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Scientific paper
A New Ceramide Isolated from Ficus lutea Vahl (Moraceae)
Herve Martial Poumale Poumale,1'2'* Aurelie Vigee Barry Songfack Djoumessi,1 Bathelemy Ngameni,3 Louis Pergaud Sandjo,1 Bonaventure Tchaleu Ngadjui1'3
and Yoshihito Shiono2
1 Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon
2 Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka,
Yamagata 997-8555, Japan
3 Department of Pharmacy and Traditional Pharmacopoeia, Faculty of Medicine and Biomedical Science, University of Yaoundé I, P.O. Box 8664, Yaoundé, Cameroon
* Corresponding author: E-mail: poumale@yahoo.fr
Received: 11-08-2010
Abstract
In addition to benjaminamide (2), jS-amyrin, jS-amyrin acetate, lupeol, betulinic acid, ^-sitosterol glucoside, a new ceramide glycoside was isolated from the woods of Ficus lutea Vahl (Moraceae). Using mass fragmentation pattern, 1 and 2D NMR spectra and by comparison with published data, the new compound was characterized as 1-O-j-D-glucopyra-nosyl-(2S,3R,5£,12£)-2N-[(2'R)-hydroxyhexadecanoyl]-octadecasphinga-5,12-dienine (1a) for which the trivial name lutaoside was proposed. Some isolated compounds were evaluated for their antimicrobial activities. Compounds 1a and 2 showed some antimicrobial activity.
Keywords: Ficus lutea, moraceae, ceramide, lutaoside, antimicrobial activity.
1. Introduction
The Moraceae family consists of about 50 genera and nearly 1400 species including important groups such as Artocarpus, Morus and Ficus.1 The genus Ficus consists of trees and shrubs that possess latex-like material within their vasculatures, affording protection and self-healing for physical assaults.2 A number of Ficus species are used as food and for medicinal properties in traditional Chinese medicine especially amongst people where these species grow.3 Ficus benjamina is used as an ornamental plant in University of Yaounde I, Cameroon.4 Previous phytochemical studies of Ficus resulted in the isolation of flavonoids, coumarins, alkaloids, steroids, triterpenes, ce-ramides and salicylic acids.4-12 The strong antioxidant and antibacterial activities exhibited by this genus,13 in addition to the search for the chemical constituents of Came-roonian medicinal plants,14 justified further attempts to isolate and identify active compounds. To the best of our
knowledge, little phytochemical research has been reported on Ficus lutea Vahl.15
2. Results and Discussion
The woods of Ficus lutea were extracted with CH2Cl2/MeOH (1:1) during two days. The extract was submitted to repeated column chromatography and monitored by TLC to afford benjaminamide (2), ¡-amyrin, /¡-amyrin acetate, lupeol, betulinic acid, /¡-sitosterol glucoside and a new ceramide glycoside (Fig. 1). The 1H and 13C NMR, and MS of the known compounds were consistent with those reported in the literature.
Lutaoside (1a) was obtained as a colourless crystalline solid in fraction F. The molecular formula C40H75NO9Na was determined by HRFABMS at m/z 736.5334 [M+Na]+ (Calcd. 736.5339). In the positive FABMS, fragment ions at m/z 714 [M+H]+, 696
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Fig. 1. Structure of compounds 1a, 1b, 1c, 1d and 2.
