Phytochemistry 70 (2009) 419–423 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Triterpenoids with antimicrobial activity from Drypetes inaequalis Simon Suh Awanchiri a, Hanh Trinh-Van-Dufat c, Jovita Chi Shirri a, Marlise Diane J. Dongfack a, Guy Merlin Nguenang b, Sabrina Boutefnouchet c, Zacharias T. Fomum a, Elisabeth Seguin d, Philippe Verite e, François Tillequin c, Jean Wandji a,* a Department of Organic Chemistry, University of Yaounde-1, Faculty of Science, P.O. Box 812, Yaounde, Cameroon Department of Vegetal Biology and Physiology, University of Yaounde-1, Faculty of Science, P.O. Box 812, Yaounde, Cameroon c Laboratoire de Pharmacognosie, UMR/CNRS N° 8638, Université Paris Descartes, Faculté des Sciences Biologiques et Pharmaceutiques, 4-Avenue de l’Observatoire, 75006 Paris, France d Laboratoire de Pharmacognosie, UFR de Médecine Pharmacie Rouen, 22-Bld Gambetta, 76183 Rouen Cedex 1, France e Laboratoire de Chimie Analytique, UFR de Médecine Pharmacie Rouen, 22-Bld Gambetta, 76183 Rouen Cedex 1, France b a r t i c l e i n f o Article history: Received 26 March 2007 Received in revised form 10 December 2008 Available online 13 February 2009 Keywords: Drypetes inaequalis Euphorbiaceae Stems Fruit Triterpenoid esters Saponins Antimicrobial activity a b s t r a c t The air-dried stems and ripe fruit of Drypetes inaequalis Hutch. (Euphorbiaceae) were studied. Four triterpene derivatives, characterized as lup-20(29)-en-3b,6a-diol, 3b-acetoxylup-20(29)-en-6a-ol, 3b-caffeoyloxylup-20(29)-en-6a-ol and 28-b D-glucopyranosyl-30-methyl 3b-hydroxyolean-12-en28,30-dioate along with 10 known compounds were isolated from the whole stems. One triterpene, characterized as 3a-hydroxyfriedelan-25-al along with six known compounds were isolated from the ripe fruit. Their structures were established on the basis of spectroscopic analysis and chemical evidence. The triterpenes were tested for antimicrobial activity against some Gram-positive and Gram-negative bacteria, and two of them appeared to be modestly active. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Drypetes inaequalis Hutch. (Euphorbiaceae) is a forest shrub growing in the Centre and East provinces of Cameroon. Therapeutic applications of the Drypetes plants in West and Central Africa concern the treatment of sinusitis, swellings, boils, gonorrhoea and dysentery (Dalziel, 1937; Irvine, 1961; Bouquet and Debray, 1974; Walker et al., 1961). In our previous study on the Drypetes genus, we have reported on the anti-inflammatory and analgesic actions of the crude extract and compounds isolated from D. molunduana (Wandji et al., 2000; Chungag-Anye et al., 2001, 2002), the phenolic constituents from D. armoracia (Wandji et al., 2003) and the antileishmanial furanosesquiterpenes and tritrepenoids from D. chevalieri (Wansi et al., 2007). As a continuation of our search for compounds with biological activities from the Drypetes species, we studied the whole stems and the ripe fruit of D. inaequalis. From the whole stems, we isolated four new and 10 known compounds, and from the ripe fruit we obtained one new and six known compounds. The known compounds from both parts of the plant were identified as serjanic acid (5) (Javasinghe et al., * Corresponding author. Tel.: +237 77 39 02 87, +237 22 31 09 57; fax: +237 22 23 44 96. E-mail address: jeanwandji@yahoo.fr (J. Wandji). 