Phytochemistry Letters 5 (2012) 524–528 Contents lists available at SciVerse ScienceDirect Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol Chemical constituents of Trilepisium madagascariense (Moraceae) and their antimicrobial activity Patrick Y. Ango a, Deccaux W.F.G. Kapche b,*, Victor Kuete c, Bonaventure T. Ngadjui a, Merhatibeb Bezabih d, Berhanu M. Abegaz d,** a Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, Cameroon Department of Chemistry, Higher Teacher Training School, University of Yaoundé I, Cameroon Department of Biochemistry, Faculty of Science, University of Dschang, Cameroon d Department of Chemistry, Faculty of Science, University of Botswana, Botswana b c A R T I C L E I N F O Article history: Received 24 February 2012 Received in revised form 11 May 2012 Accepted 22 May 2012 Available online 6 June 2012 Keywords: Antimicrobial activity Moraceae Trilepisflavan Trilepisuimic acid Trilepisium madagascariense A B S T R A C T The chemical study of Trilepisium madagascariense has led to the isolation of two previously undescribed compounds, (+)-(2S)-7-hydroxy-30 ,40 -dimethoxyflavan (trilepisflavan) and (E)-4-[3-(3,4-dihydroxyphenyl)prop-2-enoyloxy]-3-hydroxybenzoic acid (trilepisuimic acid), together with ten known compounds caffeic acid, catechin, erythrodiol-3-O-palmitate, 8-C-glucopyranosylapigenin, dihydrokaempferol, protocatechuic acid, 30 ,7-dihydroxy-40 -methoxyflavan, isoliquiritigenin, luteolin and 1,3-dimethoxybenzene. Their structures were elucidated on the basis of spectroscopic evidence. Crude extracts, trilepisflavan, dihydrokaempferol and 8-C-glucopyranosylapigenin showed significant antimicrobial activity. Crown Copyright ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. 1. Introduction Trilepisium Thouars (family: Moraceae) is a monotypic genus represented by Trilepisium madagascariense DC, a deciduous tree of up to 30 m height, of the middle storey of the high-forest, extending on to the borders of Savanna (Burkill, 1985). T. madagascariense is found in Cameroon, Democratic Republic of Congo and Madagascar (Berg et al., 1985). The leaves of T. madagascariense are used as vegetable; other parts of the plant are traditionally used as pain killers and to treat venereal diseases, arthritis, rheumatism, diarrhoea, dysentery, stomach troubles, malnutrition, debility, cutaneous and subcutaneous parasitic infections (Burkill, 1985). To the best of our knowledge, there is very little phytochemical information on T. madagascariense. Teke et al. (2010) have reported the antidiarrheal and bacteriostatic (vs Staphylococcus aureus) activities of the methanolic extract of the stem barks and leaves, respectively. These authors have also reported the isolation of isoliquiritigenin and the detection of alkaloids, flavonoids, tannins, saponins and cardiac glycosides in * Corresponding author. Tel.: +237 77 66 49 73. ** Corresponding author. Tel.: +254 73 95 45 50 99. E-mail addresses: dkapche2002@yahoo.com (Deccaux W.F.G. Kapche), b.abegaz@aasciences.org (B.M. Abegaz). these extracts. These preliminary results prompted us to undertake more investigations on this plant. 