Identification of compounds with anti-proliferative activity from the wood of Ficus elastica Roxb. ex Hornem. aerial roots

    Identification of compounds with anti-proliferative activity from the wood of Ficus elastica Roxb. ex Hornem. aerial roots Jean Emmanuel Mbosso Teinkela, Xavier Siwe Noundou, Edwige Laure Nguemfo, Franck Meyer, Alfred Djoukoue, Pierre Van Antwerpen, Silv`ere Ngouela, Etienne Tsamo, Emmanuel Albert Mpondo Mpondo, Juliette Cath´erine Vardamides, Guy Anathole Blaise Azebaze, Ren´e Wintjens PII: DOI: Reference:

S0367-326X(16)30100-9 doi: 10.1016/j.fitote.2016.05.002 FITOTE 3406

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

31 March 2016 4 May 2016 6 May 2016

Please cite this article as: Jean Emmanuel Mbosso Teinkela, Xavier Siwe Noundou, Edwige Laure Nguemfo, Franck Meyer, Alfred Djoukoue, Pierre Van Antwerpen, Silv`ere Ngouela, Etienne Tsamo, Emmanuel Albert Mpondo Mpondo, Juliette Cath´erine Vardamides, Guy Anathole Blaise Azebaze, Ren´e Wintjens, Identification of compounds with anti-proliferative activity from the wood of Ficus elastica Roxb. ex Hornem. aerial roots, Fitoterapia (2016), doi: 10.1016/j.fitote.2016.05.002

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ACCEPTED MANUSCRIPT

Identification of compounds with anti-proliferative activity from the wood of Ficus elastica

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Roxb. ex Hornem. aerial roots

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By Jean Emmanuel Mbosso Teinkela a,b,c,*, Xavier Siwe Noundou d, Edwige Laure Nguemfo b, Franck Meyer a, Alfred Djoukoue c,e,, Pierre Van Antwerpen f , Silvère Ngouela , Etienne Tsamo g, Emmanuel Albert Mpondo Mpondo e, Juliette Cathérine Vardamides c,

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g

a

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Guy Anathole Blaise Azebaze,c and René Wintjens a Laboratory of Biopolymers and Supramolecular Nanomaterials, Faculté de Pharmacie,

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Université Libre de Bruxelles (ULB), Campus Plaine (CP 206/4), Boulevard du Triomphe, B1050 Brussels, Belgium

Département des Sciences Biologiques, Faculté de Médecine et des Sciences Pharmaceutiques

D

b

Department of Chemistry, Faculty of Sciences, University of Douala, P.O. Box. 24157, Douala,

Cameroon d

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c

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(FMSP), Université de Douala, BP 2701 Douala, Cameroun

Nanomaterials and Medecinal Organic Chemistry Laboratory, Department of Chemistry,

Rhodes University, Grahamstown, 6140, South Africa. e

Département de Pharmacie, Faculté de Médecine et des Sciences Pharmaceutiques (FMSP),

Université de Douala, BP 2701 Douala, Cameroun f

Analytical Platform of the Faculty of Pharmacy, Faculté de Pharmacie, Université Libre de

Bruxelles (ULB), Campus Plaine (CP 205/5), Boulevard du Triomphe, B-1050 Brussels, Belgium g

Laboratoire de Substances Naturelles et Synthèse Organique, Département de Chimie

Organique, Faculté des Sciences, Université de Yaoundé I, BP 812 Yaoundé, Cameroun * Corresponding author: Dr. Jean Emmanuel Mbosso Teinkela, phone: +32 2 650 51 80; fax number: +32 2 650 59 29; e-mail

ACCEPTED MANUSCRIPT Abstract The natural products from plants still remain an important source of pharmaceuticals. In the

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current study, methanolic wood extracts of Ficus elastica Roxb. ex Hornem. (Moraceae) aerial roots were screened for anti-cancer activity. Using bioassay-guided fractionation, three new

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compounds, elasticamide (1), elastiquinone (2) and ficusoside B (3), together with four known

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compounds, were isolated. The structures of the new compounds were established by means of extensive spectroscopic analyses (1D and 2D NMR, HR-ESI-MS, as well as IR and UV) and by

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comparison of their spectroscopic data with those reported for structurally related molecules. The isolated compounds were afterwards evaluated for their anti-proliferative effect on six human cancer cell lines (U373n and Hs683 glioblastoma, MCF7 and A549 NSCLC carcinoma, and

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B16F10 and SK-MEL-28 melanoma) using colorimetric MTT assay. Most notably, elastiquinone (2) showed cytotoxic activity with IC50 = 14 µM against B16F10 melanoma cells, whereas the

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peracetylated form of ficusoside B (3a) displayed the lowest IC50 value (11 µM) against U373n

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glioma cell lines. Elasticamide (1) and ficusoside B (3) exhibited a weak cytotoxicity with IC50 values ≥ 88 µM. The results of this investigation demonstrate that the wood of F. elastica aerial

activity. Keywords:

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roots might be a potential source for identifying new compounds with potent anti-proliferative

ACCEPTED MANUSCRIPT 1. Introduction Every year, approximately seven million people suffer from cancer, making this disease responsible for at least 12% of deaths worldwide

. In the quest of new molecules with anti-

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cancer activities, compounds isolated from plants represent an important source of potential drugs, as already demonstrated in the past with vincristine, irinotecan, etoposide and paclitaxel

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. Despite the successful discovery of naturally occurring drugs, the search for new anticancer

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agents is still necessary. This activity could increase the number of available chemical building blocks and also enable to discover less or non-toxic and more effective ones. Approximately 6%

ones have been phytochemically evaluated

.

