Ceramides and cytotoxic constituents from Ficus glumosa Del. (Moraceae)

ARTICLE

Ceramides and cytotoxic constituents from Ficus glumosa Del. (Moraceae)

Frédéric Nana^I,II; Louis Pergaud Sandjo^*,I; Félix Keumedjio^I; Pantaléon Ambassa^I; Rizwana Malik^II; Victor Kuete^III; Vincent Rincheval^IV; Muhammad Iqbal Choudhary^III; Bonaventure Tchaleu Ngadjui^*,I,V

^IDepartment of Organic Chemistry, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon

^IIH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan

^IIIDepartment of Biochemistry, University of Dschang, P.O. Box 67, Dschang, Cameroon

^IVLaboratoire de Génétique et Biologie Cellulaire Bâtiment Fermat - Maison 4, Niveau 2, 45, Avenue des Etats-Unis, 78035 Versailles Cedex, University of Versailles St Quentin-en-Yvelines, France

^VDepartment of Pharmaceutical Sciences and Traditional Pharmacopeia, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, P.O. Box 8664, Yaoundé, Cameroon

ABSTRACT

Chemical investigation of the stem bark of Ficus glumosa (Moraceae) yielded two new ceramides (2R,7E)-2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyhexadecan-2-yl]hexacos-7-enamide and (2R)-N-{(2S,3S,4R,9Z)-1-O-[(β-D-glucopyranosyl]-3,4-dihydroxyheptadec-9-en-2-yl}-2-hydroxypentacosanamide together with twenty one known compounds. The structures were established using NMR data, mass spectrometry, chemical transformation and by comparison with the reported data. Twenty one compounds were further tested against the prostate cancer PC-3 cell line and six of them revealed cytotoxic effect. Dongnoside E was the most active compound with an IC₅₀ 0.75 µmol L^-1 against the cancer cells line PC-3 while the reference drug doxorubicin displayed 0.91 µmol L^-1. This compound also proved to inhibit the cell growth of the fibrosarcoma cancer HT1080 (IC₅₀ 0.7 µmol L^-1).

Keywords:Ficus glumosa, ceramides, glumoamide, glumoside, dongnoside E, cytotoxicity

RESUMO

A investigação química da casca do caule de Ficus glumosa (Moraceae) deu origem a duas novas ceramidas (2R,7E)-2-hidróxi-N-[(2S,3S,4R)-1,3,4-trihidróxihexadecano-2-il]hexacos-7-enamida e (2R)-N-{(2S,3S,4R,9Z)-1-O-[(β-D-glucopiranosil]-3,4-dihidróxiheptadec-9-eno-2-il}-2-hidróxipentacosanamida, em conjunto com vinte e um compostos conhecidos. As estruturas foram estabelecidas usando-se dados de RMN, espectrometria de massas, transformação química e por comparação com dados relatados. Vinte e um compostos foram posteriormente testados contra células de câncer de próstata PC-3 e seis deles revelaram efeito citotóxico. Dongnósido E foi o composto mais ativo, com IC₅₀ de 0,75 µmol L^-1 contra células de câncer PC-3, enquanto o medicamento de referência doxorrubicina, apresentou IC₅₀ de 0.91 µmol L^-1. Este composto também demonstrou inibir o crescimento de células do câncer de fibrossarcoma HT1080 (IC₅₀ 0,7 µmol L^-1).

Introduction

Ficus glumosa is a small to medium-sized tree with 5 to 10 m tall; it may become a large tree reaching 20 m with a trunk of about 2 m of girth. Ficus species also produce white latex like commonly see in the Moraceae.¹ In Ivory Coast, the aqueous decoction of the leaves of F. glumosa is prescribed in traditional pharmacopeia to relieve chest pain and to treat lung diseases such as bronchitis, pneumonia and cough.² The aqueous decoction of its stem bark is rather used in Central African Republic as oral solution to cure gingivitis, caries, and tooth aches.³ Additionally, the methanolic extract of the stem bark of F. glumosa has demonstrated in vivo antidiabetic and in vitro antioxidant activities.⁴ The positive reactions of the diluted crude extract to FeCl₃, Liebermann Burchard, and Molish reagent associated to the traditional uses prompted us to look for the secondary metabolites of this plant and to evaluate their cytotoxicity.

