Cytotoxicity of Enterolobium timbouva plant extract and its isolated pure compounds

Aims: The present study aimed to evaluate the cytotoxic activities of the aqueous alcohol extract of Enterolobium timbouva leaves as well as its isolated pure compounds. Place and duration of study: Department of Pharmacognosy, Faculty of pharmacy, Ain Shams University, between March 2010 and May 2012. Methodology: In vitro Cytotoxic study was conducted for the aqueous methanol extract and the isolated pure single compounds to determine the IC50 by sulphorhodamine B (SRB) assay Results: Phytochemical investigation of the extract resulted in the isolation and structural determination of ten phenolic compounds isolated for the first time from entitled genus viz; 3,4-Dihydroxy-Cinnamic acid (Caffeic acid) (1); Quercetin-3-O-β-D-glucopyranoside (Isoquercitrin) (2); Quercetin-3-O-β-D-galacto-pyranoside (Hyperin) (3); Kaempferol-3-O-β-D– glucopyranoside (Astragalin) (4); Hesperetin-7-O-rutinoside (Hesperidin) (5); Quercetin 3-Orutinoside (Rutin) (6); Quercetin (7); Kaempferol (8); 7-methoxycoumarin (Herniarin) (9); and Chrysin (10). The aqueous alcohol extract exhibited potent cytotoxic activity against diffferent cancer cell lines with IC50 values of 2.67 μg/ml against MCF-7 cell line, 3.89 μg/ml against HCT116 cells, 4 μg/ml against HEp2 cells, 4.5 μg/ml against HeLa cells, 1.7 μg/ml against PC-3 cells, and 5.7 μg/ml against Huh-7 cells. In vitro cytotoxic assay of the isolated pure compounds against Huh-7 cell Line showed that compounds 1, 9 and 10 are the only tested compounds exhibiting potent cytotoxic activity with IC50 of 3 μg/mL, 0.76 μg/mL, and 18.51 μg/mL respectively. The rest of tested compounds exhibited IC50 exceeding 1000 μg/mL which reflects their safety. Conclusion: The current study indicated that the phenolic compounds isolated from Enterolobium timbouva leaves are promising molecules with potentially useful cytotoxic activity profiles. This confirms that this terrestrial plant has great value as a source of lead compounds with pharmaceutical applications.


INTRODUCTION
In the last few years, the identification and development of phenolic compounds or extracts from different plants have become a major area of health-and medical-related research.Fabaceae occupies a distinguishable situation among the famous plant families which include genera embracing phenolic rich species.Fabaceae is particularly rich in flavonoids and related compounds; about 28% of all flavonoid and 95% of all isoflavonoid aglycone structures known from the plant kingdom are produced by legumes [1].
Enterolobium timbouva Mart.(synonym: Enterolobium contortisiliquum), a member of the tree family Fabaceae, sub-family Mimosoideae, commonly known in English as the earpod tree, is a tree more than 20 m high which is characterized with its black ear-shaped fruits.It is native to tropical South America, southern Brazil, and temperate South America.But, it is widely populated and cultivated in Egypt .Since nothing could be traced in literature concerning the phenolic content of the various parts of Enterolobium timbouva and as part of an ongoing study to discover potential bioactive phenolics from terrestrial plant sources [2, 3], the present study was directed to investigate the phenolics present in the aqueous alcohol extract obtained from the leaves of Enterolobium timbouva and to investigate their in vitro cytotoxic activities.

Plant material
Fresh leaves of Enterolobium timbouva (Fabaceae) were collected from plants grown in El-Orman botanical garden, Ministry of Agriculture, Giza, on March (2010).They were kindly authenticated by Mrs. Tereize Labib, agricultural engineer, El-Orman botanical garden, Giza, Egypt.A voucher specimen of the authenticated plant (ETf-5001) was deposited at the department of Pharmacognosy, Faculty of pharmacy, Ain Shams University, Cairo, Egypt.

Extraction, isolation, and purification of phenolics from Enterolobium timbouva:
The intact air dried plant material 2 kg were homogenized in MeOH-H 2 O (3:1) mixture (three extractions each with 5 L).The dried filtrate (50 g) of the homogenate was applied on a polyamide 6s column (500 g, 120 x 5 cm) and eluted with H 2 O followed by H 2 O-MeOH mixtures of decreasing polarities to yield 36 individual fractions (2L each) that were examined separately under UV light.Each fraction was investigated by two-dimensional paper chromatographic investigation (2D-PC).Similar fractions were pooled to yield seven main fractions (I-VII) that were separately dried in a vacuum and subjected to 2D-PC.

