Vol.4, No.4 (2011), 753-763 ISSN: 0974-1496 CODEN: RJCABP http://www.rasayanjournal.com ACYLSTERYL GLYCOSIDES AND OTHER CONSTITUENTS FROM CAMPYLOSPERMUM DENSIFLORUM (OCHNACEAE) 1 1 2 Dominique Serge Ngono Bikobo , Jeanne Louise Nkot , Paul Mosset , Alex de 1 1 1 ,2 Théodore Atchade , Joseph thierry Ndongo , René Pemha 1,* and Dieudonné Emmanuel Pegnyemb 1 Department of Organic Chemistry, Faculty of Science, University of Yaoundé 1, P.O Box 812, Yaoundé, Cameroon. 2 Ecole National Supérieure de Chimie de Rennes, Avenue du Général Leclerc, Campus Beaulieu CS 50837, 35708 Rennes, France. * E-mail: pegnyemb@yahoo.com ABSTRACT A mixture of two new acylsteryl glycosides named densifloside A and B was isolated from the leaves of Campylospermum densiflorum, together with serotobenine, decursivine, N-feruloyltriptamine , ent-16 ,17dihydroxykauran-19-oic acid, menisdaurin, and secoisolariciresinol. The structure elucidations were based on spectroscopic evidence. The biological assay of crude extract of this plant showed good antimicrobial activity against Gram-positive cocci, weak activity against Gram-negative bacilli, and displayed moderate activity against two strains of fungi (C. albicans and C. neoformans) among several strains. Keywords: Campylospermum densiflorum, Ochnaceae, acylsteryl glycosides, Ouratea, densifloside A and B, chemotaxonomy, antimicrobial activity © 2011 RAS YAN. All rights reserved. INTRODUCTION In the course of a systematic investigation of Ochnaceae plants, especially in the tribes Ouratea and Campylospermum, the chemical constituents of Campylospermum glaucum and Ouratea turnarea were reported1 and also, recently, those of Campylospermum flavum2. To further study of plants in the same tribe, the investigation of the constituents of Campylospermum densiflorum (De Wild & Th. Durand) Farron collected in the South region (Campo) of Cameroon constituted the main objective of this study. C. densiflorum is a shrub up to 5 m high, distributed in the Equatorial zone of African continent. The decoction of the leaves and roots of this plant is used by autochthon people in traditional medicine to treat malaria, icterus, stomach ache and for anti-fever purposes. No previous phytochemical and biological work has been performed on this species. Preliminary studies on plants in this genus [Campylospermum and Ouratea are related species3] have led to the isolation of several compounds, such as flavonoids, diterpenoids4-8, alkaloids, and cyano glycosides1-2. The present paper deals with the isolation of eight compounds, including two new acylsteryl glycosides, densifloside A (7a) and B (7b) as well as six known compounds. EXPERIMENTAL NMR spectra were recorded in DMSO and DMSO-d6 solutions and were obtained using a Bruker instrument (1H, 400 MHz; 13C, 100 MHz) spectrometer, with TMS as internal standard. 1H assignments were made using 2D-COSY and NOESY (mixing time 500 ms) while 13C assignments were made using 2D-HSQC and HMBC experiments. For this latter, the delay was 70 ms. The positive-ion mode TOF-MS were recorded on an API Q-STAR PULSAR spectrometer. EIMS was recorded on a JEOL JMSD-300 CAMPYLOSPERMUM DENSIFLORUM (OCHNACEAE) D. S. N. Bikobo et. al Vol.4, No.4 (2011), 753-763 instrument. Optical rotations, uncorrected were measured on a Perkin-Elmer 341 polarimeter. IR data were measured on a JASCO FTIR 300E spectrometer with KBr pellets. Gas-liquid chromatography