We herein report the structure elucidation of two new ceramides and the cytotoxicity of some compounds isolated from F. glumosa.

Experimental

General procedure

Melting points (mp): electro thermal IA 9000 apparatus, uncorrected; optical rotation: JESCO P-2000 polarimeter ; IR (KBr disc): JASCO A-302 spectrophotometer; HR-EI-MS and LR-EI-MS: JOEL MS and FLINNIGAN MAT SSQ-700 apparatus, respectively; 1 and 2D NMR: Brüker AM-300 and DRX-400 MHz with TMS as internal reference. Preparative TLC and analytical TLC plates silica G60 type MERCK. Columns chromatography: normal phase SiO₂ 0.063-0.200 mm, reverse phase silica RP18.

Plant material

The stem bark of F. glumosa was collected in March 2009 in Makenene, central region of Cameroon and identified by the national herbarium where a specimen was deposited under the registration 28151/SRF/Cam (Makenene 1972).

Extraction and isolation

Air-dried powder (5 kg) of the stem bark of F. glumosa was macerated in a CH₂Cl₂-MeOH (1:1, 6 L) mixture for 48 h. The diluted extract was concentrated under reduced pressure to afford 405 g of a dark residue which was dissolved in water and extracted successively with hexane (Hex), CH₂Cl₂ (DCM), ethyl acetate (EA) and n-butanol to give fractions A (12 g), B (15.3 g), C (20 g), and D (89.4 g), respectively. Fraction A was subjected to silica gel column chromatography (CC) eluted in the gradient conditions with Hex, Hex-DCM and DCM-EA, affording compounds 3 (3.5 mg), 16 (3.7 mg), 4 (4.0 mg), 5 (4.0 mg), 6 (3.2 mg), and 7 (5.3 mg), in order of elution. Compounds 1 (7.5 mg) and 2 (6.8 mg) were precipitated with acetone in the sub-fractions eluted with DCM-EA 19:5 and 9:1, respectively. Fraction B was subjected to silica gel CC and eluted in the gradient conditions with Hex-DCM, DCM and DCM-MeOH to give compounds 8 (5.4 mg), 9 (5.1 mg), 10 (6.2 mg), and 12 (4.5 mg). SiO₂ CC of fraction C, eluted with a gradient from Hex-EA to EA, afforded 15 (6 mg), 11 (5.5 mg), 13 (6.3 mg), 14 (5.7 mg), 22 (4.6 mg) and 19 (10 mg). Fraction D was loaded on reverse phase silica RP18 CC and eluted with a gradient H₂O-MeOH, yielding 20 (3.1 mg), 21 (3.8 mg), 18 (5.4 mg), 23 (12 mg), and 17 (7 mg).

(2R,7E)-2-Hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxy­hexadecan-2-yl]hexacos-7-enamide (1)

White powder; mp 127.0-127.3 °C; R_f (2/3, DCM-MeOH 19:1); [α] +10.5 (c 0.03, THF); IR (KBr disc) ν_max/cm^-1: 3330, 3203, 2918, 2850, 1620, 1544, 1263; HR-ESI-MS m/z 682. 6319 [M + H]⁺, 668.6182 [M – CH₂ + H]⁺, 654.6033 [M – C₂H₄ + H]⁺, 640.5844 [M – C₃H₆ + H]⁺; ¹H and ¹³C NMR see Table 1.