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of the world’s plants have been screened for biological activities and only 15% of the screened

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The plants investigated for medicinal use include the members of the Moraceae family where the genus Ficus is well documented for its biological activities such as antioxidant

,

anticancer

,

, antimicrobial , and gastroprotective

, antiplasmodial

, anti-pyretic

. The latex of some species of Ficus is used in traditional

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antiulcer

antidiarrhoeal

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folk medicine for its anthelmintic property in Central and South America activity was attributed to the presence of ficin

. This parasiticidal

and it was demonstrated that latex of Ficus

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elastica Roxb. ex Hornem. (Moraceae) has a significant antischistosomal activity

.

In our ongoing search for bioactive compounds in Cameroonian plants of the Moraceae family, we focused herein on F. elastica (the rubber tree) which is a widely-spread evergreen tree of up to 30 m tall. Its leaves’ extract is used for the treatment of skin infections and skin allergies, as well as diuretic agent

. The phytochemical investigation of this taxon revealed the

presence of 6,10,14-trimethyl-2-pentadecanone (25.9%) and geranyl acetone (9.9%) as main compounds in the leaf oil

. The MeOH extract of the F. elastica leaves contains emodin,

sucrose, morin and rutin with antimicrobial activities

, and many others compounds with

antioxidant properties such as feroxidin, quercitrin, kaempferin, myricitrin, syringin, citroside B, corchoionoside

C,

(6S,9R)-roseoside,

oleanolic

acid,

ursolic

acid,

benzyl

O-β-D-

glucopyranoside, icariside F2, ficuselastic acid and (1'S,6'R)-8-O-β-D-glucopyranosyl sodium abscisate

.

In our previous studies on the Ficus genus, we isolated from the bark of aerial roots of F. elastica, n-alkanes, friedelin, friedelinol, linear aliphatic primary alcohols, linear fatty acids, phytosterols, betulinic acid, ursolic, acid, sitosteryl 3-O-β-D-glucopyranoside, a ceramide

ACCEPTED MANUSCRIPT (ficusamide), a cerebroside (ficusoside) and a saponin named elasticoside with anticancer and antimicrobial activities

.

In the present project, we have examined the methanolic wood extract of F. elastica aerial

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roots. This study deals with the isolation and structural characterization of three new molecules named elasticamide (1), elastiquinone (2) and ficusoside B (3) together with four known

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compounds. Anti-proliferative testing was carried out on the methanolic extract, varied fractions

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and isolated compounds. To the best of our knowledge, this is the first report on the wood of F.

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elastica aerial roots.

Melting

points

were

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General

determined

on

an

American

Optical

(Reichert)

Forty

Stereomicroscope. UV spectra were recorded on Shimadzu-2401 PC spectrophotometer. IR

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spectra were obtained with a Perkin–Elmer 881 infrared spectrophotometer. Optical rotation was

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measured with a polarimeter (ATAGO AP 100). 1D and 2D NMR spectra were recorded using two spectrometers (Bruker Avance 300 and Varian Inova 400), respectively, in CDCl3 and

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CD3OD using the residual isotopic solvent CHCl3 and CH3OH, as references for δH = 7.26 ppm, δC = 77.16 ppm, δH = 3.31 ppm, δC = 49.00 ppm. Records of high-resolution mass spectra (positive mode) were obtained with direct infusion in a 6520 series Electrospray Ion Source (ESI)-Quadrupole Time-Of-Flight (Q-TOF) mass spectrometer (Agilent, Palo Alto, CA, USA). The difference between the observed mass was expressed in ppm. When this difference was below 3 ppm, the molecules were considered to have the predicted formula. Extracts were purified using column chromatography (SiO2, Merck 230-400 mesh and 70230 mesh). Thin layer chromatography was performed using aluminium silica gel sheets (60 F254). After spraying with an ethanolic solution (0.1% ethanol) combined with berberin HCl, spots were visualized under ultraviolet light (λ=254 and 365 nm) and dried at 100 °C. The chemical structures of isolated known compounds were determined by comparing their spectra with reported data (after dilution in chloroform, i.e. 0.5 mg in 2 mL of CHCl3). The extracts were tested for phytochemical composition using the methods described by Harborne

2.2. Plant material

.

ACCEPTED MANUSCRIPT The wood of F. elastica aerial roots was collected from Melen-Yaoundé (Cameroon) in December 2007 and stored in a fridge at -20°C. The plant’s identification was established by a member of the National Herbarium of Cameroon (NHC), where a voucher specimen (No. 65646

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HNC) was deposited. After Air-drying, the plant material (5.50 kg of the wood of F. elastica aerial roots) was crushed into a fine powder by using an electric grinder.