was run on a Hewlett-Packard 5890 (series 2 plus) equipped with FID and Hewlett-Packard 5989 B mass spectrometer. Chemical shifts ( ) are given in ppm and coupling constants (J) are reported in Hz. Column Chromatography was performed using Merck (70-230 mesh) silica gel of an appropriate particle. Preparative TLC was carried out on silica gel plates (Merck silica gel 60 F254).TLC layers were visualized by UV at 254 and 366 nm and exposure to iodine vapour. The solvent systems were the mixture of CH2Cl2/MeOH and EtOAc/MeOH of gradually increasing polarity. Plant material The leaves of C. densiflorum (De Wild & Th. Durand) Farron were collected at Campo in Southregion of Cameroon in December 2005, and identified by Mr. Nana Victor a botanist. A voucher specimen (N°48270/HNC) was deposited at the National Herbarium in Yaoundé, Cameroon. Extraction and isolation Dried and powdered leaves (1.20 kg) of C. densiflorum were extracted exhaustively with MeOH at room temperature during 48 h. The solvent was removed under vacuum giving a residue (234 g). The residue was partitioned with hexane and CH2Cl2 to remove chlorophyll and non-polar metabolites. The methanolic residue was partitioned with ethyl acetate to yield an EtOAc soluble residue (25.4 g). The EtOAc residue was analyzed by TLC and fractionated on a silica gel column eluted successively with CH2Cl2, mixtures of CH2Cl2/MeOH, and pure MeOH. Five main fractions were collected [I (5.7 g), II (3.6g), III (3.8 g), IV (4.4 g) and V (8.3 g)]. Fraction II (3.6 g) was submitted to silica gel (500 g) CC using the solvent system CH2Cl2/MeOH (30/1 to 15/1) to give three sub-fractions (IIa , IIb and IIc). Sub-fraction IIc (1.1 g) was further chromatographed on a silica gel (200 g) column using CH2Cl2/MeOH (10/1) to furnish compound 1 (37 mg). Using the same process, Fraction III (3.8 g) gave three sub-fractions (IIIa, IIIb and IIIc). Fraction IIIb (1.20 g) was further chromatographed on Sephadex LH-20 (200 g) to yield two sub-fractions (IIIb1 and IIIb2). Sub-fraction IIIb1 (0.4 g) was subjected to a silica gel (115 g) column and eluted with CH2Cl2/MeOH (10/1) to afford compound 2 (11 mg). Sub-fraction IIIb2 (0.8 g) was purified by repeated CC on silica gel (125 g) with the solvent system CH2Cl2/MeOH (10/1) to obtain compound 3 (18 mg) and compound 4 (4 mg). Sephadex LH-20 with pure MeOH was used to purify sub-fraction IIIb21 (0,15g) to afford compound 5 (11 mg). Fraction IV (4.4 g) was subjected to CC on silica gel (500 g) and eluted with the solvent system CH2Cl2/MeOH (10/1) to give two sub-fractions (IVa and IVb). Sub-fraction IVa (2.10 g) was subjected to silica gel (250 g) column chromatography with the solvent system CH2Cl2/MeOH (15/1 to 6/1), and for the dirty crystal obtained, Sephadex LH-20 (100 g) was also used to provide compound 6 (6 mg). Fraction V (8.3 g) was subjected to CC on silica gel (700 g) and eluted with the solvent system CH2Cl2/MeOH (10/1-0/100) to give four sub-fractions (Va, Vb, Vc and Vd). Sub-fraction Vc (0,90 g) was subjected to silica gel (150 g) column chromatography with the solvent system EtOAc/MeOH (15/1 to 1/1) to give two sub-fractions Vc1 (0.44 g) and Vc2 (0.34 g). The latter was subject to Sephadex LH-20 (100 g) with pure MeOH to provide compounds 7a/7b (42 mg). Densiflosides A (7a) and B (7b) White amorphous powder; [ ]D25 -24.8° (c 0.12, MeOH); IR maxKBr: 3350 (br OH), 3001(alkenes), 