[M+H-H2O]+, 678 [M+H-2H2O]+ and 552 [M+H-C6H11O5]+ were observed. The IR spectrum of 1a indicated absorption bands at v 3445 cm-1 (OH), and strong absorption bands for a secondary amide at v 1647 and 1586 cm-1. These data were supported by the signals at 5 53.0 and 175.0 in 13C NMR spectrum which confirm the presence of C-N and C=O, respectively. Compound 1a gave a positive reaction in the Molisch test, suggesting the presence of a sugar moiety. The 1H and 13C NMR spectral data (Table 1) of 1a indicated the presence of an amide linkage, two long chain aliphatic moieties, suggesting the sphingolipid (glycolipid) nature of the molecule. The 1H NMR spectral data indicated the presence of a broad singlet centered at 5 1.28 (methylene protons) and two terminal methyl protons at 5 0.90 (t, J = 6.4 Hz, 6H), which confirm the presence of two long aliphatic chain. The methylene protons close to the double bond appeared at 5 1.99-2.25 (m, 8H). A down-field doublet at 5 8.50 (d, J = 8.2 Hz), exchangeable with D2O, was assigned to the amide proton. The olefinic pro-
tons appeared at 5 5.50 (dt, J56 = 15.4 Hz, J45 = 5.3 Hz), 5.00 (dd, J56 = 15.4 Hz, J67 = 4.7 Hz), 5.54 , (dd, J1213 = 16.2 Hz, J11,12 = 4.8 Hz) and 5.40 (dd, J13,12 = 16.2Hz, J1314 = 4.6 Hz) were assigned to the C-5/C-6 and C-12/C-13 protons, respectively. Two carbinol protons were observed at 5 4.58 (dd, J = 10.7, 4.9 Hz) and 5 4.40 (dd, J = 10.7, 4.3 Hz). The anomeric proton appeared at 5 4.49 (d, J = 7.8 Hz) suggesting a /¡-configuration of sugar moiety.
From 13C NMR and DEPT spectra of compound 1a it is possible to conclude that it consists of 40 carbon atoms, including two methyl, 25 methylene, 12 methine and one quaternary carbon atoms. Four methine carbons resonating at 5 129.8, 129.1, 128.7 and 128.1 suggested the presence of two double bonds in the molecule which were further confirmed in 1H NMR spectrum with the presence of four olefinic proton signals at 5 5.54, 5.40, 5.50 and 5.00, respectively.
The glycosphingolipid skeleton was supported by the observation that a proton attached to a nitrogen appea-
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Table 1. 1H (400 MHz) and 13C (100 MHz) NMR data of lutaoside 1a in pyridine-d6
Position	5C	5h
1a	68.8 (t)	4.58 (dd, 10.7; 4.9)
10	68.8 (t)	4.40 (dd, 10.7; 4.3)
2	53.0 (d)	5.20 (m)
3	75.4 (d)	4.50 (m)
4	33.5 (t)	2.25 (m)
5	128.7 (d)	5.50 (dt, 15.4; 5.3)
6	128.1 (d)	5.00 (dd, 15.4; 4.7),
7	32.8 (t)	2.25 (m)
8-10, 15-17	24.0-26.5a	(t each) 1.28 (br s)
11	33.0 (t)	2.11 (m)
12	129.8e (d)	5.54Z (dd, 16.2; 4.8)
13	129.1e (d)	5.40Z (dd, 16.2; 4.6)
14	32.1 (t)	1.99 (m)
18	13.0 (q)	0.90 (t, 6.4)
NH	-	8.50 (d, 8.2)
1'	175.0 (s)	-
2'	72.5 (d)	4.20 (t, 7.3)
3'	31.0 (t)	1.80 (m)
4'-14'	28.0-29.1a	(t each) 1.28 (br s)
15'	21.9 (t)	1.70 (m)
16'	13.0 (q)	0.90 (t, 6.4)
1"	102.9 (d)	4.49 (d, 7.8)
2"	73.5 (d)	4.48 (m)
3"	71.9 (d)	4.00 (m)
4"	70.9 (d)	4.11 (m)
5"	70.0 (d)	4.55 (m)
6"a	61.8 (t)	3.70 (dd, 11.5; 5.5)
6"b	61.8 (t)	3.98 (dd, 11.5; 2.4)
Multiplicities and coupling constants in Hz are given in parentheses. a Overlapping signals
Resonances with the same superscripts (e, Z) in the same column may be interchanged. br s: broad singlet;
d: doublet; dd: doublet of doublet; m: multiplet; t: triplet; dt: doublet of triplet; s: singlet; t: triplet; q: quartet