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2.3. Extraction and isolation

temperature (27 ± 2°C)

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Macerate of the dried aliquot was obtained using 25 L of methanol twice for 48 h at room . After filtration (Whatman Number One) and evaporation at low

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pressure in a rotary evaporator (bath at 40°C), 15 g of extract was obtained. Concentrated extracts were obtained by drying at 40°C in a hot air oven. The dried extract was packaged in air

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proof containers and stored in a refrigerator at 4°C until further use. The crude MeOH extract (14.5 g) was subjected to silica gel column chromatography (CC) (cyclohexane/EtOAc/MeOH gradient of increasing polarity) to afford four fractions, FEBr1 to FEBr4 on the basis of TLC

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composition. The two most active fractions according to anti-proliferation assays were further

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subjected to silica gel CC to isolate the active components. Fraction FEBr2 (1.2 g) was eluted with cyclohexane/EtOAc and CHCl3/MeOH to yield a mixture of linear aliphatic alkanes (10

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mg) with n-hexacosane as major compound (NMR spectroscopy) at cyclohexane/EtOAc (95:5, v/v), β-sitosterol (10 mg) at cyclohexane/EtOAc (9:1, v/v), elasticamide 1 (2 mg) and elastiquinone 2 (4 mg) at CHCl3/MeOH (98:2, v/v), sitosteryl 3-O-β-D-glucopyranoside (3 mg) at CHCl3/MeOH (96:4, v/v)

. Fraction FEBr3 (2.3 g) underwent another CC, eluted with

cyclohexane and gradient of CHCl3/MeOH to afford a mixture of linear aliphatic alkanes (2.5 mg) at cyclohexane 100%, elasticamide 1 (4.8 mg) at CHCl3/MeOH (96:4, v/v), sitosteryl 3-O-βD-glucopyranoside (32 mg) at CHCl3/MeOH (95:5, v/v), ficusoside B 3 (25.7 mg) at CHCl3/MeOH (9:1, v/v) and biochanin A 4 (PubChem CID 5280373) (4.7 mg) at cyclohexane/EtOAc (98:2, v/v).

2.4. Data of the isolated compounds 2.4.1. Elasticamide Colorless solid, mp 121-122°C. [α]24D= +17.2 (CHCl3; c 0.4). 1H NMR, (CDCl3/CD3OD 1:1, 300 MHz) and

13

C NMR (CDCl3/CD3OD 1:1, 75 MHz) data, see Table 1; HRESIMS m/z

684.6471 [M+H]+ (calcd for C42H86NO5, 684.6505).

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2.4.2. Elastiquinone Orange powder (MeOH); mp 187–189°C; UV (CHCl3) λmax (log ) 230 (3.31), 252 (3.40),

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287 (3.35) and 442 (3.59) nm; IR (KBr) max 1563, 1614, 3418 cm-1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, see Table 2; HR-ESI-MS m/z 337.1126 [M+H]+

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(calcd for C20H17O5, 337.1075). 2.4.3. Ficusoside B

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Colorless solid; mp 146-148°C. [α]24D: -9.0 (CH3OH; c 0.22). 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, see Table 3; HR-ESI-MS m/z 738.5452 [M+Na]+

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(calcd for C40H77NO9Na, 738.5495). 2.4.4. Acetylation of Ficusoside B

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Dry pyridine (0.5 mL) and Ac2O (0.5 mL) were added to compound 3 (2 mg) and left H NMR and HR-ESI-MS.

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1

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overnight. The usual workup yielded 3a (3.5 mg) which was identified without ambiguity with

2.4.5. Ficusoside B hexaacetate

Colorless solid; mp 49-51 °C. [α]24D= +11.7 (CHCl3; c 0.2 ). 1H NMR (CDCl3, 400 MHz): δH 0.90 (6H, t, J = 6.3 Hz, H-16’, H-17), 1.21-1.36 (42H, br. s, H-4’-7’, H-12’-15’, H-4-16), 1.62 (2H, m, H-3), 1.85 (2H, m, H-3’), 2.09 (4H, m, H-8’, H-11’), 2.05-2.13 (18H, s, 6-OAc), 4.03 [1H, dd, J = 3.0, 11.7 Hz, CH2(a)], 4.36 (1H, dd, J = 6.3, 11.7 Hz, CH2(b), 4.97 (1H, dd, J = 2.7, 12.0 Hz, H-2’), 5.12 (1H, m, H-2), 5.39 (1H, m, H-1), 4,48 (1H, d, J = 7,9, H-1”), 3.64 (1H, m, H-2”), 4.87 (1H, m, H-3”), 4.91 (1H, m, H-4”), 5.13 (1H, m, H-5”), 4,48 (1H, dd, J = 2.2, 12.4, Ha-6”), 4,48 (1H, dd, J = 4.4, 12.4, Hb-6”); HR-ES-IMS m/z 990.6142 [M+Na]+ (calcd for C52H89NO15Na, 990.6129). 2.5. Determination of in vitro anticancer activities The in vitro growth inhibitory activities on cancer cell lines of the wood methanolic extract of F. elastica aerial roots, fractions FEBr1-FEBr4, biochanin A (4), elasticamide (1), elastiquinone (2), ficusoside B (3) and peracetylated of 3 (3a), were determined with a 3-[4,5]-