2938 (alkanes), 1738 (ester), 1598 (sugar), 1472 (alkanes), 1100 (C-O) cm-1; TLC Rf: 0.19 (EtOAc/MeOH 85/15); for 1H and 13C NMR (400 and 100 MHz, DMSO-d6), see Table 1; MS positive HR-ESI-TOF, m/z 997,6963 and 995,6798 [M+Na]+(calcd. for C57H98O12, 997.6958 and C57H96O12, 995.6801). Antimicrobial assays The determination of the antimicrobial activities of the extracts was done by the disc diffusion test according to the methods of the National Committee for Clinical Laboratory Standards (NCCLS) in the USA. The minimum inhibitory concentrations of strains which exhibit an inhibition diameter more than 9 mm were determined by the agar dilution method. The MICs of Gentamicin (antibiotic drug) and Econazole (antifungal drug) were also determined in parallel experiments in order to control the sensitivity of the test microorganisms. All tests were performed in triplicate. The dried plant extracts were dissolved in MeOH-H2O (1/1) to a final concentration of 200 mg/ml and sterilized by filtration using 0.22 CAMPYLOSPERMUM DENSIFLORUM (OCHNACEAE) 754 D. S. N. Bikobo et. al Vol.4, No.4 (2011), 753-763 µm Millipore filters. Antimicrobial test was then carried out by the disc diffusion method using 100 µl of saline suspension containing 107 CFU/ml of bacteria, 105 CFU/ml of yeast on Mueller Hinton agar (MH) and Sabouraud dextrose agar medium, respectively. The discs (6 mm in diameter) were impregnated with 15 µl of extracts and placed on the inoculated agar plate. Negative controls were prepared using the same solvents employed to dissolve the plant extracts. The inoculated plates were incubated at 37°C for 24 h bacteria strains and 48 h for yeasts. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organism. Table-1: 1H and 13C-NMR spectroscopic data of compounds 7a and 7b (DMSO-d6)a; (ppm). Compd. Position 7a 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. Glc' 1. 2. 3. 4. 5. 36.5 31.6 76.2 39.8 140.3 121.0 28.6 31.0 50.5 36.1 21.1 38.9 41.9 55.9 24.8 28.3 55.3 11.9 19.0 35.1 18.2 33.5 28.4 n.o. 31.2 18.7 20.8 23.7 12.0 6. 64.2 Glc 1. 2. 98.2 76.6 7b H (J[Hz]) C 100.7 72.8 74.2 71.5 76.6 1.81, m 1.88, m 3.52b 1.98b 5.32, brs 1.76, m 1.52b 1.01b 1.88, m 0.99b 1.13b 1.21b 1.12, m 0.66, s 0.94, s n.o. 0.91b n. o. 1.24, m n.o. 1.61, m 0.76b 0.82b n. o. 0.81, t(J = 6.1) C 36.5 31.6 76.2 39.8 140.3 121.0 28.6 31.0 50.5 36.1 21.1 38.9 41.9 55.9 24.8 28.3 55.3 11.9 19.0 39.6 20.9 137.9 128.7 50.5 31.2 18.7 20.8 24.8 12.0 4.22, d (7.5) 2.88, m 3.08b 3.20b 3.32b 4.89/4.39, dd (J =12.1; 2.3) 100.4 73.4 76.1 70.0 76.6 4.52, d (7.6) 4.18, m 98.2 76.6 CAMPYLOSPERMUM DENSIFLORUM (OCHNACEAE) 755 66.5 H (J[Hz]) 1.81, m 1.88, m 3.52b 1.98b 5.32, brs 1.76, m 1.52b 1.01b 1.88, m 0.99b 1.13b 1.21b 1.12, m 0.66, s 0.94, s 1.98, m 0.97 b 5.15, m 5.03, m 1.98, m 1.61, m 0.76b 0.82b 1.39b 0.81, t (J = 6.1) 4.25, d (7.5) 2.90, m 3.10, m 3.01, m 3.24b 4.78/4.23, dd (J =12.0; 4.0) 4.52, d (7.6) 4.08, m D. S. N. Bikobo et. al Vol.4, No.4 (2011), 753-763 3. 4. 5. 76.6 68.4 76.6 3.06, m 3.72 m 3.28b 4.35/4.13, dd (J =10.0; 2.0) 76.6 69.6 76.6 6. 62.5 Ac ' 1. 2. 3. 172.3 33.3 24.3 2.23 m 1.48 m 4-7 28.4-28.9 1.23-1.28 brs 8. 9. 10. 11. 33.9 137.8 128.6 33.8 n. o. 5.10, m 5.01, m 1.99, m 12-13 28.4-28.9 1.23-1.26, m 14. 15. 16. 