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2008.12.017 1993), oleanolic acid (6) (Mahato and Kundu, 1994), hederagenin (7) (Mahato and Kundu, 1994), queretaroic acid (8) (Agrawal and Jain, 1992), serragenic acid (9) (Agrawal and Jain, 1992), 28-b-Dglucopyranosyl 3b-hydroxyolean-12-en-28-oate (10) (Srivastava and Jain, 1989), friedelin (12) (Li et al., 2006), 3,7-dioxofriedelane (13) (Mahato and Kundu, 1994), 3a-friedelanol (14) (Salazar et al., 2000), 3-oxofriedelan-25-al (15) (Anjaneyulu and Narayana, 1980), stigmasterol (16) (Wandji et al., 2000), 3b-D-glucopyranosylstigmasterol (17) (Wandji et al., 2000), sitosterol (18) (Wandji et al., 2003) and 3b-D-glucopyranosylsitosterol (19) (Wandji et al., 2003). The structures of the five new compounds have been determined as lup-20(29)-en-3b,6a-diol (1), 3b-acetoxylup20(29)-en-6a-ol (2), 3b-caffeoyloxylup-20(29)-en-6a-ol (3), 28-bD-glucopyranosyl-30-methyl 3b-hydroxyolean-12-en-28,30-dioate (4) and 3a-hydroxyfriedelan-25-al (11), on the basis of spectroscopic analysis and chemical evidence. In the present paper, the isolation, structural determination and antimicrobial activity of the new compounds will be described. 2. Results and discussion The whole stems and the ripe fruit of D. inaequalis were sundried, ground into a powder form, macerated with a mixture of solvents and chromatographed on silica gel to afford 19 compounds 420 S.S. Awanchiri et al. / Phytochemistry 70 (2009) 419–423 1–19. Compounds 5–10 and 12–19 were identified as known compounds by comparison of their 13C NMR data and other physical properties with reported values. Compounds 1–4 and 11 were characterized as five new triterpene derivatives. H 3 RO 6 H H OH 1 R=H OH 2 R = COCH3 4' 8' 3 R= 1' 9' 3' OH 7' O 30 29 CO2CH3 20 O 28 O O H HO H HO H OH H Table 1 13 C NMR data for compounds 1a, 2a, 3b, 4c, 11c and 15c (100 MHz)d. N° C 1 2 3 4 11 15 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 38.5 27.0 78.7 39.1 60.6 68.8 46.7 42.1 49.9 39.3 20.8 25.0 37.6 43.0 27.4 35.5 42.9 48.3 47.9 150.8 29.8 39.9 30.9 15.5 17.1 17.5 14.5 18.0 109.4 19.3 38.1 23.3 80.8 38.0 60.6 68.5 46.7 42.0 49.8 39.1 20.8 24.9 37.5 43.0 27.4 35.5 42.8 48.2 47.9 150.7 29.7 39.9 30.6 16.5 17.2 17.5 14.5 18.0 109.4 19.2 Ac 38.3 23.3 81.2 38.2 60.5 67.6 45.8 41.9 49.9 39.1 20.5 25.2 37.9 42.9 27.5 35.4 42.8 48.2 47.9 150.6 29.6 39.8 30.1 16.5 16.0 16.8 13.8 17.2 109.0 18.3 Caffeoyl 126.5 113.9 145.6 148.4 115.3 121.7 145.5 114.4 168.1 39.2 28.1 78.8 39.5 55.9 I9.0 33.4 39.8 48.2 37.6 24.4 123.1 144.3 41.4 28.3 24.0 46.4 43.1 42.2 44.2 30.7 33.8 29.3 17.1 16.2 17.7 26.5 175.8 28.8 177.3 Glc 95.1 73.4 77.7 70.6 77.4 61.7 21.0 37.0 71.2 53.3 38.3 41.9 18.0 52.2 51.5 58.2 28.2 31.2 39.2 38.2 31.4 35.7 30.0 42.8 35.5 28.1 32.8 38.9 10.6 14.7 204.7 19.6 18.5 31.9 35.1 31.6 22.4 40.9 211.7 59.0 41.6 40.2 17.2 52.5 51.4 57.0 28.8 31.6 39.4 38.4 31.7 35.1 30.1 42.7 34.9 28.1 31.9 39.0 7.2 15.9 204.9 19.7 18.4 31.7 35.1 31.6 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 3-OCOCH3 21.2 171.0 52.7 30-CO–OCH3 a H CDCl3. CD3OD. c C5D5N. d Assignments were made on the basis of DEPT, 1H–1H COSY, NOESY, HMQC and HMBC experiments. b HO HO H 4 carbons (Table 1). The signals exhibited at dC 109.4 and 150.8 confirmed 1 to be a lupane-type triterpene bearing two hydroxyl 26 H 25 CHO HO H H 27 CH3 H 3 CH3 24 23 CH3 CH3 11 RO Compound 1 was obtained as a colourless amorphous solid. The pseudo molecular ion peaks at m/z 443 [M+H]+ and 460 [M+NH4]+ in its CI/NH3 MS, and the HR TOF MS ES+ at m/z 442.3825 suggested its molecular formula to be C30H50O2. The IR spectrum indicated the presence of hydroxyl (3400 cm 1) and olefinic (1660 cm 1) groups. The 13C NMR and DEPT spectra of 1 showed 30 carbon signals including seven methyls, nine methylenes, seven methines (two of which are oxygenated (dH 3.20 and 4.10)) and six quaternary H3C H CH3 HO H H Fig. 1. Important NOESY correlations in compounds 1; 2 and 3. 