2. Results and discussion The EtOAc extracts of the stem bark and the leaves of T. madagascariense on repeated column chromatography and further purification by Sephadex LH-20 yielded two previously undescribed compounds, namely, trilepisflavan (1) and trilepisuimic acid (2) (Fig. 1) in addition to ten known compounds 30 ,7dihydroxy-40 -methoxyflavan (Masaoud et al., 1995), isoliquiritigenin (Reyes-Chilpa et al., 1998; Teke et al., 2010), dihydrokaempferol (Lee et al., 2003), catechin (Seto et al., 1997), erythrodiol-3-Opalmitate (Nakano and Hasegawa, 1975), luteolin (Lopez-Lazaro, 2009), 8-C-glucopyranosylapigenin (Zhang et al., 2008a), caffeic acid (Agarwal and Rastogi, 1974), 1,3-dimethoxybenzene (Rogerson et al., 2002), protocatechuic acid (Scott, 1972). Trilepisflavan (1) was obtained as brown oil, [a]D25 = +8.33. Its molecular formula, C17H18O4, was determined on the basis of HREIMS, m/z 286.1207 (calcd. 286.1205). Its IR spectrum showed absorption bands at 3437 cm 1 (OH) and 1622–1460 cm 1 (aryl absorptions and overtones). The 1H and 13C NMR data of 1 (Table 1) showed one oxymethine, and two methylene groups together with two trisubstituted benzene rings suggesting according to the coupling pattern and the chemical shifts that 1 is a trisubstituted 1874-3900/$ – see front matter . Crown Copyright ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2012.05.006 P.Y. Ango et al. / Phytochemistry Letters 5 (2012) 524–528 Table 1 1 H (d, CDCl3, 300 MHz) and Position 1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 30 -OCH3 40 -OCH3 –COOH 13 C (d, CDCl3, 75 MHz) NMR data of compounds 1 and 2. 1 1 525 2 H dH (m, J in Hz) 13 HMBC (H ! C) – 84.7 34.1 – – – – C-9 C-9 C-7, C-9, C-4 C-10 – C-6, C-7, C-9, C-10 – – – – – – C-30 , C-20 , C-40 , C-50 C-30 C-40 – C dC – 5.03 (m) a: 2.89 (m) b: 3.11 (m) a: 2.89 (m) b: 3.11 (m) 6.96 (d, 8.4) 6.29 (dd, 2.4; 8.4) – 6.32 (d, 2.4) – – – 6.80 (d, 1.8) – – 6.84 (d, 8.2) 6.81 (dd, 1.8; 8.2) 3.89 (s) 3.88 (s) – 41.5 125.0 106.9 156.0 97.6 160.5 118.7 129.9 112.6 148.9 147.8 121.3 111.2 55.9 55.8 – 4' OCH3 9 O 2 5 10 4 7.53 (d, 2.1) – 123.1 115.8 145.6 – 150.7 6.90 7.47 – 6.27 7.54 – – 7.17 (d, 8.4) (dd, 2.1; 8.4) (d, 15.9) (d, 15.9) (d, 2.1) HO HO 2' 9 1 C-1, C-3, C-4, C-6 C-1, C-3, C-4, C-6 C-4, C-5, –COOH C-10 , C-7 C-10 , C-20 , C-60 – C-9, C-40 C-10 , C-30 , C-60 C-40 , C-8, C-9 – – – 3 7 O 4 3 HMBC (H ! C) OH 8 1' C dC 117.5 123.5 168.2 115.6 145.9 – 127.6 115.0 146.3 148.7 116.3 122.4 – – 167.6 – 6.87 (d, 8.4) 7.03 (dd, 2.1; 8.4) – – – 1' 3' OCH3 13 H dH (m, J in Hz) 4' HO 7 1 1 O 2 COOH 6 Fig. 1. Chemical structures of compounds 1 and 2. flavan (Garo et al., 1996). Further analysis of the spectral data allowed us to conclude that 1 is the 30 -O-methyl derivative of 30 ,7dihydroxy-40 -methoxyflavan which had been isolated for the first time from Dracaena cinnabari (Masaoud et al., 1995). The absolute configuration at C-2 was deduced to be (S) from CD measurements (negative Cotton effect at 285 nm; cf data for 40 -hydroxy-30 ,5,7trimethoxyflavan (Garo et al., 1996). Therefore, 1 was identified as (+)-(2S)-7-hydroxy-30 ,40 -dimethoxyflavan, a new natural product for which the trivial name trilepisflavan is proposed. Trilepisiumic acid (2) was obtained as colourless powder. The molecular formula C16H12O7 was determined from HR-EIMS: m/z 316.0594 (calcd. 316.0583). Its IR spectra also showed absorptions at ca 3400 (–OH stretch) and the typical aryl absorptions and overtones from 1610 to 1450 cm 1. Compound 2 gave a dark green colour with methanolic ferric chloride confirming the presence of free phenolic hydroxyl substituents. The NMR data of 2 (Table 1), exhibited 16 carbons and 8 vinyl/aryl protons, of which nine carbons and five protons were assigned to the caffeic acid unit (A) (Zhang et al., 2008b) and the remaining seven carbons and three protons to a di-oxygenated benzoic acid unit (B). 