31.2 21.9 13.8 1.27, m 1.22, m 0.86, t(J = 6.3) 172.5 33.3 24.3 28.428.9 33.9 137.8 128.6 33.8 28.428.9 31.2 21.9 13.8 60.9 3.06, m n. o. 3.28b 3.67/3.42, dd (J =10.0; 2.0) 2.24 m 1.48 m 1.23-1.28 brs n. o. 5.10, m 5.01, m 1.99, m 1.23-1.26, m 1.27, m 1.22, m 0.86, t (J = 6.3) a Assignement based on HSQC and HMBC experiments. Measured at 100 MHz Overlapped signals are reported without designated multiplicities. Measured at 400 MHz Glc=glucose; Ac= acetyl moiety n.o.: not observed b MIC values of the Gram-positive organisms and the yeast isolated were studied, based on the agar dilution method according to NCCLS. The extract was added aseptically to sterile melted MH agar medium at the appropriate volume to produce the concentration range 1-10 mg/ml. The resulting MH agar solutions were immediately poured into Petri plates after vortexing. The plates were spot inoculated with 2 µl of each Gram-positive bacteria and yeast isolate. Gentamicin was used as the reference antibiotic drug and Econazole as the reference antifungal drug. The inoculated plates were incubated at 37°C for bacteria and the yeast for 24 h and for 48 h, respectively. At the end of the incubation period, the plates were evaluated for the presence or absence of growth. MIC values were determined as the lowest concentration of the extract where absence of growth was recorded. Hydrolysis of 7a/7b The mixture (8 mg) was dissolved in7% H2SO4 (0.5 ml) and refluxed on an aqueous bath at 100°C for 4 hours. The reaction mixture was diluted with H2O and extracted with CH2Cl2. The CH2Cl2 layer was evaporated to dryness and purified by preparative TLC over silica gel with CH2Cl2-MeOH (9/1) as eluent. A mixture of two aglycons (4 mg) were isolated and identified as -sitosterol and stigmasterol through direct comparison with authentic samples (TLC, MP, and IR). The neutralized and lyophilized aqueuous hydrosylates of the mixture gave only glucose. GC-MS (Column: 5% phenyl and 95% methyl silicone on ultra 2, 0.2× 46 m, column temp.: 250°C, carrier gas: He 0.8 ml/min, sample: trimethylsilyl derivatives: tR (min) glucose (19.75 for 7a/7b). Acetylation of 7a/7b The mixture (6 mg) was dissolved in pyridine (2 ml) in a 5 ml round-bottom flask, and AC2O (5 ml) was added. The flask was well corked and left in the oven at 50°C for 4 hours, after which the solvent was removed under vacuum and the powder obtained purified over Sephadex LH-20 with MeOH as eluent to give a mixture of hexa-acetate acylsteryl compounds (3 mg): IR max KBr: 3000, 2928, 1736, 1729 cm-1; 1H and 13C-NMR (400 and 100 MHz, DMSO-d6): H: 2.01; 2.03; 2.05; 2.08; 2.10; 2.13 (each 3H, s, OCOCH3 × 6). C: 173.8; 19.9 (OCOCH3 × 6). CAMPYLOSPERMUM DENSIFLORUM (OCHNACEAE) 756 D. S. N. Bikobo et. al Vol.4, No.4 (2011), 753-763 Aggressive alkaline hydrolysis and GC-MS analysis of 7a/7b Compounds 7a/7b (10 mg) was hydrolyzed with 3% NaOH (2 ml) and MeOH (0.4 ml) for 10 hours at room temperature. After adjusting the pH to 5.0 with 2 M HCl, the reaction mixture was concentrated in vacuum to dryness. The residue was dissolved in water and partitionated successively with n-hexane and EtOAc. The n-hexane extract containing a C16:1 fatty acid was removed, dried, dissolved in 1 N anhydrous methanolic HCl in a screw-cap test tube under N2, and subjected to methanolysis at 80°C for one day. Then this mixture was added to 1 ml of n-hexane. Finally, supernatant fluid (FAME) was injected into the GC-MS system (tR, 36.43 min).The EtOAc was separated by preparative TLC using repeatedly EtOAc-MeOH (9/1) to afford a mixture of -sitosterol and