421 S.S. Awanchiri et al. / Phytochemistry 70 (2009) 419–423 groups. The EIMS of 1 exhibited key peaks at m/z 218, 205, 203 and 189, indicating that the two hydroxyl groups are located on rings A and B. The determination of the positions of both hydroxyl groups is based on the HMBC and NOESY experiments. The HMBC spectrum of 1 showed correlations between the oxymethine proton signal at dH 3.20 and the carbon signals at dC 27.0 (C-2), 39.1 (C-4), 60.6 (C-5), 30.9 (C-23), and 15.5 (C-24), confirming the location of one hydroxyl group at C-3. Interactions were also exhibited between the second oxymethine proton signal at dH 4.10 and the carbon signals at dC 39.1 (C-4), 60.6 (C-5), 46.7 (C-7), 42.1 (C-8), and 39.3 (C10) suggesting that the second hydroxyl group was connected to C6. The NOESY (Fig. 1) cross-peaks exhibited between both protons H-3a and H-5 (d 0.78), in addition to the coupling constant of H3a (J = 10.7, 5.6 Hz) confirmed the b-orientation of the 3-hydroxyl group; the NOESY spectrum also showed correlations between the proton signal at dH 4.10 (H-6b) and the methyl proton signals at dH 0.98 (CH3-24), 0.85 (CH3-25) and 1.08 (CH3-26), confirming the a-equatorial orientation of the 6-hydroxyl group. The configuration of C-6 was also supported by the coupling constant of H-6b (J = 9.6, 4.3 Hz) and the 13C NMR spectrum of 1 which exhibited a key and characteristic signal at dC 60.6 for carbon C-5 as deduced from the HMQC spectrum; this value was 4 ppm higher than that of some reported data for C-5 (56.6 ppm) in similar lupane-type triterpenes with a 6b-OH group (Núnez et al., 2005). Therefore, the structure of compound 1 was established as, lup-20(29)-ene-3b,6a-diol. The same 3b,6a-structure was postulated before for a metabolite isolated from Periploca aphylla (Ghulam et al., 2000). However, the configuration of C-6 subsequently corresponded to lup-20(29)-en3b,6b-diol (Núnez et al., 2005). Compound 2 was obtained as a colourless amorphous solid, and was deduced to have the molecular formula C32H52O3 on the basis of CI/NH3 MS (m/z 485 [M+H]+, 502 [M + NH4]+, and the HR TOF MS ES+ (m/z 484.3934). The IR spectrum of 2 showed signals assigned to hydroxyl (3400 cm 1), ester (1740 cm 1) and olefinic (1668 cm 1) groups. On comparison, the 1H NMR spectra of compounds 2 and 1 were almost identical, apart from the change of chemical shift of H-3 from dH 3.20 in 1 to dH 4.42 in 2, and the presence of one additional acetyl proton singlet at dH 2.04 in 2. The second oxymethine proton at dH 4.10 in 1 did not change in 2 (dH 4.03). In the 13C NMR (Table 1) the carbon C-3 signal, dC 78.7 in 1, changed to dC 80.8 in 2. Thus, compound 2 was deduced to be the 3-monoacetylated derivative of 1. On the basis of the 1H–1H COSY and NOESY (Fig. 1) spectra, the stereochemistry of the two oxymethine carbons C-3 and C-6 in both compounds 2 and 1 were confirmed to be the same. Accordingly, the structure of 2 was established as 3b-acetoxylup-20(29)-en-6a-ol. Compound 3 was obtained as colourless crystals. The pseudo molecular ion peaks at m/z 605 [M+H]+ and 622 [M + NH4]+ in its CI/NH3 MS, and the HR TOF MS ES+ at m/z 604.4105 suggested its molecular formula to be C39H56O5. The IR spectrum showed absorption bands assigned to hydroxyl (3500, 3310 cm 1), ester (1680 cm 1), and aromatic (1600 cm 1) groups. The 1H NMR spectral data of 3 showed two sets of signals: the first set of signals were analysed as an (E)-caffeoyl moiety [dH 6.22 (H-70 ), 7.50 (H-80 ), and three aromatic protons at dH 6.75 (H-50 ), 7.01 (H-20 ) and 6.92 (H-60 )]; the second set of signals in 3 were almost similar to those of 1, except few modifications observed on the oxymethine protons, dH 3.20 in 1 and dH 4.50 in 3. The second oxymethine proton at dH 4.10 in 1 did not change in 3 (dH 4.00). The 13C NMR (Table 1) confirmed the presence of an (E)-caffeoyl moiety in 3 (dC 113.9, 114.4, 115.5, 121.7, 126.5, 145.5, 145.6, 148.4 and 168.1). The