ACCEPTED MANUSCRIPT dimethylthiazol-2yl-diphenyl tetrazolium bromide (MTT, Sigma, Belgium) assay . MTT assay allows the determination of the in vitro concentration that reduces by 50% (IC50) the

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growth or proliferation of cancer cells after having cultured the cells in our case for 72 h compared to control. Six human cancer cell lines with various levels of resistance to pro-

carcinoma

the MCF7 and

and the B16F10 and SK-MEL-28 for melanoma

.

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A549 NSCLC

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apoptotic stimuli were used, i.e. the U373n and Hs683 for glioblastoma

The cells were cultured in RPMI (Invitrogen, Merelbeke, Belgium) media supplemented with

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10% heat inactivated fetal calf serum (Invitrogen). All culture media were supplemented with 4 mM glutamine, 100 μg/mL gentamicin, and penicillin-streptomycin (200 U/mL and 200 μg/mL,

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respectively) (Invitrogen). Briefly, MTT viability assay is based on the capacity of living cells to reduce MTT into purple formazan crystals within their mitochondria. After dissolution of the crystals formed in dimethylsulfoxide, optical densities are measured by a plate reader at 570 nm

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(Biorad 680XR, Nazareth, Belgium) with a reference wavelength lecture at 630 nm. The assay

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was performed once in sextuplicate on each cell line over a 72 h period of incubation with the

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compound/extract at nine concentrations (from 0.01 to 100 μg/mL, with semilog increases).

3. Results and Discussion

The CHCl3/MeOH 1:1 crude extract of wood of F. elastica aerial roots was subjected to silica gel column chromatography. Some fractions with similar TLC patterns were combined to yield a total of four fractions, denoted FEBr1 to FEBr4. The most active fractions according to the MTT cell proliferative assay (FEBr2 and FEBr3) were further purified by silica gel column chromatography (CC). The purification of FEBr2 yielded a mixture of linear aliphatic alkanes with n-hexacosane as major compound (NMR spectroscopy), β-sitosterol 3-O-β-D-glucopyranoside A

and sitosteryl

. FEBr3 afforded a mixture of linear aliphatic alkanes, biochanin

, and sitosteryl 3-O-β-D-glucopyranoside

. In addition, three new products, i.e.

elasticamide (1) and elastiquinone (2) were isolated from FEBr2 while elasticamide (1) and ficusoside B (3) were from FEBr3. Their structures were elucidated by extensive spectroscopic methods including 1D and 2D NMR experiments as well as HR-ESI-MS analysis and comparison with reported data.

ACCEPTED MANUSCRIPT Elasticamide (1) was obtained as a colorless solid (m.p. 121-122°C). The positive mode HR-ESI-MS analysis showed a protonated molecular ion at m/z 684.6471 [M + H]+ (calcd for C42H86NO5+, 684.6505), which accounted for one degree of unsaturation. Subsequently, the 13

C NMR spectroscopy. The

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structure was fully elucidated by 1H and

13

C NMR spectrum

revealed a signal at δC 52.4 (C-N) and a carbonyl signal at δC 176.5, suggesting the presence of

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an amide group (Table 1). Thus, unsaturation of 1 could be readily assigned to a carbonyl group.

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The 1H NMR spectral (Table 1) of 1 displayed a broad signal in the range of δH 1.22-1.35 (brs, CH2 group) and a triplet at δH 0.86 (6H; J = 6.3 Hz, two terminal CH3 groups) assigned to two

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long aliphatic chains. Finally, the presence of two carbinol H-atoms at δH 3.73 (dd, J = 12.6 Hz, J = 4.4 Hz) and 3.79 (dd, J = 12.6 Hz, J = 4.4 Hz) made compound 1 consistent with the structure .

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of a ceramide

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δ

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δ

δ

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Detailed 1H NMR analysis confirmed the presence of five protons geminal to the hydroxy

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groups at δH 3.53 (m, H-2’), 3.73 (dd, J = 12.6 Hz, J = 4.4 Hz), 3.79 (dd, J = 12.6 Hz, J = 4.4 Hz), 3.53 (m, H-2) and 4.02 (m, H-3). Further confirmation of these hydroxy functionalities was made by a peracetylation reaction with acetic anhydride, yielding the tetraacetate 1a. A

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comparison of 1H NMR spectra of 1 and 1a suggested that 1 contains four free hydroxy groups

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on the backbone, due to the presence of four CH3CO fragments at δH 2.05, 2.08, 2.10, 2.20. On the 1H NMR spectrum of 1a, a fifth signal at δH 4.13 (m, H-1) was also identified as a methine