red as a doublet at 5 8.50 in the 1H NMR spectrum and that a tertiary carbon at 5 53.0 and a quaternary carbon at 5 175.0 were correlated with this nitrogen proton in the HMBC spectrum (Fig. 2).16
The positions of the sugar moiety and the double bonds in the long chain sphingosine part were determined by 2D NMR experiments. The 1H-1H COSY (Fig. 2) displayed spin systems of -HOCH-CH2-CH2-CH2, -CH-HOCH-CH2-CH=CH-CH2, -CH2-CH=CH-CH2 -CH2, -OCH2-CH-, -CH2-CH2- and two -CH2-CH3. In the HMBC spectrum (Fig. 2) the anomeric proton H-1" showed a correlation with C-1 suggesting the position of the sugar moiety to be at the sphingosine part. Accordingly, the HMBC spectrum of 1a did not show any correlations of the methyl group to the olefin carbons (5 129.8, 129.1, 128.7 and 128.1). Further investigation of the EIMS showed some important fragment ions at m/z 71 and 97 suggesting one of the double bond to be located at
C-12,17 while the second was clearly located at C-5 due to the correlation of H-2 with C-3, C-4 and one small correlation with C-5, and H-3 with C-4 and C-5/C-6. The geometry of the C-5/C-6 and C-12/C-13 alkenyl bonds were evident to be trans based on the vicinal coupling constants (J56 = 15.4 Hz and J1213 = 16.2 Hz). These E-geometries were also deduced from the chemical shifts of the allylic carbons C-4, C-7, C-11 and C-14 (5 33.5, 32.8, 33.0 and 32.1, respectively).18 The chemical shifts for the corresponding allylic carbons for the Z configuration are usually less than 5 29.0.19,20
Fig. 2. Important HMBC and H- H COSY correlations for compound 1a.
The above spectral data revealed compound 1a to be a glycosphingolipid of the C18-sphinga-5,12-dienine type.21 The absolute stereochemistry at C-2 and C-3 were proposed as 2S and 3R (identical to that of D-sphingosine) on the basis of 13C NMR spectral data, since the chemical shifts of C-2 (5 53.0) and C-3 (5 75.4) were in agreement with those of the reported natural product hylodendroside-I (5 52.8 and 75.9).21
The length of the long chain base (LCB) and the fatty acid were determined by EIMS, which showed fragment ions at m/z 254 and 459 (Fig. 3). On the other hand, the peak at m/z 551 in EIMS is due to the elimination of the sugar moiety which is also confirmed in FABMS at m/z 552 [M+H-C6H11O5]+. Based on the 13C NMR chemical shifts of the chiral center (Table 1) and optical rotation compared with those of the natural and synthetic ceramine,22 it is clear that compound 1a has a D-glucopyranosylsphingosine moiety with (2S,3R, 5E,12E geometry. Thus, the structure of lutaoside (1a) was assigned as 1-O-0-D-glucopyranosyl-(2S,3R,5E, 12E)-2N-[(2'R)-hydroxyhexadecanoyl]-octadecasphinga-5,12-die-nine, which is described here for the first time.
Acetylation of compound 1a gave 1b (C52H87NO15). Methanolysis of 1a gave 1c and 1d. The structure of compounds 1b, 1c and 1d (Fig. 1) were confirmed by MS and 1H NMR spectra.
The antifungal and antibacterial activities of compounds 1a and 2 were determined using the agar diffusion method with 8 mm paper disks loaded with 40 ^g of each compound (See Table 2). Compound 1a exhibited in vitro good antimicrobial activity against Mucor mie-hei and Bacillus subtilis compared to the nystatin as reference.
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m/z 197
m/z 237 m/z 207
Fig. 3. Important mass fragmentation pattern of compound 1a.