stigmasterol 3-O-gentiobioside (4 mg) through direct comparison with literature data (1H and 13C-NMR, MS, IR). Table-2: MIC of the crude extract of C. densiflorum against Gram-positive and negative cocci Microorganisms Minimal Inhibitory Concentration E.hirae ATCC 9790 Enterococcus sp. P054 S. aureus ATCC 25923 S. aureus U271 S. saprohyticus Escherichia coli ATCC 25922 E .coli ATCC 35218 Enterobacter aerogenes ATCC 13048 E. aerogenes ATCC 29751 E. cloacae HGY 18 Klebsiella pneumoniae HGY 6 K. pneumoniae HGY 19 K. oxytoca U271 Serratia marcescens HGY4 S. marcescens HGY10 Acinobacter baumanii HGY 12 A. baumanii HGY 13 Pseudomonas aeruginosa ATCC 27823 a C. densiflorum (mg/ml) (leaves) 1.25a 10 5 5 1.25 >10 >10 >10 Gentamicin (µg/ml) >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 1 1 >32 >32 >32 >32 >32 0.5 >32 1 16 2 16 < 0.125 < 0.125 0.5 0.5 0.5 Minimum Inhibitory Concentration was determined because inhibitory zone diameters by disc diffusion test were >8 mm RESULTS AND DISCUSSION Dried and powdered leaves of C. densiflorum were extracted with MeOH and evaporated to yield a residue. The MeOH extract was dissolved in H2O and partitioned between hexane and CH2Cl2 to remove chlorophyll and non-polar constituents. The aqueous phase was partitioned with EtOAc to produce an ethyl acetate-soluble residue. The EtOAc phase was fractionated on a silica gel column, eluting with a gradient solvent system (CH2Cl2-MeOH) to give five main fractions. The fractions II, III, IV and V were further chromatographed over silica gel, eluting with the same solvent system as above to afford eight compounds, among which six (Fig. 1) were identified as ent-16 ,17-dihydroxykauran-19-oïc acid (1) previously isolated from Ouratea Semiserrata9, serotobenine (2), decursivine (3) and Nferuloyltriptamine (4) previously isolated from Carthamus tinctorius10 and Rhaphidophora decursiva11, menisdaurin (5) isolated from Ouratea reticulata12, and secoisolariciresinol (6) from Stereospermum personatum13. These Known compounds were characterized by direct comparison of their physical and CAMPYLOSPERMUM DENSIFLORUM (OCHNACEAE) 757 D. S. N. Bikobo et. al Vol.4, No.4 (2011), 753-763 spectroscopic characteristics with those published in the literature. The new compounds were characterized by different spectroscopic methods, as follow: Characterisation of densifloside A and B 7a/7b Compounds 7a and 7b, named densifloside A and B were obtained as inseparable mixture with close molecular formulas C57H98O12 and C57H96O12 determined from their ESI-Q-TOF-HRMS at m/z 997,6963 and 995,6798 [M+Na]+, respectively. The IR spectrum exhibited a broad hydroxyl absorption band at 3350 cm-1 and absorption band at 1738 cm-1 which is consistent with the presence of ester. 12 1 16 17 14 9 2 OH 13 11 20 OH 10 8 3 15 7 5 4 6 COOH 18 19 1 R1O 4' 5' 3' RO 6' 2' H O 9' 1' 7' H N 8' 9 H O 4 8 5 3a 3 6 2 7a 7 N H 2: R= H, R1= CH3 3: R= R1= -CH23' OH 4' 2' 8 4 3a 5 8' H N 3 5' 1' 9' OCH3 7' 9 2 6' O 6 N H 7a 7 4 1 CN 2 6' HO HO 5' 4' O 1' 2' HO O 8 4 OH 3' 7 758 5 6 H CAMPYLOSPERMUM DENSIFLORUM (OCHNACEAE) 3 OH D. S. N. Bikobo et. al Vol.4, No.4 (2011), 753-763 5 2 H3CO 7 3 9 8 1 OH OH 6 4 HO 8' 7' 5 9' 1' 2' 6' 3' 5' OCH3 4' OH 6 29 21 20 22 18 12 6" HO HO HO 4" 5" 3" 2'' O 1'' 5' 2 O 1' 3' 7a: R= palmitelaidoyl7b: R= palmitelaidoyl-; 8a: R=H 8b: R=H; 22 1 6' O OR HO 4' HO 2' OH 19 11 10 9 3 O 4 5 13 8 28 24 17 23 16 26 25 27 14 15 7 6 22 Fig.