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proton vicinal to nitrogen atom. These results were corroborated by the 13C and DEPT 135 NMR spectra where a signal at δC 176.5 attributed to the amide carbonyl, four methines at δC 76.1 (CHOH), 72.6 (CHOH), 73.0 (CHOH) and 52.4 (CHNH) and a methylene group at δC 61.8 (CH2OH) were depicted. The length of the fatty acid chain (FAC) was determined by MS/MS, which emphasized a significant fragment ion peak at 384.3814 [CH3(CH2)21CH(OH)CONH3]+. The length of the long chain base (LCB) was elucidated by the characteristic ion at m/z 300.2865 [CH2-CH(NH2)CH(OH)CH(OH)(CH2)13CH3]+

and

318.2935

[HO-CH2-

+

CH(NH)CH(OH)CH(OH)(CH2)13CH3] . The typical fragment ion at m/z 666.6371 was formed by elimination of a hydroxyl group from [M]+. The positions of the hydroxyl functions at C-2 and C-3 were ascertained by the mass fragmentation pattern (Fig. 1), as usually in other ceramides and especially from 1H-1H COSY (Fig. 1) and HMBC spectra (Fig. 2).

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282.2778 O 1'

300.2865

OH 3'

NH OH 2 1 4 HO

17

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HO

24'

666.6371

318.2935

MS-MS

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COSY

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-H2O

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384.3814

OH O

24'

17

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H H H OH

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H H OH

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HO

HN H

3'

The absolute configuration of the chiral centers at C1, C2, C3, and C2’ could not be precisely established without chemical transformations that would require much more material . However, considering the biogenesis and steric hindrance of sphingolipids, the chemical shift of the H-2 signal and the chemical shifts of C-1 to C-4, C-1’ and C-2’ are generally acknowledged to determine the absolute configuration of the phytosphingosine

. The

chemical shifts of C-atoms at δC 62.8 (CH2OH), 52.4 (C-1), 76.1 (C-2), 72.6 (C-3), 176.5 (C-1’), and 73.0 (C-2’) in 1 were closely identical with those of (2R)-2-hydroxy N-[(1S,2S,3R)-1,2,3trihydroxypentadec-8-en-2-yl] tetracosanamide

. By similarity of the NMR data, the

absolute configuration at C-1, C-2, C-3, and C-2’ was inferred to be (S), (S), (R) and (R). In consequence, the LAC and the FAC of 1 correspond to 1-amino-[(1S,2S,3R)-1-(hydroxymethyl)2,3-dihydroxy-heptadecane

and

(2R)-2-hydroxy-hexadecanoic

acid,

respectively.

Thus,

ACCEPTED MANUSCRIPT compound 1 was determined to be (2R)-2-hydroxy N-[(1S,2S,3R)-1-(hydroxymethyl)-2,3dihydroxy-heptadecanyl] hexadecanamide and the trivial name elasticamide was given. Elastiquinone (2) was obtained as an orange powder (m.p. 187–189°C) and showed a

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positive reaction to the Borntrager’s test, characteristic of anthraquinone derivatives. Compound 2 also positively reacted with FeCl3, indicating its phenolic nature. The positive mode HR-ESI-

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MS exhibited a protonated molecular ion at m/z 337.1126 [M + H]+ (calcd for C20H17O5+,

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337.1075), accounting for 13 degrees of unsaturation. The UV spectrum showed specific absorptions with maxima at 230 (3.31), 252 (3.40), 287 (3.35) and 442 (3.59) nm. The IR

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spectrum exhibited absorption bands at 1614 and 1563 cm-1 due to the free and chelated carbonyl groups, respectively, and an important band at 3418 cm-1 due to the hydroxyl substituent. Both . The 13C

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UV and IR data indicated that compound 2 was a 9,10-anthraquinone derivative

NMR spectrum (Table 2) displayed 20 carbon signals which were classified by DEPT experiments into two methyl and one methoxy groups, six sp2 methines and eleven quaternary

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carbon atoms including two carbonyl moieties (δC 190.5, 181.4). These results indicated clearly . The 1H NMR data (Table 2) of 2 showed

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that the methoxy fragment was attached to C-9

the characteristic signals of the dimethylpyran ring [δH 6.68 (1H, d, J = 10.0 Hz), 5.72 (1H, d, J =

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10.0 Hz) and 1.46 (6H, s)].



δ



H NMR spectrum of 2 gave other signals, corresponding to one methoxy substituent [δH

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1

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ACCEPTED MANUSCRIPT

3.35 (3H, s)], and three aromatic protons [δH 8.05 (1H, s), 7.53 (1H, d, J = 8.2 Hz) and 6.78 (1H, d, J= 8.2 Hz)]. Pertinent HMBC correlations of 2 (Fig. 3) were observed between the cis olefinic proton at δH 6.68 (H-4) and C-2 (δC 78.2), C-5 (δC 157.7), C-12a (δC 160.0), and between the olefinic proton at δH 5.72 (H-3) and C-2 (δC 78.2), C-4a (δC 106.0), establishing thus the fusion of the dimethylpyran ring at C-4a and C-12a with an ether linkage at C-12a.