Table 2. Antimicrobial activity of compounds 1a and 2
Micro-organisms tested	Sample		
	Compo-	Compo-	Nysta-
	und 1a	und 2	tin
Chlorella vulgaris	14	-	-
Scenedesmus subspicatus	11	10	-
Chlorella sorokiniana	13	11	-
Mucor miehei	17	13	15
Bacillus subtilis	16	14	14
Candida albicans	12	13	15
Streptomyces viridochromogenes	-	-	14
Escherichia coli	-	-	-
Staphylococcus aureus	-	-	-
Diameter of inhibition zone in mm. Nystatin was used as the reference.
3. Conclusion
This research study reports a new ceramide 1a isolated in fraction F (EtOAc/10%MeOH). The new compound was fully characterized and it is one of the rare reported phytochemical study on F. lutea. Ceramide has already being reported in other Ficus species.4,12 Two isolates (1a and 2) were evaluated for their antimicrobial activities. Compound 1a exhibited in vitro good antimicrobial activity against Mucor miehei and Bacillus subtilis. However, the chemical constituents as well as the biological activities of Ficus species still remain unclear. Therefore, the secondary metabolites and the biological activities reported from F. Lutea seem to be worth for further studies.
4. Experimental
4. 1. General
Melting point is uncorrected and was obtained with a micro melting point apparatus (Yanaco, Tokyo-Japan). Optical rotation values were measured with a Horiba
SEPA-300 Polarimeter, and IR spectra were recorded with JASCO J-20A spectrophotometer. 1H and 13C NMR spectra were acquired with a Jeol EX-400 spectrometer. Chemical shifts are given on a 5 (ppm) scale with TMS as an internal standard. Mass spectra were obtained with a Jeol JMS-700 instrument. Column chromatography was conducted on silica gel 60 (Kanto Chemical Co., Inc., Japan), Sephadex LH-20 (Pharmacia, Sweden) and ODS (Fuji Silysia, Japan). TLC analysis was carried out by using precoated silica gel plates (Merck), and the spots were detected by spraying with H2S04/10% vanillin and then heating. Flash chromatography was carried out on silica gel (230-400 mesh). Rf values were measured on Polygram SIL G/UV254 (Macherey-Nagel & Co.).
4. 2. Plant Material
The woods of Ficus lutea Vahl were collected in July 2008 at Kribi, South Cameroon. A voucher specimen has been deposited in the National Herbarium, Yaounde, Cameroon (Ref. No. 3471/SRFK).
4. 3. Hydrolysis Experiments
Compound 1a (1.9 mg) was dissolved in a mixture of MeOH (1 mL) and distilled H20 (1 mL). Then 7% HCl (1 mL) solution was added and the solution was refluxed for 10 h at 50 °C. MeOH was evaporated in vacuum after cooling. The reaction mixture was extracted three times with chloroform (8 mL each). The residue obtained after removal of acid was compared with standard sugar units on silica gel plate using EtOAc:H2O (9:1) as the solvent system and the sugar was found to be D-glucopyranoside.
4. 4. Extraction and Isolation
The powdered woods of Ficus lutea (3.6 kg) were soaked in 15 L of CH2Cl2/MeOH (1:1) during two days at room temperature. Solvent was removed under reduced
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pressure and 100 g of organic extract was obtained. Part of this dark-green residue (95 g) was subjected to vacuum liquid chromatography (VLC) on silica gel and eluted with pure n-hexane (Fraction A), followed by mixture of n-hexa-ne/p% ethyl acetate in incremental steps (p% = 25%, 50%, 75%, 100% for fractions B, C, D, E, respectively) and finally the mixture of ethyl acetate/10% methanol (Fraction F). Six main fractions (A-F) were obtained and, based on analytical TLC, fractions D and E were combined.