Acetylation and oxidative cleavage of 1

An amount of 3 mg of 1 was dissolved in 3 mL of pyridine and treated with 3 mL acetic anhydride under magnetic stirring for 6 h at room temperature. The acetylated compound was obtained by concentration of medium under vacuum. Comparative TLC revealed the formation of a non polar compound which was further dissolved in 4 mL of THF-H₂O (9:1) and treated with 5 equiv. of KMnO₄ and NaIO₄ each. The medium was stirred at room temperature overnight and poured onto water. The organic layer obtained by extraction with n-butanol was subjected to de-acetylation using MeONa in MeOH overnight at room temperature. The medium was concentrated under vacuum, treated with aqueous solution of 1 mol L^-1 HCl and extracted with EA yielding compound 1a which was characterized by ESI-MS which gave the pseudo-molecular ion at m/z 448.

(2R)-N-{(2S,3S,4R,9Z)-1-O-[( β -D-glucopyranosyl]-3,4-dihydroxyheptadec-9-en-2-yl}-2-hydroxypentacosanamide (2)

White powder; mp 199.6-199.8 °C; R_f (2.5/3, DCM-MeOH 9:1) [α] +17 (c 0.035, THF); IR (KBr disc) ν_max/cm^-1: 3402, 1620, 1544; HR-ESI-MS m/z 844.6897 [M+H]⁺; ¹H and ¹³C NMR see Table 1.

Methanolysis of 1 and 2

An amount of 1.5 mg of 1, and 2 mg of 2 were separately refluxed in 2 mL of MeOH and 2 mL of aqueous solution of 1 mol L^-1 HCl for 18 h under magnetic stirring at 70 °C. An aqueous solution of NaHCO₃ was used to neutralize the medium which was further extracted with DCM. The organic fractions were purified by CC on silica gel eluted with Hex-EA (4:1). The residual oil of both compounds were identified by EI-MS from peaks at m/z 424 [C₂₇H₅₂O₃]^+. and 426.3 [C₂₇H₅₄O₃]^+., corresponding to the fatty acid methyl esters (2R)-2-hydroxyhexacos-7-enoic acid methyl ester 1b and (2R)-2-hydroxypentacos-7-enoic acid methyl ester 2a.

Disulfenylation of 2

To a solution of 2 (1 mg) dissolved in toluene (2 mL) were added 5 mg of FeCl₃ and 2 mL of dimethyl disulfide; the reaction mixture was stirred at room temperature and monitored by TLC. After 24 h, the medium was concentrated under reduced pressure and the residue was treated with a saturated aqueous solution of NaHCO₃ and extracted with EA. The colorless amorphous solid 2b obtained was analyzed by EI-MS.

Cytotoxicity assays

The cytotoxicity of the compounds was evaluated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)⁵ assay for the prostate cancer PC-3 cell line and flow cytometry for the fibrosarcoma cancer HT1080 cell line.⁶ The compounds were dissolved with DMSO and incubated with the cells for 48 h. The drug references used were doxorubicin and etoposide for PC-3 and HT1080, respectively. Furthermore, the IC₅₀ were calculated to evaluate the cytotoxicity of each natural product. The concentration of sample required to inhibit 50% of the cell proliferation (IC₅₀) was calculated from a calibration curve by a linear regression⁷ using Microsoft Excel. Three independent experiments are carried out for each sample.

Results and Discussion

The crude organic extract was subjected to repeated columns chromatography yielding two new ceramides (1, 2) together with twenty one known compounds namely lanosta-7,24-dien-3-one (3),⁸ lanosta-8,24-dien-3-one (4),⁸β-amyrine (5),⁹ lupeol (6),⁹ 6-prenylpinocembrin (7),¹⁰ bergapten (8),¹¹ chiricanine A (9),¹² genistein (10),¹³ wighteone (11),¹³ 6-prenylapigenin (12),¹⁴ 3,4-dihydroxybenzoic acid (13),¹⁵ 2,4,5-trihydroxybenzoic acid (14),¹⁶ alpinumisoflavone (15),¹⁷ 4'-O-methylalpinumisoflavone (16),¹³ luteolin (17),¹⁸ catechine (18),¹⁹β-sitosterol-3-O-(6'-O-heptadecanoyl)-β-D-glucopyranosyl (19),²⁰ polystachyol (20),²¹ lyoniresinol-2a-O-β-D-xylopyranoside (21),²¹β-sitosterol-3-O-β-D-glucopyranoside (22),²² and dongnoside E (23).²³