carbon C-3 signal at dC 78.7 in 1 changed to dC 81.2 in 3. Thus, compound 3 was deduced to be the 3-(E)-caffeoyl derivative of 1. On the basis of the 1H–1H COSY and NOESY (Fig. 1) spectra, the stereochemistry of the two oxymethine carbons (C-3 and C-6) in both compounds 3 and 1 were confirmed to be the same. Therefore, the structure of 3 was established as 3b-caffeoyloxylup-20(29)-en-6a-ol. The molecular formula of compound 4 was deduced as C37H58O10 from the FAB MS and 13C NMR data. The positive FAB MS of 4 revealed a quasi-molecular ion at m/z 669.5 [M + Li]+. The 1H NMR of 4 showed the presence of six tertiary methyl singlets at dH 0.80–1.19 (each, 3H, s), a doublet of doublets at dH 2.73 (1H, J = 3.65, 14.35 Hz, H-18) and a triplet at dH 5.36 (1H, J = 3.42 Hz, H-12). These signals and the 13C NMR signals (Table 1) at dC 123.1 and 144.3 were in agreement with reported data of olean-12-ene type triterpenes. The 13C NMR and DEPT spectra of 4 showed an oxymethine carbon signal at dC 78.8 and two C@O groups at dC 175.8 and 177.3. The 1H and 13C NMR signals at dH 3.73 (3H, s) and dC 52.7 indicated that one of them was present as a carbomethoxyl group. Moreover, the 13C NMR resonances of the carbons C-12, C-13, C-14, C-17 and C-20 were identical with spectral values of compounds having carboxyl functions at C-17 and C-20 (Hassanean and Mohamed, 1998). The HMBC spectrum showed key correlations between the proton H-18 (dH 2.73) and carbons C-28 (dC 177.3), C-20 (dC 44.2), and between the proton H-10 (dH 5.37) and carbon C-28 (dC 177.3), confirming the position of connectivity of the b-glucopyranosyl ester to be to the 28-carboxyl group. Consequently, the carbomethoxyl was deduced to be connected at C-20, and its position was established to be C30, as deduced from the 13C NMR spectrum of 4 which exhibited resonance for C-29 methyl at dC 28.8 in agreement literature (Hassanean and Mohamed, 1998). In addition, alkaline saponification of 4 gave glucose and the corresponding sapogenin which was identical to compound 5, isolated from the same plant and identified as serjanic acid (5) (Javasinghe et al., 1993). Accordingly, compound 4 was elucidated as, 28-b-D-glucopyranosyl-30-methyl 3bhydroxyolean-12-en-28,30-dioate. Compound 11 was obtained as colourless amorphous powder. Its molecular formula C30H50O2 was established on the basis of the HR TOF MS ES+, m/z 442.3821, the CI/NH3 MS, m/z 443 [M+H]+, 460 [M+NH4]+ and NMR data. The 13C NMR (Table 1), 1H NMR, HMQC, HMBC and DEPT spectra suggested a friedelin-type triterpene skeleton containing one oxymethine (dC 71.2 and dH 3.49) and one aldehyde function (dC 204.4 and dH 10.19). From the GC-SM, the fragment ion at m/z 205 suggested the absence of oxygen function on rings D and E. Also, the fragments at m/z 125 and 315 resulting from the cleavage of ring B suggested the location of one oxygen function on ring A. The HMBC spectrum showed correlations between the proton signal (dH 3.49) and carbons C-4 (dC 53.3), C-5 (dC 38.3) and C-23 (dC 10.6), and between the aldehyde proton signal at dH 10.19 and the carbons C-8 (dC 52.2), C-9 (dC 51.5), C-10 (dC 58.2).and C-11 (dC 28.2). These data confirmed the position of the hydroxyl group at C-3 and the aldehyde function (C-25) at C-9. The NOESY spectrum of 11 (Fig. 2) showed interactions between the aldehyde proton (dH 10.19) and the methyl groups CH3-24 (dH 0.65) and CH3-26 (dH 0.95). The NOESY crosspeaks from H-3 (dH 3.49) to CH3-23 (dH 1.01) and CH3-24 (dH 0.65), in addition to the coupling constant of H-3 (J = 10.0, H 24 CH3 3 HO H3C 25 CHO H 26 CH3 CH3 H 28 CH3 CH3 H CH3 27 Fig. 2. Important NOESY correlations in compound 11. 