-1: Structure of compounds isolated from Campylospermum densiflorum Table-3: Disc diffusion assay of the crude extract of C. densiflorum and compound 2 against fungi. Fungi strains Candida albicans NCPF 3153 C. tropicalis CBS 94 C. parapsilosis NCPF 8658 C. kefyr NCPF 3931 C. glabatra CBS 7904 Cryptococcus neoformans sero DIP 1173 Aspergillus fumigatus A 12 A. flavus GM 4895 A. parasiticus GM 4898 Trichophyton canis T. rubrum T. mentagrophytes A 42863 Inhibition diameter (mm) ent-16 , 17dihydroxykauran-19oic acid (2) 8 n.a. n.a. n.a. n.a. 8 n.a. n.a. C. densiflorum Econazole, 50 µg/ml (leaves) 16 n.a. n.a. n.a. n.a. 15 n.a. n.a. (antifungal drug) 22 12 23 25 22 29 25 28 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 34 26 27 25 n.a : not active The 1H-NMR spectrum of this mixture contains proton signals characteristic of -sitosterol and stigmasterol. Thus, the Me-21, Me-26, and Me-27 appear at H 0.91/0.97, 0.76 and 0.82, respectively are CAMPYLOSPERMUM DENSIFLORUM (OCHNACEAE) 759 D. S. N. Bikobo et. al Vol.4, No.4 (2011), 753-763 overlapped where as the Me-29 at H 0.81 appears as triplet. The Me-18 and Me-19 resonate as singlets at H 0.66 and 0.94, respectively. The vinylic proton H-6 gives a broad singlet at H 5.32. The structure of the aglycone of the free sterol was confirmed by acid hydrolysis of the mixture and identified by comparative TLC with authentic samples and comparison of 13C-NMR chemical shift from the literature14,15. The appearance of the molecular peaks at m/z 414, 412, and 397 proves that the aglycone is a C29-sterol as shown in the literature15. Furthermore, the structures of the aglycone were confirmed by comparing the 1H and 13C-NMR spectra of this mixture of acylsteryl glycosides with the known characteristics of related compounds15-18. Spectral data are consistent with the presence of fatty acid residues in the mixture of these two molecules. Thus, broad singlets at H 1.23-1.28, a triplet at H 0.86, and broad multiplets at H 2.23/2.24 were observed and assigned to protons of the methylenes, of the terminal methyl group and the -(COCH2)respectively. Signals of two carbonyl carbons at C 172.3 and 172.5 and a large number of methylene carbons atoms in 13C-NMR spectrum (Table 1) belong to fatty acid residues. This is also confirmed by the remaining band absorption of ester function. Except the double bond of the side chain of stigmasterol moiety at H 5.15 and 5.03, a supplementary double bond was observed in the fatty acid (Table 1). This is confirmed by the HMBC experiment between the methylene protons at H 1.26 with the carbons at C 28.4, 33.8 in the first hand and, the methylene protons at H 1.28 with the carbons at C 137.8, 28.9 and 33.9 in the other hand (Fig. 2). The specific chemical shift of the methylene groups adjacent to an aliphatic double bond system ( C8 '33.9 and C-11 ' 33.8) established the E-configuration of the double bond in accordance with previous studies on alkyl phenol compounds19 which indicated a higher value for this configuration ( C 32.0). The double bond of the alkyl side chain, was originally located between the seventh and the eighth carbons from the terminal methyl group, namely at C-9 '-C-10 '. Furthermore, aggressive alkaline hydrolysis of the mixture 7a and 7b with 3% NaOH in aqueous MeOH at room temperature produced steryl glycosides [ -sitosterol 3-O-gentiobioside (8a) and stigmasterol 