H

OH O

H

H H3C H2C H

H O H

O

H

O CH3

ACCEPTED MANUSCRIPT The HMBC correlations observed between the aromatic protons at δH 8.05 (H-10) and C12 (δC 181.4), C-9 (δC 157.8), C-8 (δC 115.2), at δH 7.53 (H-7) and C-6a (δC 115.1), C-8 (δC 111.2), C-10a (δC 123.6), C-9 (δC 157.8) and at δH 6.78 (H-8) and C-6a (δC 115.1), C-9 (δC

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157.8), C-10 (δC 122.1) respectively, clearly indicated that the methoxy group was attached to C9 (Fig. 3). On the basis of the above results, compound 2 was determined as 9-methoxy-5-

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dihydroxy-6,11-dioxo-6,11-dihydro-2H-anthra[2,3-b]pyrane, which was given the trivial name

OH O

4

H3C

4a 2

5a

6a

7

8

6

11 O 12a 12 11a 10a 1 O

9 10

O

CH3

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H3C

5

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3

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of elastiquinone (Fig. 4).

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Ficusoside B (3) was isolated as a colorless solid (m.p. 146-148°C). The positive mode HR-ESI-MS analysis showed a sodium adduct ion at m/z 738.5452 [M + Na]+ (calcd for

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C40H77NO9Na +, 738.5495). The existence of the two signals at δC 50.0 (C-N) and 177.1 (C=O) in the 13C NMR spectrum of (3) suggested the presence of an amide function (Table 3).

δ

δ

δ

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ACCEPTED MANUSCRIPT

Compound 3 gave a positive Molisch’s test, indicative of the presence of a sugar moiety. While the 1H NMR spectrum of 3 exhibited the anomeric proton signal at δH 4.29 (1H, d, J = 8.4 Hz), its

13

C NMR spectrum displayed a signal at δC 103.6 and a set of signals (δC 73.5, 76.3,

70.5, 76.4, and 61.8), consistent with a β-D-glucopyranoside [5]. The value of the coupling

ACCEPTED MANUSCRIPT constant between H-1” [δH 4.29 (1H, d, J = 8.4 Hz)) and H-2” (δH 3.17 (1H, dd, J = 4.4 Hz, J = 8.4 Hz)] corresponded to two trans diaxial protons, and suggested a chair conformation of the carbohydrate moiety. The C-1” and C-2” substituents occupied an equatorial position indicating

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the presence of a β-glycoside.

The 1H NMR spectrum provided additional information on the structure of 3, showing two

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aliphatic chains as evidenced by an intense signal at δH 1.30 (br s), and the triplet at δH 0.92 (t, J

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= 7.6 Hz). Moreover, two signals of olefinic protons at δH 5.37 (m) and 5.39 (m) matched well with a double bond. The three unsaturations of 3 could thus be readily assigned to a double bond,

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a carbonyl and a glucopyranosyl group. The Δ9’ double bond was found to be cis, as evidenced by the chemical shift of C-8’ at δC 27.0 and C-11’ at δC 26.3

. Furthermore, a CH2O

segment [δH 3.83 (Ha, dd, J = 4.0 Hz, J = 10.4 Hz) and δH 3.73 (Hb, dd, J = 4.0 Hz, J =10.4 Hz;

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δC 68.9)] and CH-O groups [δH 4.02 (m) and δH 3.60 (m); δC 71.9 and 73.9] were also observed. From the foregoing data, a polyunsaturated β-D-glucopyranosyl ceramide structure was 13

C NMR

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suggested for compound 3. This hypothesis was subsequently confirmed by the

glucose moiety (Fig. 5).

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spectrum and a MSMS analysis which further highlighted a peak at m/z 180.0634 due to the

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OH

H H

H H

O

3' 2' 1' H NH O H 12 HO 2" O HO 3 OH 1" 3" OH H H OH H 6"

16'

9' 10'

COSY

17

HMBC

The position of the glucose cycle at C-1 was evidenced by the downfield chemical shift observed for the hydroxymethylene carbon at δC 68.9

and further corroborated by the

HMBC spectrum in which the anomeric proton H-1” correlated with the C-atom C (Ha, Hb). Pertinent HMBC correlations (Fig. 5) were also observed between H-1” and C-2” and also between the multiple H-atom signals at 1.30 and the CH3 group at δH 0.92. H-1” displayed correlations with CHaHb, H-8’ with C-9’ and C-7’, H-11’ with C-10’ and C-12’. The COSY

ACCEPTED MANUSCRIPT spectrum (Fig. 5) exhibited correlations between proton H-1” and Ha, Hb and H-2”, H-2” and H3”, H-8’ and H-9’, H-9’ and H-10’ and H-10’ and H-11’. The former data suggest that the FAC is unsaturated while LCB side is saturated. The length of the long chain acid was determined by

254.2246 180.0634

147.1114

OR (CH2)4

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O OR NH

O RO RO

(CH2)4CH3

(CH2)14CH3

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O

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the loss of a characteristic ion at m/z 254.2246 [CH3(CH2)5CH=CH(CH2)6CH(OH)CO]+ (Fig. 6).

