Lutamide, a New Ceramide Isolated from the Leaves of Ficus lutea

The Moraceae family consists of about 50 genera and nearly 1400 species including important group 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 ornamental plant in University of Yaounde I, Cameroon.4 Previous phytochemical studies on the wood of Ficus lutea resulted in the isolation of benjaminamide (2), β-amyrin, β-amyrin acetate, lupeol, betulinic acid, βsitosterol glucoside and lutaoside.5 The strong antioxidant and antibacterial activities exhibited by this genus6 in addition to the search for the chemical constituents of Cameroonian medicinal plants7 justified further attempts to isolate and identify active compounds. The few differences between the secondary metabolites isolated from the wood and the leaves of F. lutea are may be related to the real specific differences or more probably to a geographic or environmental influence on biosynthesis.


Results and discussion
The leaves of F. lutea were extracted with MeOH during 30 hours. The extract was submitted to repeated column chromatography to afford benjaminamide (2), betulinic acid, 9,19-cycloart-25-ene-3β,24-diol, vitexin as well as one new ceramide (1a). The 1 H and 13 C NMR, and MS of the known compounds were consistent with those reported in the literature.
Lutamide (1a) was obtained as an amorphous solid. The molecular formula C 34 H 64 NO 4 was determined by HRFABMS at m/z 550.48348 [M-H] -(Calcd. 550.48351). The IR spectrum of 1a indicated absorption bands at ν 3405 cm -1 (OH), and strong absorption bands for a secondary amide at ν 1639 and 1590 cm -1 . These data were supported by the signals at δ 52.5 and 174.8 in 13 C NMR spectrum which confirm the presence of C-N and C=O, respectively. The 1 H and 13 C 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. 1D and 2D NMR spectral data of 1a were nearly superimposable to that of lutaoside (1c) which was further isolated from the wood extract of this plant. 5 A careful comparison of the spectra data of 1a and lutaoside (1c) let to the conclusion that, the structure of lutamide (1a) was (2R)-2-hydroxy-N-((2S,3R,5E,12E)-1,3-dihydroxyoctadeca-5,12-dien-2-yl)hexadecanamide (1a), which is reported here for the first time.  Multiplicities and coupling constants in Hz are given in parentheses Resonances with the same superscripts (ε, ζ) in the same column may be interchanged. The antifungal and antibacterial activities of compounds 1a, 1b and 2 were determined using the agar diffusion method with 8 mm paper disks loaded with 40 μg of each compound (See Table 2). Compound 1a and 1b exhibited in vitro good antimicrobial activity against Mucor miehi and Bacillus subtilis compared to the nystatin as reference.

Materials and method
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. 1 H and 13 C NMR spectra were acquired with a Jeol EX-400 spectrometer. Chemical shifts are given on a δ (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 H 2 SO 4 /10% vanillin and then heating. Flash chromatography was carried out on silica gel (230-400 mesh). R f values were measured on Polygram SIL G/UV254 (Macherey-Nagel & Co.).

Plant material
The leaves of Ficus lutea Vahl were collected in July 2008 at Kribi, South Cameroon. A voucher specimen (Ref. N o . 3471/SRFK) has been deposited in the National Herbarium, Yaoundé, Cameroon.

Extraction and isolation
The powdered leaves of Ficus lutea (2 Kg) were soaked in 10 l of MeOH during 30 hours at room temperature. Solvent was removed under reduced pressure and 60 g of organic extract were obtained. Part of this dark-green residue (58 g) was subjected to vacuum liquid chromatography (VLC) on silica gel and eluted with pure n-hexane (Fraction A), followed by mixture of n-hexane/ethyl acetate in incremental steps 50%, 100% (Fractions B, C respectively) and finally 10% of the mixture of ethyl acetate/methanol (Fraction D). Four main fractions (A-D) were obtained and, basis of analytical TLC, fractions C and D were combined.
Fraction C and D (21 g) was passed through a Sephadex LH-20 column and subjected to silica gel column chromatography and preparative TLC to afford benjaminamide (2, 19 mg) and lutamide (1a, 34 mg). © 2012 The Author(s). Licensee IntechOpen. This is an open access article distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.