Compound 1 was obtained as a white powder. Its positive mode HR-ESI-MS spectrum gave a pseudo-molecular ion at m/z 682.6319 (calc. 682.6344) corresponding to the molecular formula [C₄₂H₈₄O₅N+H]⁺ accounting for 2 double bond equivalents. The IR spectrum exhibited characteristic absorption bands for free OH group (3330 cm^-1), olefinic function (1649 cm^-1) and secondary amide (3203, 1620 and 1544 cm^-1). The ¹H and ¹³C NMR spectra (Table 1) of 1 displayed a triplet of 6H at δ_H/δ_C 0.82, (t, 6.3 Hz)/14.0 assigned to the two terminal CH₃ groups, a broad singlet at δ_H/δ_C 1.10-1.30/(29.3-29.6) corresponding to a sequence of CH₂ groups, an exchangeable nitrogen-attached proton appearing as doublet at δ_H 7.38 (J 8.7 Hz) and a proton geminated to the amide at δ_H/δ_C 4.03 (m)/51.8. Furthermore, an oxymethylene group was observed at δ_H/δ_C 3.66 (dd, 4.8, 11.4), and 3.75 (dd, 4.2, 11.7)/61.3, along with three oxymethine groups at δ_H/δ_C 3.44 (br s) 75.8, 3.47 (br s)/72.4, and 3.98 (dd, 3.6, 8.4)/72.0, and a crbonyl at δ_C 175.5 suggesting a phytoceramide structure.²⁴ The ceramide core was confirmed by long-range correlations exhibited on the HMBC spectrum between the proton at δ_H 7.38 and the carbonyl at δ_C 175.5. Moreover, the proton of oxymethine group at δ_H 3.47 (H-4) correlated with the carbons at δ_C 61.3 (C-1), 51.8 (C-2), and 75.8 (C-3) which matched with a trihydroxylamine-sphingosine in the phytoceramide skeleton. The sphingosine was attached to a α-hydroxyfatty acyl by an amide function since both protons at δ_H 4.03 (H-2) and 3.98 (H-2') showed correlations with the carbonyl at δ_C 175.5.

The resonances of two overlapped olefin protons in trans-geometry²¹ were further observed at δ_H/δ_C 5.34 (br s)/129.7 and 5.34 (br s)/130.8 and located in the fatty acid chain, as deduced from the mass spectrum of the methanolysis product of 1, which displayed a molecular ion at m/z 424 [C₂₇H₅₂O₃]^+.. The position Δ^7' of this double bond was assigned from its oxidative cleavage (1a) which formed a fragment with a pseudo-molecular ion at m/z 448 [M+H]⁺ (Scheme 1). NOESY correlations and comparison of ¹H and ¹³C NMR data with those of the literature allowed to deduce the relative and absolute configuration at C-2, C-3, C-4, and C-2' to be (S), (S), (R), and (R) respectively.²⁴ The foregoing data led to identify 1 as (2R,7E)-2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyhexadecan-2-yl]hexacos-7-enamide, trivially named glumoamide.