Fraction A (4 g) gave mainly ¡-amyrin (65.0 mg)23,24 and /¡-amyrin acetate (25.1 mg).25 Fraction B (12 g) was passed through a Sephadex LH-20 column and subjected to silica gel column chromatography and preparative TLC to afford a mixture of sterol (160 mg),26 lupeol (87.8 mg)27 and betulinic acid (35.0 mg).28 Fractions D and E (17 g) were chromatographed on silica gel and elu-ted with a mixture of n-hexane/ethyl acetate of increasing polarity to yield 98 fractions (ca. 100 mL each). Fractions 1-58 (3 g), subjected to column chromatography over silica gel, yielded mainly ¡-sitosterol glucoside (165.3 mg),29 while benjaminamide (2, 38.9 mg)4 was obtained in fractions 59-98 (6 g) eluted with CHCl3/MeOH (3:1). Fraction F (13 g) was purified by silica gel column chromatography eluting with CHCl3/MeOH (15:4) to yield lutaosi-de (1a, 16.2 mg).
Lutaoside or 1-0-/-D-Glucopyranosyl-(2S,3^,5£',12£') -2N-[(2'_R)-hydroxyhexadecanoyl]-octadecasphinga-5,12-dienine (1a)
Colourless crystalline; mp 203-205 °C; Rf = 0.11 (CHCl3/10% MeOH); [a]D22 +8.12 (c 0.04, MeOH); IR (film) (vmax/cm-1): 3445 (OH), 3209 (NH), 2924, 2853, 1647, 1586, 1427, 1388, 1217, 1167, 1078, 1047, 1040, 881; 1H NMR (400 MHz, C5D5N, 30 °C, TMS) and 13C NMR (100 MHz, C5D5N): see Table 1; EIMS: see Fig. 3; FABMS: m/z 736 [M+Na]+, 714 [M+H]+, 696 [M+H-H2O]+, 678 [M+H-2H2O]+, 552 [M+H-C6H11O5 (sugar moiety)]+; HRFAB MS: Calcd for C40H75NO9Na 736.5339 [M+Na]+, found 736.5334.
Acetylation of Lutaoside (1a)
Lutaoside (1a, 3.0 mg) was dissolved in pyridine (0.6 mL) and acetanhydride (0.9 mL). The solution was stirred for 7 h at 47 °C. The usual work-up gave hexaace-toxylutaoside (1b) (2.2 mg, 93%) as an amorphous solid with Rf = 0.91 (CHCl3/10% MeOH); [a]D22 +7.67 (c 0.06, pyridine); IR (film) (vmax/cm-1): 3200 (NH), 2915, 2853, 1653, 1580, 1500, 1452, 1419, 1217, 1100, 890; 1H NMR (400 MHz, C5D5N, 30 °C, TMS) 5 0.83 (t, J = 6.3 Hz, 6H, H-18, H-16'), 1.20-1.30 (br s), 1.50-1.55 (m, 4H, H-3', H-15'), 1.90 (m, 6H, H-6, H-11, H-14), 2.00, 2.05, 2.06, 2.08, 2.12, 2.13 (s, 3H each, CH3-C=O), 4.09 (dd, J = 11.3, 3.7 Hz, 1H, H-1a), 4.12 (m, 2H, H-6"), 4.25 (dd, J = 11.3, 5.4 Hz, 1H, H-1b), 4.26 (m, 1H, H-5"), 4.46 (d, J = 7.8 Hz, 1H, H-1"), 4.92 (dd, J = 10.7, 7.8 Hz, 1H, H-2"), 5.40-5.47 (m, 5H, H-3", H-4", H-6, H-12, H-13), 5.48 (m,
1H, H-2'), 5.50 (m, 1H, H-3), 5.71 (dd, J = 15.8, 4.5 Hz, 1H, H-5), 8.53 (d, J = 8.2 Hz, 1H, NH); FABMS m/z 967 [M+H]+; HRFAB MS Calcd for C^H^NO^ 966.6154 [M+H]+, found 966.6138.