3-O-gentiobioside (8b)]15,17 by comparison with reported data (13C-NMR, IR, SM) and palmitelaidic acid which was further submitted to methanolysis, the identity of which was confirmed by GC-MS and through reported data20. The 1H-NMR spectrum also, exhibits four signals due to the anomeric protons at H 4.25 (1H, J=7.5 Hz), 4.22 (1H, J=7.5 Hz), 4.52 (2H, J=7.6 Hz), and the corresponding carbon resonances at C 100.4, 100.7, and 98.2 respectively. The glycosidic linkage was established to be by the coupling constants of the 1H-NMR spectral signal due to anomeric protons 21. The interglycosidic linkage was established by NOESY experiment (Fig. 3). The key Nuclear Overhauser Effect was observed between proton H-2 ( H 4.18) and the proton H-6' ( H 4.39), indicating that this mixture bears two consecutive sugars as gentiobioside derivative compounds. A comparison of the 13C-NMR spectrum for the mixture of compounds 7a and 7b with known data for substituted glucopyranoses confirmed that the fatty acid is attached through the hydroxyl group of the second glucose at C-2 atom, because of the inhabitual weak-field shift of this carbon ( C 76.6) before strong alkaline hydrolysis. The value of this carbon undergoes to strong-field shift ( C 73.2) after hydrolysis as shown in literature15. The acetylation of this mixture leading to the formation of hexaacetates indicates that only one fatty acid (palmitelaidic acid) is present. As described above, and obtained after acid hydrolysis of 7a and 7b, only D-glucose is present in the mixture, as sugar, and it was analysed as trimethylsilyl derivative by GC-MS using authentic sample as reference. Consequently, the structures of 7a and 7b were concluded to be -sitosterol-3-[2 -Opalmitelaidoyl- -D-glucopyranosyl (1 -6')- -D-glucopyranoside] and stigmasterol-3-[2 -Opalmitelaidoyl- -D-glucopyranosyl (1 -6')- -D-glucopyranoside]. Chemotaxonomy A number of phytochemical investigations of Ochnaceae have shown the family to be a rich source of flavonoids7,8,22. Moreover, they are taxonomic markers of some genera such as Luxemburgia, Ochna, and Ouratea which is close to Campylospermum3,22,23. The alkaloids isolated in this study are CAMPYLOSPERMUM DENSIFLORUM (OCHNACEAE) 760 D. S. N. Bikobo et. al Vol.4, No.4 (2011), 753-763 mainly indole alkaloids. Previous phytochemical studies on Campylospermum species, or related species like Ouratea, do not mention the isolation of these compounds, except for the study of Campylospermum glaucum and Ouratea turnarea and recently, Campylospermum flavum which provided alkaloids like serotobenine 10 and another analogue like flavumindole 2 and cyanoglycosides1,2,12. These indole akaloids seem to be exceptional1,2 in Ochnaceae family. Their appearance in this family through this study reflects probably the deficiency of exhaustive studies on species of the Campylospermum genus. H H O H O H H H H Fig.-2: HMBC correlations observed for the fatty acid moiety. HO H H O HO HO 2'' 1'' H O OR HO HO 6' O R1 OH O R1: sitosteryl-3- and stigmasteryl-3Fig.