1H chemical shifts and HMBC correlations (Table 1) allowed us to establish the 3,4-dioxygenated patterns for the benzoic acid unit. The linkage between the two units was deduced from the upfield chemical shifts of the carbon bearing the hydroxyl group (C-3, d 145.6) and the downfield chemical shift of the adjacent carbon (C-4, d 150.5) and by comparison of the NMR data with those published for a structural analogue (Siddiqui et al., 2007). All the proton and carbon signals were assigned from analyses of the 1H, 13C, COSY, HMQC, and HMBC data (Table 1). Compound 2 was, thus, assigned the structure of (E)-4-[3-(3,4-dihydroxyphenyl)prop-2-enoyloxy]-3-hydroxybenzoic acid, a new natural product for which the trivial name trilepisiumic acid is proposed. This compound is an ester of caffeic acid and protocatechuic acid isolated in the same plant. The antibacterial activities of the two crude extracts and 10 of the isolated compounds were tested for activities and against Candida albicans. The results are reported in Tables 2 and 3. The minimal inhibitory concentrations (MICs) summarized in Table 2, showed that the crude extracts of the stem bark (TMB) and leaves (TML), trilepisflavan, dihydrokampferol and 8-C-glucopyranosylapigenin were able to prevent the growth of all tested microbial species at different concentrations. All other compounds showed selective activities. It is worth noting that threshold MIC values of 100 and 10 mg/ml have been recommended for plant extracts and pure compounds, respectively, to rate them as having significant antimicrobial activity (Kuete, 2010). Thus the MIC values measured for the activities of the crude extracts TMB and TML on Salmonella typhi (64 mg/ml) and E. coli (64 mg/ml) and for dihydrokampferol and 8-C-glucopyranosylapigenin on E. coli (8 mg/ml) may be considered significant. Other workers have suggested higher MIC thresholds of 8 mg/ml for extracts (Fabry et al., 1998) and 1 mg/ml for natural products (Gibbons, 2004). According to these less stringent criteria the extracts as well as compounds dihydrokampferol and 8-C-glucopyranosylapigenin may be considered as important, highlighting the antimicrobial potency of T. madagascariense. Some of the MIC values recorded with 8-C-glucopyranosylapigenin were closer or equal to that of the reference antibiotic chloramphenicol. The results of Table 3 showed detectable minimal microbicidal concentration (MMC) values for some of the studied samples on the tested microbial strains. The MMC/MIC < 4 observed for the crude extracts as well as for dihydrokampferol and 8-C-glucopyranosylapigenin on most of the studied samples, suggesting that killing effects could be expected (Carbonnelle et al., 1987). P.Y. Ango et al. / Phytochemistry Letters 5 (2012) 524–528 526 Table 2 MIC (mg/ml) values of extracts and compounds from the stem bark (TMB) and the leaves (TML) of T. madagascariense on eight microbial species and strains. Tested samples TMB TML Trilepisflavan Trilepisiumic acid 30 ,7-Dihydroxy-40 -methoxyflavan Isoliquiritigenin Dihydrokaempferol Erythrodiol-3-O-palmitate 8-C-glucopyranosylapigenin Caffeic acid 1,3-Dimethoxybenzene Protocatechuic acid RA Microorganisms, strains and MIC (mg/ml) Providencia smartii Pseudomonas aeruginosa Klebsiella pneumoniae Staphylococcus aureus Salmonella. typhi Escherichia. coli Candida albicans ATCC29916 PA01 ATCC11296 