74 (d, 12.2) 4.56 (d, 12.2) – 1.37; 1.63 (m) 1.99; 1.26 (m) 0.84 (s) 0.84 (s) 0.95 (s) 0.85 (s) 1.12 (s) – 0.96 (s) 0.92 (s) – 2.13 (s) T. Maffo et al. / Phytochemistry Letters 12 (2015) 328–331 fractions (A–D). Fraction B (23.2 g, eluted with hexane/EtOAc (39:1–9:1), was applied to multiple column chromatography (CC) [silica gel (50 g), column (50  3 cm)], to afford 3a-acetyl-14ahydroxyoleana-19,28-olide (2) (5.3 mg), 3-acetyl aleuritolic acid (3) (10.0 mg), sitosterol (8) (3 g), 3-acetylolean-12-enoic acid (4) (15 mg) and olean-12-enoic acid (5) (15 mg). Fraction C (148.3 mg, eluted with hexane/EtOAc, 4:1–3:2) was subjected to CC [silica gel (70 g), column (60  3 cm) eluting with mixtures of hexane/EtOAc] to purify neoboutomannin A (1) (4.7 mg) and 3,30 ,4-tri-Omethylellagic acid (6) (25.3 mg). Sitosterol 3-O-b-D-glucopyranoside (7) (75 mg) was obtained as MeOH insoluble material from fraction D. 3.4. Antimicrobial screening Microbial strains of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Streptomyces viridochromogenes, C. albicans and M. miehei were used for the antimicrobial testing following the disk diffusion method (Bauer et al., 1966). The compounds (2 and 3) as well as the crude extract were tested against these bacterial and fungal species, at concentrations varying from 200 to 0.78 mg/mL. Compound 1, obtained in small amount, was not tested. An inhibition zone of 14 mm or greater was considered as high antimicrobial activity. 3.5. Brine shrimp lethality assay The assay was done according to the method described by Meyer et al. (1982) with some modifications. The crude extract together with compounds 2 and 3 was evaluated. Brine shrimps (Artemia salina) were hatched from brine shrimp eggs in a conical shaped vessel (1 L), filled with sterile artificial sea water (prepared using sea salt 38 g/L and adjusted to pH 8.5 with 1 N NaOH) under constant aeration for 48 h. After hatching, active nauplii, free from egg shells were collected from brighter portion of the hatching chamber and used for the assay. To determine the LC50, several concentrations (100, 50, 25, 12.5, 6.25 and 3.25 mg/mL) of the crude extract or compound in DMSO were prepared. Then 10 mL of each concentration was added to 990 mL of artificial sea water containing more than 20 brine shrimps in each of the 24-well tissue culture plate. After incubation for 24 h, the mean percentage lethality per test concentration of the crude extract or compound was determined. The percentage was plotted against the logarithm of concentration. The concentration, at which 50% of the brine shrimps died (LC50), was determined from the graph. The highly cytotoxic actinomycine D was used as positive control while DMSO was used as negative control. Brine Shrimp (Artemia sp.) Lethality Assay (BSLA) is a general bioassay that appears capable of detecting a broad spectrum of bioactivity present in plant crude extracts (Pisutthanan et al., 2004). According to Meyer et al. (1982), it is used as an indicator for general toxicity and also as a guide for the detection of antitumor and pesticidal compounds. Crude plant extract is toxic (active) if it has an LC50 value of less than 1000 mg/mL while non-toxic (inactive) if it is greater than 1000 mg/mL. View publication stats 331 3.6. Spectroscopic data 3.6.1. Neoboutomanin A (1) Yellow solid (hexane-EtOAc); m.p. > 310  C; IR nmax cm 1: 3411.00, 2966.00, 1681.00 1645.00, 1579.00, 1479.00, 1382.00, 1360.00; 1H NMR (DMSO-d6, 400.13 MHz) and 13C NMR (DMSO-d6, 100.6 MHz) data: see Table 1. HR-ESI-MS spectra gave the positive ion at m/z 255.1015 [M + H]+ Analysis found: C 75.87, H 5.18%; C16H14O3 requires C 75.88, H 5.17%; 3.6.2. 3a-acetyl-14a-hydroxytaraxan-19,28-olide (2) White amorphous powder (hexane-EtOAc); ½aŠ20 D = 9.9 (c = 0.10 in MeOH); IR nmax cm 1: 3430.32, 3300.92, 1730.73, 1680.52, 1469.49, 1450.21, 1386.57; EI-MS: m/z: 45 (100%); 55 (25%); 189 (42%); [M + Na]+ peak at m/z 537.3564 (calcd. for [M + Na]+ 514.3550); 1H NMR (CDCl3, 300 MHz) and 13C NMR (CDCl3, 75 MHz) data: see Table 2. Acknowledgements The authors are thankful to Mr. Nana, the botanist at the National Herbarium Cameroon for identification of plant material. References Bauer, A.W., Kirby, W.M., Sherris, J.C., Turck, M., 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45, 493–496. Khac, D.D., Tran-Van, S., Campos, A.M., Lallemand, J.Y., Fetizon, M., 1990. Ellagic compounds from Diplopanax stachyanthus. Phytochemistry 29, 251–256. 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