422 S.S. Awanchiri et al. / Phytochemistry 70 (2009) 419–423 Table 2 Antimicrobial activities of compounds 1, 2, 3, 4 (each conc. 200 mg/l in DMSO). Micro-organisms used Inhibition zone diameter (mm) 1 Staphylococcus aureus Gram-(+) Escherichia coli Gram-( ) Salmonella typhi Gram-( ) Shigella dysenteriae Gram-( ) Klebsiella pneumoniae Gram-( ) Pseudomonas aeruginosa Gram-( ) 15 2 3 4 Gentamicin (control) 11 14 13 34 35 42 30 40 43 4.0 Hz) confirmed the b-axial orientation of H-3 and consequently the a-equatorial orientation of the hydroxyl group in agreement with reported data (Salazar et al., 2000). Therefore, the structure of 11 was established as 3a-hydroxyfriedelan-25-al. From the antimicrobial test results (Table 2), it appears that compound 1 exhibits antimicrobial activity against Staphylococcus aureus. Compounds 2 and 3 which are the 3-acylated derivatives of compound 1 showed no inhibitory activity on the six bacterial strains. Compound 4 reveals antimicrobial activity against S. aureus, Escherichia coli and Salmonella typhi. The activities of both compounds 1 and 4 were lower in comparison to that of gentamicin which was used as control. 3. Experimental 3.1. General MPs were determined using a Kofler microhot stage apparatus. IR spectroscopy was performed on a Perkin–Elmer 257 spectrometer. Specific rotations were measured on a Perkin–Elmer 241 polarimeter. MS were registered on a Micromass Q-Tof instrument, on a Nermag R10-10C spectrometer and a HP-5973 Mass Selective Detector. NMR experiments were performed on a Varian Gemini 400 MHz instrument and a Bruker AC 400 spectrometer, the residual solvent signal was taken as reference in each case (CDCl3, CD3OD and C5D5N). Si gel 60 (240–400 mesh) was used for CC at normal pressure while Si gel 60 H (5–40 lm) and Si gel 60 C (20– 40 lm) were used for CC under compressed air (300 mbar). Precoated Si gel 60 F254 aluminium plates were used for TLC. 3.2. Plant material The whole stems and the ripe mature fruit of D. inaequalis Hutch. (Euphorbiaceae) were collected from Eloundem (Centre province of Cameroon) in August 2004. The herbarium specimen documenting the collection has been deposited in the National Herbarium, Yaoundé, Cameroon (Ref 4981/SRFK). 13–20 (hexane–CH2Cl2 45:55 to 25:75)]; D (27 g) [Fr. 21–27 (hexane–CH2Cl2 20:80 to 0:100)]; E (25 g) [Fr. 28–35 (CH2Cl2–MeOH 100:0 to 95:5)]; F (30 g) [Fr. 36–45 (CH2Cl2–MeOH 93:7 to 90:10)]; G (36 g) [Fr. 46–57 (CH2Cl2–MeOH 88:12 to 80:20)] and H (50 g) [Fr. 58–75 (CH2Cl2–MeOH 75:25 to 50:50)]. Further CC over Si gel 60 C (20–40 lm) of group C fractions using hexane – CH2Cl2 (50:50) yielded compounds 2 (30 mg), 12 (40 mg), 13 (30 mg) and 16 (50 mg). Further CC over Si gel 60 C (20–40 lm) of group D fractions using hexane–CH2Cl2 (75:25) afforded compounds 1 (22 mg), 5 (15 mg) and 6 (70 mg). Further CC over Si gel 60 H (5–40 lm) of group E fractions by using CH2Cl2 (100%) yielded compounds 7 (20 mg), 8 (12 mg) and 9 (10 mg). Further CC over Si gel 60 H (5–40 lm) of group F fractions using CH2Cl2– MeOH (95:5) yielded compounds 3 (20 mg), 4 (45 mg), 10 (15 mg) and 17 (20 mg). The ground fruit (2.0 kg) was macerated at room temperature with a mixture of EtOAc–MeOH (3:1), (3  4 l) for 6 days. The solvents were evaporated under reduced pressure to yield the total crude extract (157.0 g) which was subjected to CC over Si gel [60 (230–400 mesh), 500 g]. A total of 144 fractions (200 ml each) were eluted with hexane, EtOAc and MeOH in increasing polarity. TLC permitted the combination the resulting fractions into five series of fractions coded A, B, C, D and E, obtained as follows: series A (10.0 g) [Fr. 1–10 (hexane– EtOAc, 100:0 to 75:25)]; series B (15 g) [Fr. 11–19 (hexane–EtOAc 70:30 to 50:50)]; series C (10 g) [Fr. 20–50 (hexane–EtOAc 45:55 to 25:75)]; series D (50 g) [Fr. 51–102 (hexane–EtOAc 