ATCC25922 ATCC6539 ATCC8739 AG100 ATCC 9002 128 128 512 >512 >512 256 512 >512 32 >512 >512 512 32 1024 512 512 512 >512 >512 128 >512 64 256 512 >512 64 64 64 128 >512 512 128 16 512 16 >512 >512 >512 4 128 128 128 >512 512 >512 64 256 32 128 512 >512 4 64 256 512 512 >512 >512 16 512 128 >512 256 >512 4 128 64 16 512 >512 >512 8 128 8 >512 256 >512 4 128 128 256 512 >512 >512 64 512 64 >512 128 >512 4 512 128 512 64 512 >512 64 >512 128 >512 >512 >512 16 RA: reference antibiotics – chloramphenicol for bacteria and nystatin for C. albicans. Table 3 MMC (mg/ml) values of extracts and compounds from the stem bark (TMB) and the leaves (TML) of T. madagascariense on eight microbial species and strains. Tested samples TMB TML Trilepisflavan Trilepisiumic acid 30 ,7-Dihydroxy-40 -methoxyflavan Isoliquiritigenin Dihydrokaempferol Erythrodiol-3-O-palmitate 8-C-glucopyranosylapigenin Caffeic acid 1,3-Dimethoxybenzene Protocatechuic acid RA Microorganisms, strains and MMC (mg/ml) P. smartii P. aeruginosa K. pneumoniae S. aureus S. typhi E. coli ATCC29916 PA01 ATCC11296 ATCC25922 ATCC6539 ATCC8739 AG100 C. albicans ATCC 9002 512 256 >512 – – >512 >512 – 64 – – >512 64 – >1024 >512 >512 – – 256 – 128 >512 >512 – 128 256 256 256 – >512 512 32 >512 32 – – – 8 256 256 256 – >512 – 128 512 64 512 >512 – 8 128 1024 >512 >512 – – 32 >512 256 – 512 – 8 256 128 64 >512 – – 32 256 64 – >512 – 8 256 512 512 >512 – – 128 >512 128 – 512 – 8 1024 256 >512 256 >512 – 128 – 256 – – – 32 –: not determined as the MIC was >512 mg/ml for compounds or 1024 mg/ml for the crude extracts. RA: reference antibiotics – chloramphenicol for bacteria and nystatin for C. albicans. The overall results of this study indicated that the antimicrobial activity of the crude extract from T. madagascariense could be due to the presence of both antibacterial and anti-candidal compounds. Previously, Teke et al. (2010) provided evidence that the methanol extract, fractions and isoliquiritigenin from T. madagascariense stem bark possess antidiarrheal activities for Shigella-induced diarrhoea. The present study gives more information on the antimicrobial activities of the plant. We are not aware of any previous report on the antimicrobial activities of erythrodiol-3-Opalmitate, 30 ,7-dihydroxy-40 -methoxyflavan and 8-C-glucopyranosylapigenin. Nevertheless, apigenin (Basile et al., 1999) and apigenin-7-O-glucoside (Akroum et al., 2010) are known to have good antibacterial activities. The antimicrobial activity of dihydrokaempferol, on bacteria such as S. aureus has been well documented (Malterud et al., 1985) and corroborates the results reported herein. Catechin reported to have antimicrobial activities (Kuete et al., 2008a) and luteolin obtained in low amount were not tested in this work. 3. Experimental 3.1. General Optical rotations were measured on a Jobin–Yvon–Isa Dichrograph in chloroform. NMR experiments were performed with a Brucker 300-Ultra Shield spectrometer. ESI-MS spectra were recorded by GCT Premier-Waters manufactury. IR spectra were recorded by Perkin Elmer-FTIR spectrometer. UV spectra were recorded in acetone. CD spectrum was measured on a Jasco J-600 Spectropolarimeter in chloroform (c = 0.0035 mol l 1; cell length 0.1 cm) at 25 8C; l (De) in nm. Column chromatography was carried out with silica gel 60 Merck (0.040–0.063 mm). 