OR

OR

484.3229

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558.4885

D

576.4886

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The typical fragment ion at m/z 147.1114 was formed by elimination of [CH3(CH2)6CH=CH2]+ from [M]+, through a McLafferty rearrangement. This, together with the

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correlations described above, also confirmed the position of the double bond in the FAC. By NMR data comparison, the absolute configuration at C-1, C-2 and C-2’ was deduced to be (S), (S) and (R), respectively, identical to that of D-sphingosine on the basis of 13C NMR data, since the chemical shifts of C-1 (δC 50.0), C-2 (δC 73.9) and C-2’ (δC 71.9) were in agreement with those reported in the literature

. From

the foregoing data, the structure of 3 was characterized as (2R,9Z)-2-hydroxy N-[(1S,2S)-1-[(βD-glucopyranosyloxy)methyl]-2-hydroxy-heptadecanyl]-hexadec-9-enamide, which was given the trivial name of ficusoside B (Fig. 6). Subsequently, the biological properties of aforementioned samples were examined. Elasticamide (1) and ficusoside B (3) displayed some activity against all tested human cancer cell lines with a mean IC50 value higher than 100 μM for most of the cell lines (Table 4). In contrast, the other samples exhibited better cytotoxic activities against the human cancer cell lines with mean IC50 values in the range 5-8 μg/mL for the crude wood methanol extracts of F. elastica aerial roots FEBr and 11-83 μM for compounds 2, biochanin A and peracetylated derivative of 3 (3a). This latter product 3a (IC50=11 μM) showed the lowest IC50 value against

ACCEPTED MANUSCRIPT U373n, followed by compound 2 (IC50=14 μM) against B16F10. Biochanin A (Fig. 7), a natural plant isoflavone and phytoestrogen, has been reported to possess anti-carcinogenic, antiproliferative, antiallergic and anti-inflammatory properties, using both in vivo and in vitro

O

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HO

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.

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experiments

OH O

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OCH3

. Other

studies

showed

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It was also found to protect against L-glutamate-induced cytotoxicity and apoptosis in vitro that

this

molecule

inhibits

iNOS

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lipopolysaccharide (LPS)-induced nitric oxide production in macrophages with other published bioactivity results

expression

and

. In agreement

, biochanin A, isolated here from the fraction

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FEBr3, displayed a moderate anti-proliferative effect with IC50 in the range 47- 69 µM against

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the six tested cancer cell lines.

We carried out the acetylation of 3 (isolated from MeOH extract of F. elastica) and this afforded its peracetylated derivative 3a. A structure-activity relationship investigation was therefore performed on 3 and 3a to determine their antitumor activity on the studied cell lines. This study revealed that, ficusoside B hexaacetate (3a) is more active than ficusoside B (3). The peracetylated derivative (3a) exhibited anti-proliferative activity against cancer cell lines U373, MCF7, Hs683, A549 and SK-MEL-28, with IC50 of 11, 34, 48, 64 and 83 µM, respectively (Table 4).

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ACCEPTED MANUSCRIPT

More importantly, ficusoside B hexaacetate (3a) was the most active compound with an IC50 of 11 µM against the cells U373n of glioblastoma cancer. It has been reported that the peracetylation modification can significantly enhance the anti-proliferative activity

. It may

therefore be suggested that the anti-proliferative activity of compound 3 could be improved by the presence of acetyl groups. However, it cannot rule out that the overall anti-proliferative activity of the crude extract and fractions might be due to the presence of several antiproliferative compounds with moderate activities. Previous studies have already stated the presence of various compounds belonging to different classes in plants of the genus Ficus (Table 5). Several compounds were isolated from the leaves [4] and bark of aerial roots of F. elastica [5]. Here, we elucidated several chemical structures of molecules isolated from the wood of F. elastica aerial roots: a mixture of linear

ACCEPTED MANUSCRIPT aliphatic alkanes

similarly isolated from bark of aerial roots of F. elastica [5]; β-

sitosterol previously obtained from bark of aerial roots of F. elastica, from F. Bubu fruits, F. carica, F. sycomorus and F. salicifolia [5,6,56]; sitosteryl 3-O-β-D-glucopyranoside found in the

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bark of aerial of F. elastica and in F. Bubu fruits [5,6]; biochanin A already isolated from F. Bubu fruits [6]; and finally elasticamide 1, elastiquinone 2 and ficusoside B 3, reported here for

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the first time. Taken altogether, the present study increases our knowledge on phytochemical of

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Ficus elastica. Indeed, the extraction and the identification of seven different structures from the wood of F. elastica aerial roots represent the first phytochemical work on this plant part and may

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be used as foundation for further chemotaxonomic.