20:80 to 0:100)]; series E (55 g) [Fr. 103–144 (EtOAc–MeOH 100:0 to 50:50)]. Further CC over Si gel 60 C (20–40 lm) of series A using hexane–EtOAc in increasing polarity yielded compounds 12 (50 mg) and 14 (17 mg). Repeated CC of series B over Si gel 60 C (20–40 lm) using hexane–EtOAc in increasing polarity afforded compounds 11 (7 mg) and 15 (18 mg). Further CC of series C over Si gel 60 C (20–40 lm) using hexane–EtOAc in increasing polarity yielded compound 18 (60 mg). Repeated CC over Si gel 60 C (20 40 lm) of series D using EtOAc–MeOH in increasing polarity afforded compounds 10 (65 mg) and 19 (200 mg). 3.3.1. lup-20(29)-ene-3b,6a-diol (1) Colourless amorphous solid; [a]D20 = +24.5° (CHCl3, c 0.60); IR (KBr) mmax cm 1: 3400, 3030, 1660, 1260, 1180, 890; 1H NMR spectral data (400 MHz, CDCl3): d 3.20 (1H, dd, J = 5.6, 10.7 Hz, H-3), 4.10 (1H, dt, J = 4.3, 9.6 Hz, H-6), 4.58 (1H, d, J = 2.3 Hz, H-29), 4.68 (1H, d, J = 2.3 Hz, H-29), 2.35 (1H, m, H-19), 1.68 (3H, s, CH3-30), 1.32 (3H, s, CH3-23), 1.08 (3H, s, CH3-26), 0.98 (3H, s, CH3-24), 0.96 (3H, s, CH3-27), 0.85 (3H, s, CH3-25), 0.78 (m, H-5), 0.75 (3H, s, CH3-28), 1.40 and 1.67 (m, H-7); 13C NMR spectral data (100 MHz, CDCl3), see Table 1; CI/NH3 MS, m/z: 443 [M+H]+, 460 [M+NH4]+; EIMS (70 eV) m/z 442 [M]+ (10), 424 [M H2O]+ (20), 409 [M H2O CH3]+ (5), 406 [M 2H2O]+ (3), 236 (6), 218 (20), 205 (40), 203 (12), 189 (50); HR TOF MS ES+ (calcd. for C30H50O2 442.3811, found 442.3825). 3.3. Extraction and isolation The whole stems and the ripe fruit of D. inaequalis were sundried and ground separately into a powder form. The ground stems (10.0 kg) were macerated at room temperature with a mixture of CH2Cl2–MeOH (1:1) (3  25 l) for 9 days. The solvents were evaporated under reduced pressure to yield the total crude extract (390.0 g). Part of the extract (250.0 g) was subjected to CC over Si gel [60 (240–400 mesh), 800 g]. A total of 75 fractions (400 ml each) were eluted with hexane, CH2Cl2 and MeOH in increasing polarity. TLC permitted the combination the resulting fractions into 8 groups of fractions coded A, B, C, D, E, F, G and H, obtained as follow: A (5.0 g) [Fr. 1–5 (hexane–CH2Cl2 100:0 to 75:25)]; B (30 g) [Fr. 6–12 (hexane–CH2Cl2 70:30 to 50:50)]; C (35 g) [Fr. 3.3.2. 3b-acetoxylup-20(29)-en-6a-ol (2) Colourless amorphous solid; [a]D20 = +18.5° (CHCl3, c 0.80); IR (KBr) mmax cm 1: 3400, 3000, 1740, 1668, 1255, 880; 1H NMR spectral data (400 MHz, CDCl3): d 4.42 (1H, dd, J = 5.5, 10.5 Hz, H-3), 4.03 (1H, dt, J = 4.2, 9.5 Hz, H-6), 4.56 (1H, d, J = 2.3 Hz, H-29), 4.68 (1H, d, J = 2.3 Hz, H-29), 2.35 (1H, m, H-19), 1.68 (3H, s, CH3-30), 1.16 (3H, s, CH3-23), 1.10 (3H, s, CH3-26), 1.04 (3H, s, CH3-24), 0.95 (3H, s, CH3-27), 0.91 (3H, s, CH3-25), 0.78 (3H, s, CH3-28), 2.04 (3H, s, COCH3-3); 13C NMR spectral data (100 MHz, CDCl3), see Table 1; CI/NH3 MS, m/z 485 [M+H]+, 502 [M+NH4]+; EIMS (70 eV) m/z 484 [M]+ (7), 466 (8), 426 (10), 408 (5), 383 (5), 218 (100), 205 (40), 203 (12), 189 (12); HR TOF MS ES+ (calcd. for C32H52O3 484.3916, found 484.3934). S.S. Awanchiri et al. / Phytochemistry 70 (2009) 419–423 3.3.3. 3b-caffeoyloxylup-20(29)-en-6a-ol (3) Colourless crystals; mp 272.2–273.7 °C; [a]D20 = +105.4° (CHCl3, c 0.08); IR (KBr) mmax cm 1: 3500, 3310, 2930, 2860, 1680, 1600, 1255, 1180, 880; UV (CH3OH) kmax nm log(e) 303.6 (0.504), 327.6 (0.636); 1H NMR spectral data (400 MHz, CD3OD): triterpene moiety: d 4.50 (1H, dd, J = 5.70, 10.50 Hz, H-3), 4.00 (1H, (1H, dt, J = 4.10, 9.50 Hz, H-6), 4.55 (1H, d, J = 2.30 Hz, H-29), 4.68 (1H, d, J = 2.30 Hz, H-29), 2.39 (1H, m, H-19), 1.68 (3H, s, CH3-30), 1.17 (3H, s, CH3-23), 1.14 (3H, s, CH3-26), 0.96 (3H, s, CH3-24), 1.02 (3H, s, CH3-27), 1.12 (3H, s, CH3-25), 0.82 (3H, s, CH3-28); caffeoyl moiety: d 7.01 (1H, d, J = 1.83 Hz, H-20 ), 6.75 (1H, d, J = 8.06 Hz, H-50 ), 6.92 (1H, dd, J = 2.00, 8.24 Hz, H-60 ), 7.50 (1H, d, J = 15.74 Hz, H-70 ), 6.22 (1H, d, J = 15.74 Hz, H-80 ); 13C NMR spectral data (100 MHz, CD3OD), see Table 1; CI/NH3 MS, m/z: 605 [M+H]+, 622 [M+NH4]+; EIMS (70 eV) m/z 604 [M]+ (25), 239(10), 221 (15), 203 (65), 163 (100); HR TOF MS ES+ (calcd. for C39H56O5 604.4128, found 604.4105). 