3.2. Plant material The leaves and stem bark of T. madagascariense were collected in Mbankomo, Centre region of Cameroon in March 2010. The plant was identified by Mr. Victor Nana of the National herbarium, Yaoundé, Cameroon, where a voucher specimen (number 1778/ SRFK) is deposited. 3.3. Extraction and isolation The air-dried and powdered stem bark (1.5 kg) was soaked in MeOH for 48 h, at room temperature. The methanolic extract was concentrated under reduced pressure to give a brown residue (70 g). This extract, found to contain a lot of tannins was successively extracted with petrol, EtOAc and n-butanol. Removal of the solvents gave 10.0, 36.0 and 9.0 g of residues, respectively. The EtOAc extract (TMB, 30 g) containing much compounds than the others was subjected to CC over silica gel 60 (150.0 g) and eluted with petrol–CHCl3 and CHCl3–MeOH of increasing polarity. P.Y. Ango et al. / Phytochemistry Letters 5 (2012) 524–528 Forty-nine fractions of 300 ml each were collected, concentrated, monitored by TLC and similar fractions were combined. The first fractions (1–18, 2.1 g) contained mostly hydrocarbons and were not investigated further. Fractions 19–22 (1.2 g), obtained with petrol–CHCl3 3:7 was passed through Sephadex LH-20 and eluted with CHCl3–MeOH (7:3) to give trilepisflavan (5 mg). 1,3Erythrodiol-3-O-palmitate 7 (50.6 mg) and a mixture of stigmasterol glucoside and sitosterol glucoside (3.0 g) were directly obtained as crystals from fractions (23–29) obtained with pure CHCl3. Passage through Sephadex LH-20 eluting with CHCl3–MeOH (7:3) of fractions 30–33 (1.5 g, CHCl3–MeOH 97.5:2.5) gave 30 ,7dihydroxy-40 -methoxyflavan (8.9 mg). Fractions 34–45 (15.0 g, CHCl3–MeOH 95:5 to 85:15) were subjected to CC (silica gel) eluting with CHCl3–MeOH of increasing polarity to give 6 fractions (J1–J6). J2 and J4 were purified on Sephadex LH-20 eluting with CHCl3–MeOH (7:3) to give isoliquiritigenin (47.8 mg) from J2, dihydrokaempferol (7.7 mg) and catechin (8.5 mg) from J4. Other fractions (46–49) were found to contain mostly tannins and were not investigated further. The air-dried and powdered leaves (3 kg) were extracted with 5 l of CH2Cl2–MeOH (1:1) for 48 h followed by MeOH for 12 h at room temperature. The extracts (TML) were combined, freed of solvent in vacuo to yield a brown residue (210.0 g); part of this extract (160 g) was subjected to vacuum column chromatography (VCC, silica gel 60, 300.0 g) and eluted successively with n-hexane, n-hexane/EtOAc (1:1), EtOAc, EtOAc/MeOH. Fractions of 500 ml each, were collected, concentrated, monitored by TLC and similar ones pooled to give a total of six fractions (A–F). Fraction E (50.0 g), obtained with EtOAc, was subjected to CC (silica gel 60, 150.0 g) eluting with CHCl3– MeOH of increasing polarity to give 10 fractions (E1–E10). E3 (CHCl3 to CHCl3–MeOH 95:5) and E4 (CHCl3–MeOH 96:4 to 90:10) were repeatedly passed through Sephadex LH-20 (CHCl3– MeOH 7:3). 1,3-Dimethoxybenzene (6.5 mg), trilepisiumic acid (11.2 mg) and luteolin (3.5 mg) were obtained from E3, caffeic acid (4.5 mg), protocatechuic acid (9.0 mg) and 8-C-glucopyranosylapigenin (24.0 mg) from E4. Others fractions were found to contain mostly hydrocarbons or tannins and were not investigated further. (+)-(2S)-7-hydroxy-30 ,40 -dimethoxyflavan (Trilepisflavan, 1): Brown oil, [a]D25 = +8.33 (c 1, CHCl3); CD (c 0.0035 M): 285 MeCOMe nm (log e): 285 (1.29), 263 ( 11.79); 252 ( 12.62). UV lmax (1.18), 252 (1.21). IR: nmax cm 1: 3210, 1678, 1598, 1514, 1443, 1358, 1275, 1186, 1114, 979, 943, 814, 769. NMR data are given in Table 1. HR-EIMS: m/z 286.1207 (calcd. for C17H18O4, 286.1205). (E)-4-[3-(3,4-dihydroxyphenyl)prop-2-enoyloxy]-3-hydroxybenzoic acid (Trilepisiumic acid, 2): Colourless powder, UV lMeCOMe nm (log e): 365.8 (3.15), IR: nmax cm 1: 3437, 2970 max 1738, 1622, 1515, 1462, 1365, 1261, 1231, 1139, 1026, 959, 804, 766. NMR data are given in Table 1. HR-EIMS: m/z 316.0594 (calcd. for C16H12O7, 316.0583). 