-3: Key NOE correlation observed for compound 7a Steroidal glycosides were previously isolated from Ouratea species, like O. parviflora, O. semiserrata, O. sulcata5,9,24 and recently from Campylospermum flavum2. They are ubiquitous in Ochnaceae family, but it is the first report for the isolation of acylsteryl glycosides compounds from Ochnaceae native from Cameroon. At first sight, these results seem to question the accumulation of biflavonoids and terpenoids as major secondary metabolites of species belonging to Ouratea, which is a taxon closely related to Campylospermum which provided mainly indole alkaloids. The presence of steroidal glycosides in this study and an indole alkaloid in occurrence serotobenine which was previously isolated from Ouratea and Campylospermum species1,2 confirms that these two genera may be closely chemotaxonomically. Biological assay on the crude extract and the pure compounds In vitro antimicrobial tests were carried out on the crude extract and isolated compounds, such as serotobenine, decursivine , ent-16 ,17-dihydroxykauran-19-oic acid and the mixture of acylsteryl glycosides on Gram-positive bacteria [Staphylococcus aureus ATCC 25923, Staphylococcus aureus U 271, Staphylococcus saprophyticcus, Enterococcus hirae ATCC 9790, Enterococcus sp P054] [Escherishia coli ATCC 25922, Escherishia coli ATCC 35218, Enterobacter aerogenes ATCC 13048, Enterobacter aerogenes ATCC 29751, Enterobacter cloacae HGY 18, Klebsiella pneumoniae HGY6, Klebsiella pneumoniae HGY 19, Klebsiella oxytoca U 271, Serratia marcescens HGY 4, Serratia marcescens HGY 10, Acinobacter baumannii HGY 12, Acinobacter baumannii HGY 13 and Pseudomonas aeruginosa ATCC 27823] and fungi (Table 3) in accordance with the traditional uses of the plant. CAMPYLOSPERMUM DENSIFLORUM (OCHNACEAE) 761 D. S. N. Bikobo et. al Vol.4, No.4 (2011), 753-763 The tests revealed that the crude extract of C. densiflorum showed considerable activity against Gram-positive with MIC values range to 1.25-5 mg/ml but showed low activity against Gram-negative bacteria with MIC values up to 10 mg/ml (Table 2). Also in disc diffusion assay, methanolic extract showed moderated activity against two kind of fungi (C. albicans and C. neoformans) whereas ent-16 , 17-dihydroxykauran-19-oic acid showed lowest activity against these same microorganisms (Table 3). This can be explained by the fact that the outer membrane of Gram-negative bacteria is known to present a barrier to the penetration of numerous antibiotic molecules, and the periplasmic space contains enzymes which are able to degrade exogenous molecules25. Neither indole alkaloids nor the mixture of steroidal glycosides isolated exhibited any activity against the same microorganisms tested. However, seretobenine and decursivine exhibited antibacterial and antimalarial activities11. The finding of indole alkaloids derivatives compounds especially decursivine, corroborates the traditional use of Campylospermum densiflorum’ leaves against malaria. The present results regarding the antibacterial property of C. densiflorum methanol extract indicate that compounds of this plant could be used against the most common Gram-positive pathogens. ACKNOWLEDGMENTS This research was supported by the International Foundation for Science (IFS), stockhlom, Sweden, and the Organization for Prohibition of Chemical Weapons (OPCW), the Hague, the Netherlands, through a grant to Pr. Pegnyemb N°/3330-2F. We thank Mr. Nana Victor (National Herbarium of Cameroon) for his assistance in the collection and identification of the plant material and Mr. N. Leyondre for the mass spectra. We are undebtful to Dr. J. Gangoué Pieboji for all the biological assay on the crude extract and the pure compounds. 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