Source/reference F. microcarpa [49]

Ptiloepoxide Moretenolactone Rhoiptelenol 3-acetylursa-14-en-16-one Lanosta-8,24-dien-11-on-3-ol acetate 3-Acetoxycycloart-25-en-24-ol Campesterol

F. microcarpa [51] F. insipida [52] F. thunbergii [53]

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Apocarotenoïd γ-lactones derivatives

compound Ficuscarpanoside Guaiacylglycerol Ficusone Ficuspirolide Ficosoline Oleanolic acid Friedelin Friedelinol Betulinic acid Ursolic acid

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class Phenylpropanoïds

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Table 5 Class compounds previously reported from the Ficus genus

Triterpenes

Sterols

Glycosides

F. microcarpa [50] F. elastica [4] F. elastica [5] F. elastica [4,5]

F. fistulosa [54] F. pumila [55]

Mixture β-Sitosterol, stigmasterol

F. carica, F. sycomorus [56] F. salicifolia F. bubu [6]

Sitosteryl -3-O-β-D-glucopyranoside

F. elastica [5]

Bergenine Racemosic acid Icariside F2 Benzyl-O-β-D-glucopyranoside (1S,6R)-8-O-β-D-glucopyranosyl sodium abscisate Rutin Kaempferin

F. racemosa [57]

β-Sitosterol

F. elastica [4]

F. elastica [16]

ACCEPTED MANUSCRIPT

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F. microcarpa [59] F. maxima [60] F. nymphaefolia [61] F. pantoniana [62] F. bubu [6]

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Morin Quercitrin n-hexacosanol

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Flavonoïds

F. formosana [58]

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Chromenes

Syringin Citroside B Corchoionoside C (6S,9R) roseoside Ficuformodiol A Ficuformodiol B Ficuisoflavone 5,7,3’,4’,5’-Pentamethoxyflavone 5,7-Dihydroxy-4’-methoxy-3’-(2,3dihydroxy-3-methylbutyl) isoflavone Chrysine Biochanin A

F. elastica [16]

Mixture of linear alkanes Fatty acids

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Mixture of linear alcohol

F. elastica [5]

Tetracosanoic acid

Phenolic compound

Sesquiterpenes Stylbenes Saponin

Ficuselastic acid

F. elastica [4]

Pumilaside B trans-resveratrol piceid Elasticoside Ficusoside

F. pumila [68] F. bubu [6]

Ficusamide

F. elastica [5]

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F. septica [63] F. hispida [64] F. pantoniana [62] F. microcarpa [65] F. microcarpa [66]

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Lignanes α-tocopheroïds

Antofine O-Methyltycophorinidine Ficine Ficusal α-tocopherol Threo-2,3-bis(4-hydroxy-3methoxyphenyl)-3-ethoxypropan-1-ol Feroxidin

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Alcaloïds

F. beecheyana [67] F. elastica [16]

F. elastica [12]

Sphingolipids

F. bubu [6] Coumarins

S)-(-) oxypeucedanin hydrate (R)-(+) oxypeucedanin hydrate bergapten

F. exasperate [69]

Anthraquinone

emodin

F. elastica [16]

ACCEPTED MANUSCRIPT 4. Conclusion Seven compounds, including three new molecules, namely elasticamide, elastiquinone and ficusoside B (1-3), were isolated and characterized from the methanolic wood extract of F.

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elastica aerial roots. These components showed some activity against six tested human cancer cell lines. Particularly, the lowest IC50 values were obtained for the methanolic wood extracts of

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F. elastica aerial roots FEBr against U373n, compound 3a against U373n and 2 against B16F10.

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To the best of our knowledge, F. elastica is not used in traditional medicine in Cameroon as anticancer. Our study has demonstrated the anti-proliferative activity of the methanolic wood

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extract of F. elastica aerial roots and some isolated compounds on six human cancer cell lines. Standardized extracts of F. elastica could be tested in traditional medicine for the treatment of

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cancer. Our results also suggest ficusoside B hexaacetate (3a) and elastiquinone (2) as potential leads for natural product-based candidate for further studies.

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Conflicts of interest

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Acknowledgments

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The authors have no conflicts of interest to declare.

Support of this project was provided by the pilot studies grant from Wallonie-Bruxelles International (WBI) awarded to J.E.T. Mbosso. We thank the support of the “Laboratoire de cancérologie et toxicologie expérimentale, Faculté de Pharmacie, Université Libre de Bruxelles (ULB)” for antitumor tests and Victor Nana (National Herbarium of Cameroon) for the botanical identification. RW is Research Associate at the National Fund for Scientific Research FNRSFRS (Belgium).

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Graphical abstract Phytochemical investigation using bioassay-guided fractionation has led to the isolation of three

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new compounds (1-3) from the wood of Ficus elastica aerial roots. MeOH extract and fractions 2 and 3 showed cytotoxic activities against U373n, A549 NSCLC and SK-MEL-28 cancer cell

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lines with IC50 ranging from 3 to 8 μg/mL. Compound 2 (IC50 of 14 µM against B16F10 melanoma cells) and 3a (IC50 of 11 µM against U373n glioma cells) had moderate activity

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while compounds 1 and 3 exhibited weak cytotoxicity with IC50 value ≥ 88 µM.