Methanolysis of Lutaoside (1a)
Lutaoside (1a, 5.0 mg) was subjected to methanolysis in 0.1 mL of 0.9 M HCl in methanol at 60 °C for 15 h. Methyl ester (1c, 2.0 mg, 78 %) was extracted from the methanol solution with hexane. After removal of MeOH, the residue was purified using reverse phase (RP-18) eluting with MeOH/H2O (3:2) to yield 1-0-/-D-glucopyranosylsphingo-sine (1d, 0.9 mg, 85 %) as an amorphous solid.
2_R-Hydroxyhexadecanoic Methyl Ester (1c)30
Amorphous solid; Rf = 0.95 (CHCl3/10% MeOH); [a]D25 -2.3 (c 0.08, MeOH); 1H NMR (400 MHz, MeOH, 30 DC, TMS) 5 0.85 (t, J = 6.4 Hz, 3H, H-16'), 1.25 (br s), 1.40 (m, H-15'), 1.70 (m, H-3'), 3.88 (s, 3H, OCH3), 4.25 (m, 1H, H-2'); FABMS m/z 287 [M+H]+; HRFAB MS Calcd for C17H35O3 287.2586 [M+H]+, found 287.2579.
1-0-/-D-Glucopyranosylsphmgosine (1d)
Amorphous solid; Rf = 0.19 (CHCl3/10% MeOH); [a]D25 +6.8 (c 0.1, MeOH)); 1H NMR (40(3 MHz, MeOH, 30 °C, TMS) 5 0.81 (t, J = 6.4 Hz, H-18), 120 (br s), 1.99-2.12 (m, 6H, H-6, H-11, H-14), 3.20 (br m, 2H, NH2), 3.40-3.78 (m, 10H, H-Gluc, H-1, H-2, H-3), 4.22 (d, 2= 7.9 Hz, 1H, H-1"), 5.17 (m, 4H, H-5, H-6, H-12, H-13); FABMS m/z 460 [M+H]+; HRMS (FAB) Calcd for C24H46NO7 460.3274 [M+H]+, found 460.3268.
5. Antimicrobial Assay
Agar diffusion tests were performed in the usual manner10 with Bacillus subtilis and Escherichia coli (on peptone agar), Staphylococcus aureus (Bacto nutrient broth), Streptomyces viridochromogenes (M Test agar), the fungi Mucor miehei and Candida albicans (Sabouraud agar), and three microalgae (Chlorella vulgaris, Chlorella sorokiniana and Scenedesmus subspicatus).
Compounds were dissolved in an azeotrope chloro-form/MeOH (87:13) and 40 pg pro paper disks (0 8 mm) were impregnated with each using a 100 pL syringe, dried for 1 h under sterile conditions and placed on the pre-ma-de agar test plates. Bacteria and fungi plates were kept in an incubator at 37 °C for 12 h, micro algae plates for three days at room temperature in a day light incubator. The diameter of inhibition zones was measured.
6. Acknowledgements
The authors are grateful to the Japan Society for the Promotion of Science (JSPS) for the fellowship (No.
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P08430) awarded to Dr. Herve M. P. Poumale at the University of Yamagata, Japan.
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Povzetek
Iz lesa Ficus lutea Vahl (Moraceae) smo poleg benjaminamida (2), jS-amirina, jS-amirin acetata, lupeola, betulinske kisline in jS-sitosterol glukozida, izolirali tudi nov ceramid glikozid. Z uporabo fragmentacije v masnem spektrometru, 1 in 2D NMR spektrov in s primerjavo z že objavljenimi podatki, smo novo spojino karakterizirali kot 1-O-j-D-glukopi-ranozil-(2S,3^,5_E,12_E)-2N-[(2'.R)-hidroksiheksadekanoil]-oktadekasfinga-5,12-dienin (1a) ter zanj kot trivialno ime predlagali lutaozid. Nekaterim izoliranim spojinam smo tudi določili antimikrobne aktivnosti. Spojini 1a in 2 sta pokazali določeno antimikrobno aktivnost.
Poumale et al.: A New Ceramide Isolated from Ficus lutea Vahl (Moraceae)