Compound 2 was isolated as a white solid. Its molecular formula C₄₈H₉₃NO₁₀ was determined on the basis of HR-ESI-MS which displayed a pseudo-molecular ion peak at m/z 844.6897 [M+H]⁺, and the NMR data (Table 1) accounting for three double bond equivalents. The presence of OH and NH functions with absorption bands on the IR spectrum at 3402, 1620 and 1544 cm^-1 was characteristic of cerebrosides.²⁵

This compound gave a positive test to Molish reagent indicative of glycosides. The NMR spectra of 2 presented similar signals to those of 1, with additional shifted resonances at δ_H/δ_C 4.22 (d, 7.6)/102.9, 3.21 (br d, 7.2)/73.2, 3.36 (t, 8.0)/76.1, 3.36 (t, 8.0)/69.5, 3.22 (br s)/76.0, 3.69 (m), and 3.78 (br d, 11.2)/61.1 characteristic of a β-D-glucopyranosyl moiety.²⁵ Methanolysis of 2 led to a saturated fatty acid methyl ester (2a) identified by the EI-MS molecular ion at m/z 426 ([C₂₇H₅₄O₃]^+.). The position of the double bond observed on the NMR spectra at δ_H/δ_C 5.27 (2H, br s)/129.2, 130.1 was determined to be Δ¹⁰ from the ESI-MS of the sulfenylated derivative (2b), which exhibited a pseudo-molecular ion at m/z 145 (Scheme 2). The olefin cis-geometry was assigned on the basis of the carbon chemical shifts of allylic methylene groups at δ_C 27.0 and 27.1 which are around 33 ppm in the trans configuration.²⁵ Further correlation between the anomeric proton at δ_H 4.22 and the carbon at δ_C 68.6 allowed to connect the sugar moiety at C-1. The relative configuration was deduced by NOESY correlations and the absolute configuration at C-2, C-3, C-4, and C-2' was determined as being (S), (S), (R), and (R) according to the ¹H and ¹³C NMR data compared with those of the literature.^{24, 25} The above mentioned data led to characterize 2 as (2R)-N-{(2S,3S,4R,9Z)-1-O-[(β-D-glucopyranosyl]-3,4-dihydroxyheptadec-9-en-2-yl}-2-hydroxypentacosanamide, trivially named glumoside.

The structures of known compounds were identified by using their NMR data and by comparison of those reported in the literature.

Some of compounds were tested against the prostate cancer PC-3 cells (Table 2). Dongnoside E (23) showed a significant antiproliferative activity with an IC₅₀ of 0.75 µmol L^-1, while the value obtained with the reference drug doxorubicin was 0.91 µmol L^-1. In contrast, lanosta-7,24-dien-3-one (3), β-amyrine (5), lupeol (6), 6-prenylapigenin (12) and luteolin (17) showed moderated activities, whereas other compounds were not active. Compound 23 was further tested against fibrosarcoma cancer HT1080 cells lines and presented an interesting antiproliferative cells growth with 0.7 µmol L^-1 as IC₅₀ after 48 h. This natural product was more active than the reference drug etoposide which showed an IC₅₀ 1 µmol L^-1.

Conclusions

The higher concentration of flavonoids, benzoic acid derivatives, saponine, steroids and triterpenes highlights the use of this plant in traditional remedy against lung diseases, since several compounds belonging to the above-mentioned classes are present in plants having antitussive and expectorant activities.²⁶ Besides, some polyphenols are active against viral respiratory infections, bacteria and fungi.²⁷ The foregoing results clearly indicated that Ficus glumosa is a promising source of drug development, and 23 can be a potential antitumour lead.

Acknowledgments

The authors thank T. W. A. S. (The Academy of Sciences for the Developing World) for the fellowship providing to Frederic Nana.

Supplementary Information

Spectra (1D/2D NMR and MS) and NMR data of compounds (1-23) are available free of charge at http://jbcs.sbq.org.br as PDF file.

Submitted: September 7, 2011

Published online: January 24, 2012

[Supplementary material]

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Publication Dates

• Publication in this collection
  04 Apr 2012
• Date of issue
  Mar 2012

History

• Received
  07 Sept 2011
• Accepted
  24 Jan 2012