3.4. Antimicrobial assay 3.4.1. Microbial strains and culture media The studied microorganisms included reference strains of Providencia smartii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella typhi, Escherichia coli and Candida albicans obtained from the American Type Culture Collection. They were maintained on agar slant at 4 8C and sub-cultured on a fresh appropriate agar Plates 24 h prior to any antimicrobial test. Nutrient agar and Sabouraud glucose agar were used for bacteria and fungi, respectively. The Mueller Hinton broth (MHB) was used for the MIC and MMC determinations. The Mueller Hinton agar (MHA) was also used for the determination of the MMC on these species. 527 3.4.2. Chemicals for antimicrobial assay Chloramphenicol (Sigma–Aldrich, St. Quentin Fallavier, France) and nystatin (Sigma–Aldrich) were used as reference antibiotics (RA), respectively, against bacteria and Candida albicans. pIodonitrotetrazolium chloride (INT, Sigma–Aldrich) was used as microbial viability indicator. 3.4.3. MIC and MMC determinations The determinations of MICs on bacteria and C. albicans were conducted according to Eloff (1998) and Pettit et al. (2005) using INT. Briefly, the test samples were first dissolved in 10% (v/v) DMSO/MHB to give a final concentration of 2048 mg/ml for the crude extracts, 1024 mg/ml for compounds, 256 mg/ml for the RA. These were serially diluted twofold to obtain concentration ranges of 8–1024 mg/ml, 4–512 mg/ml and 1–128 mg/ml, respectively. 100 ml of each concentration was added in a well (96-well microplate) containing 95 ml of MHB and 5 ml of inoculum (standardized at 1.5  106 CFU/ml by adjusting the optical density to 0.1 at 600 nm SHIMADZU UV-120-01 spectrophotometer) (Tereschuk et al., 1997). The highest concentration of DMSO in the well was less than 3% (preliminary analyses with 3% (v/v) DMSO do not alter the growth of the test organisms). The negative control well consisted of 195 ml of MHB and 5 ml of the standard inoculum (Zgoda and Porter, 2001). The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a plate shaker and incubated at 37 8C for 24 h. The assays were repeated three times. The MIC of samples was detected following addition (40 ml) of 0.2 mg/ml p-iodonitrotetrazolium chloride and incubation at 37 8C for 30 min (Eloff, 1998; Pettit et al., 2005). Viable microorganisms reduced the yellow dye to a pink colour. MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of bacterial growth. For the determination of MMC, a portion of liquid (5 ml) from each well that showed no change in colour was plated on MHA and incubated at 37 8C for 24 h. The lowest concentration that yielded no growth after this sub-culturing was taken as the MMC (Kuete et al., 2008b). Acknowledgements A.Y.P. and K.W.F.G.D. are grateful to the Third World Academic of Science (TWAS), the Organization for the Prohibition of Chemical Weapons (OPCW) and the Network of Analytical and Bioassay Services in Africa (NABSA) for a 3-months travel and maintenance grant to the University of Botswana. 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