In vitro assay for cytotoxic activity
Enterolobium timbouva aqueous methanol extract and its isolated compounds were screened for their cytotoxicity to determine the IC 50 (50% growth inhibition) by sulphorhodamine B (SRB) assay according to the method of Skehan [4].Cytotoxicity of the aqueous methanol extract of Enterolobium timbouva was evaluated on human breast (MCF-7), colon (HCT 116 ), larynx (HEp 2 ), cervica (HeLa), prostate (PC -3), and liver (Huh-7) carcinoma cell lines.The cytotoxic activities of the isolated compounds caffeic acid (1), isoquercitrin (2), hesperidin (5), rutin (6), quercetin (7), herniarin (9) and chrysin (10) were evaluated against Huh-7 cell line.Doxorubicin was used as positive control.Exponentially growing cells were collected using 0.25% Trypsin-EDTA.Viability was determined by trypan blue exclusion using the inverted microscope.Cells were seeded in 96-well plates at 4000 cells/well in Dulbecco's Modified Eagle's Medium (DMEM) supplemented medium.After 24 hours, cells were incubated with the appropriate concentration ranges of drugs, completed to total of 200 µl volume/well using fresh medium and incubation was continued for 72 hours.Control cells were treated with vehicle alone.For each drug concentration, 4 wells were used.Following 72 h treatment, the cells were fixed with 10% trichloroacetic acid for 1 h at 4 ºC.Wells were stained for 10 min at room temperature with 0.4% SRB dissolved in 1% acetic acid.The plates were air dried for 24 h and the dye was solubilized with Tris-HCl (10 mM, pH 7.4) for 5 minutes on a shaker at 1600 rpm.The optical density (OD) of each well was measured spectrophotometrically at 564 nm with the ELIZA microplate reader.The percentage of cell survival was calculated as follows: The IC 50 values were calculated according to the equation for Boltzman sigmoidal concentration response curve using the nonlinear regression fitting models (Graph Pad, Prism Version 5).The experiment was repeated 3 times for each cell line.(6); Quercetin (7); Kaempferol (8); 7methoxycoumarin (Herniarin) (9); and Chrysin (10) (Fig. 1).The structures of these compounds were unambiguously determined by their chromatographic behaviors as well as spectroscopic analysis via UV, 1 H-NMR and
According to the American National Cancer Institute, the IC 50 limit to consider a crude extract active against cancer cells should be lower than 20 µg/ml [15].It can be concluded that the aqueous alcohol extract of Enterolobium timbouva possess potential cytotoxic activity against breast, colon, larynx, cervical, prostate, and liver cancer cells and most potent against human prostate cancer.
Results also suggest that caffeic acid (1), herniarin (9) and chrysin (10) are probably the compounds responsible for this potent cytotoxic activity of the aqueous methanol leaf extract.The inhibitory activity of caffeic acid on cell viability and cell proliferation was in accordance with the previous report by Chung et al, 2004 [16] who confirmed that the anti-metastatic and anti-tumor effects of caffeic acid are mediated through the selective suppression of matrix metalloproteinase (MMP)-9 enzyme activity and transcriptional down-regulation by the dual inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) as well as MMP-9 catalytic activity.Also the cytotoxicity of herniarin (9) in the present study matched that obtained by [17] who confirmed that herniarin induces its cytotoxic effect through apoptosis induction and chrysin cytotoxicity was in accordance with the previous report by Choo et al, 2010 [18] who confirmed that chrysin inhibits proliferation and induces apoptosis via caspase activation and inactivation of the Akt signaling.These findings consequently merit further exploration of the extract in subsequent in-vivo studies and later in controlled clinical trials.

CONCLUSIONS
Intensive phytochemical investigation of Enterolobium timbouva leaf extract confirmed that it is capable of synthesizing and accumulating appreciable amounts of several phenolics, thus leading to the isolation and characterization of ten of these constituents.The current study indicated that the phenolic compounds isolated from Enterolobium timbouva leaves are promising molecules with potentially useful cytotoxic activity profiles.This confirms that this terrestrial plant has great value as a source of lead compounds with pharmaceutical applications.

Fig. 2
Fig. 2 Concentration response plots of Enterolobium timbouva extract on different cancer cell lines.Error bars are the standard deviations from three experiments.