3.3.4. 28-b-D-glucopyranosyl-30-methyl 3b-hydroxyolean-12-en28,30-dioate (4) White crystals from CH2Cl2; mp 230-232 °C; [a]D20 = +45.5° (MeOH, c 0.75); IR (KBr) mmax cm 1: 3450, 1720 (COOR), 1710 (COOCH3); 1H NMR spectral data (400 MHz, C5D5N) d: 5.37 (1H, d, J = 8.00 Hz, H-10 ), 5.36 (1H, t, J = 3.42 Hz, H-12), 3. 73 (3H, s, COOCH3), 3.19 (1H, t, J = 8.00 Hz, H-3), 2.73 (1H, dd, J = 3.65, 14.35 Hz, H-18), 1.19 (3H, s, CH3), 1.17 (3H, s, CH3), 1.00 (3H, s, CH3), 0.98 (3H, s, CH3), 0.82 (3H, s, CH3), O.80 (3H, s, CH3); 13C NMR spectral data (100 MHz, C5D5N) data, see Table 1. CI/NH3 MS, m/z 663 [M+H]+, 680 [M+NH4]+; FAB/NBA + Li: ion mode FAB+, m/z: 669.5 [M+Li]+,630, 625, 581.6, 580.6, 460.3, 397.5, 307.0, 292.1, 291.1, 290.0, 154.1. Alkaline hydrolysis of compound (4): The glycoside 4 (15 mg) was refluxed with 5% KOH for about 5 h. After completion, the reaction mixture was neutralized with diluted H2SO4 and extracted with n-BuOH. Work up of the n-BuOH soluble portion yielded glucose identified by 1H and 13C NMR to the available authentic compound. The purification of the aqueous fraction afforded the aglycone, identical to serjanic acid (5), also isolated from the same plant. 3.3.5. Serjanic acid (3b-hydroxyolean-12-en-28,30-dioic acid 30methyl ester) (5) White crystals from CH2Cl2; mp 280–281 °C; CI/NH3 MS, m/z: 501 [M+H]+, 518 [M+NH4]+; EI MS (probe) 70 eV, m/z: 500 [M]+ (1.5), 454 (15.4), 292 (64.6), 247 (41.5), 246 (100.0), 233 (15.4), 232 (17.7), 207 (33.8), 187 (93.8), 186 (46.2), 173 (23.1), 159 (21.5). 3.3.6. 3a-hydroxyfriedelan-25-al (11) Colourless amorphous powder; [a]D20 = +14.5° (CHCl3, c 0.60); IR (KBr) mmax: 3400, 3030, 1720, 1260, 1180, 890 cm 1; 1H NMR spectral data (400 MHz, CDCl3): d 10.19 (1H, s, H-25), 3.49 (1H, dt, J = 10.0, 4.0 Hz, H-3ax), 2.16 (qd, J = 13.0, 3.0, H-2eq), 1.19 (3H, s, CH3-28); 1.07 (3H, s, CH3-30); 1.01 (3H, d, J = 6.0 Hz, CH323); 0.95 (3H, s, CH3-26); 0.94 (3H, s, CH3-29); 0.93 (3H, s, CH327); 0.65 (3H, s, CH3-24); 13C NMR spectral data (100 MHz, CDCl3), see Table 1. GC–SM m/z: [M]+ 442, 315, 205, 125. CI/NH3 MS, m/z: 443 [M+H]+, 460 [M+NH4]+. 3.4. Antimicrobial activity 3.4.1. Microbial strains A total of six micro-organisms belonging to one Gram-(+) bacterial species (S. aureus) and five Gram-( ) bacteria (E. coli, S. typhi, Shigella dysenteriae, Klebsiella pneumoniae and Pseudomonas aeruginosa) were clinically isolated from patients in the ‘‘Centre Pasteur de Yaoundé” Cameroon. They were maintained on agar slants at 423 4 °C in the Laboratory of the Applied Microbiology and Molecular Pharmacology (Faculty of Science, Yaounde). 3.4.2. Antimicrobial assays Antimicrobial activity was evaluated using the agar diffusion method, according to the NCCLS (2002) protocol with slight modifications. Briefly, sterile cylinders of 6 mm were used to make wells inside Mueller-Hinton agar plates. The plates were inoculated with 2  10 4 l of the test micro-organisms equivalent to 5  105 CFU/ml. All the compounds were dissolved in DMSO or in heated sterilized distilled water at a concentration 200 mg/l. Wells were filled with 15  10 5 l of solution of each test compound, the positive control drug (gentamicin) and the negative control DMSO, and allowed to diffuse for 45 min at 4 °C. The plates were incubated at 37 °C for 24 h. The sensitivity was recorded by measuring the clear zone of growth inhibition around the wells (mm diameter). Each set was tested in triplicate. Acknowledgements One of the authors (J. WANDJI) is grateful for a grant (F/26243F) from the International Foundation for Science (Sweden), and for the sponsorship of ‘‘Université Paris Descartes, France” during his multiple research visits in France. References Agrawal, P.K., Jain, D.C., 1992. 13C NMR Spectroscopy of oleanane triterpenoids. Progress in NMR Spectroscopy 24, 1–90. Anjaneyulu, A.S.R., Narayana, R.M., 1980. Phytochemistry 19, 1163. Bouquet, A., Debray, L., 1974. Plantes Médicinales de la Côte-d’Ivoire. Travaux et documents de l’ORSTOM 32, 82–87. Chungag-Anye, N.B., Njamen, D., Dongmo, A.B., Wandji, J., Nguelefack, T.B., Wansi, J.D., Kamanyi, A., Fomum, Z.T., 2001. Anti-inflammatory and analgesic properties of the stem extract of Drypetes molunduana Pax and Hoffm. (Euphorbiaceae) in rats. Pharmaceutical and Pharmacological Letters 11, 61–63. Chungag-Anye, N.B., Njamen, D., Dongmo, A.B., Wandji, J., Fomum, Z.T., Nguelefack, T.B., Kamanyi, A., 2002. Anti-inflammatory and analgesic effects of Drypemolundein A, a sesquiterpene lactone from Drypetes molunduana. Pharmaceutical Biology 41 (1), 26–30. Dalziel, J.M., 1937. The Useful Plants of West Tropical Africa. The Crown Agents for the Colonies. Westminster, London, pp. 140-141. Ghulam, M., Erum, A., Saeed, A., Itrat, A., Habib, A., Abdul, M., Syed, S-u-H., Choudhary, M.I., 2000. Lupene-type triterpenes from Periploca aphylla. Journal of Natural Products 63, 881–883. Hassanean, H.A., Mohamed, M.H., 1998. Novel oleanene saponins from Taverniera aegyptiaca Boiss. Pharmazie 53, 195–199. Irvine, R.F., 1961. Woody Plant of Ghana . Oxford University Press, London. pp. 223– 226. Javasinghe, U.L.B., Wannigama, G.P., MacLeod, J.K., 1993. Saponins of Diploclsia glaucescens. Natural Product Letters 2, 249–253. Li, L., Huang, X., Sattler, I., Fu, H., Grabley, S., Lin, W., 2006. Structure elucidation of a new friedelane triterpene from the mangrove plant Hibiscus tiliaceus. Magnetic Resonance in Chemistry 44, 624–628. Mahato, S.B., Kundu, A.P., 1994. 13C NMR spectra of pentacyclic triterpenoids. A compilation and some salient features. Phytochemistry 37, 1517–1575. National Committee for Clinical Laboratory Standards (NCCLS), 2002. Methods for Dilution Antimicrobial Susceptibility Test for Bacteria that Grow Aerobically, third ed. Approved standard NCCLS document M100-812, Wayne, PA. Núnez, M.J., Carolina, P. Reyes, Ignacio, A. Jiménez, Laila, M., Bazzochi, Isabel L., 2005. Lupane triterpenoids from Maytenus Species. Journal of Natural Products 68, 1018–1021. Salazar, G.C., Silva, G.D.F., Duarte, L.P., Vieira Filho, S.A., Lula, I.S., 2000. Two epimeric friedelane triterpenes isolated from Matenus truncate Reiss: 1H and 13C chemical shift assignments. Magnetic Resonance in Chemistry 38, 977–980. Srivastava, S.K., Jain, D.C., 1989. Triterpenoid saponins from plants of Araliaceae. Phtochemistry 28, 644–647. Walker, A.R., Sillans, R., Trochain, J.L., 1961. Les plantes utiles du Gabon. In: Paul Lechevalier (Ed.), 12-Rue de Tournon Paris VI, pp. 165–166. Wandji, J., Wansi, J.D., Fuendjiep, V., Dagne, E., Mulholland, A.D., Tillequin, F., et al., 2000. Sesquiterpene lactone and friedelane derivative from Drypetes molunduana. Phytochemistry 54, 811–815. Wandji, J., Tillequin, F., Mulholland, A.D., Temgoua, D.A., Wansi, J.D., Seguin, E., Fomum, Z.T., 2003. Phenolic constituents from Drypetes armoracia. Phytochemistry 63, 453–456. Wansi, J.D., Wandji, J., Lallemand, M.-C., Chiozem, D.D., Samreen, Iqbal, M.C., Tillequin, F., Fomum, Z.T., 2007. Antileishmanial furansesquiterpene and triterpenoids from Drypetes chevalieri Beille (Euphorbiaceae). BLACPMA 6 (1), 5–10.