Antioxidant, 5-Lipoxygenase Inhibitory and Cytotoxic Activities of Compounds Isolated from the Ferula lutea Flowers

A phytochemical investigation of the Ferula lutea (Poir.) Maire flowers has led to the isolation of a new compound, (E)-5-ethylidenefuran-2(5H)-one-5-O-β-d-glucopyranoside (1), designated ferunide, 4-hydroxy-3-methylbut-2-enoic acid (2), reported for the first time as a natural product, together with nine known compounds, verbenone-5-O-β-d-glucopyranoside (3), 5-O-caffeoylquinic acid (4), methyl caffeate (5), methyl 3,5-O-dicaffeoylquinate (6), 3,5-O-dicaffeoylquinic acid (7), isorhamnetin-3-O-α-l-rhamnopyranosyl(1→6)-β-d-glucopyranoside, narcissin (8), (−)-marmesin (9), isoimperatorin (10) and 2,3,6-trimethylbenzaldehyde (11). Compounds 3–10 were identified for the first time in Ferula genus. Their structures were elucidated by spectroscopic methods, including 1D and 2D NMR experiments, mass spectroscopy and X-ray diffraction analysis (compound 2), as well as by comparison with literature data. The antioxidant, anti-inflammatory and cytotoxic activities of isolated compounds were evaluated. Results showed that compound 7 exhibited the highest antioxidant activity with IC50 values of 18 ± 0.5 µmol/L and 19.7 ± 0.7 µmol/L by DPPH radical and ABTS radical cation, respectively. The compound 6 exhibited the highest anti-inflammatory activity with an IC50 value of 5.3 ± 0.1 µmol/L against 5-lipoxygenase. In addition, compound 5 was found to be the most cytotoxic, with IC50 values of 22.5 ± 2.4 µmol/L, 17.8 ± 1.1 µmol/L and 25 ± 1.1 µmol/L against the HCT-116, IGROV-1 and OVCAR-3 cell lines, respectively.


Introduction
Plants are still used as a source of large-scale original and novel chemical structures. New compounds serve as an index to promote new medicines herbal and dietary supplements. Phenolic substances, which are largely found in most plants, exhibit a wide range of biological effects including anti-inflammatory, antimicrobial and anticancer effects [1] Secondary metabolites such as alkaloids, flavonoids, tannins, saponins, generally produced by plants for their defense mechanisms, have been implicated in the therapeutic properties of most medicinal plants [2].
The Ferula genus includes 170 species [3]. Just four species of Ferula were found in Tunisia (F. communis, F. lutea, F.tunetan aand F. tingitana) [4]. Some varieties are very advantageous to humankind since they are used in regular ordinary nutrition and traditional medicine. According to our own survey in different regions in Tunisia, it has been indicated that some of these species are also useful against delirium and convulsion, activities probably associated with the presence of antioxidants [5]. A successful chemical survey of the roots of the F. lutea has contributed to the isolation of new dihydrofuranocoumarins, together with eight known compounds [6]. It is well known that the Ferula genus has a variety of coumarins [7], phenolics such as chlorogenic acid, gallic acid and pyrogallol [8]. Moreover, the Ferula genus is referred to be a good source of biologically active compounds like sesquiterpene derivatives [9,10], daucanes [11], germacranes [9] and sesquiterpene coumarins identified in the roots of the plants. Therefore, theroots are a better source for isolating sesquiterpene coumarins than the aerial parts [12,13].
The anti-inflammatory effect was also examined via 5-lipoxygenase inhibitory activity. Finally, the cytotoxic activity was evaluated using the MTT assay on the HCT-116 human colon cancer cell line and IGROV-1, OVCAR-3 human ovary cells lines.

Structure Determination
Then-butanol and ethyl acetate extracts of the flowers of F. lutea were fractionated by successive column chromatography to afford two new compounds 1 and 2, together with eight known compounds 3-10 not previously identified in the Ferulagenus (Table 1).

11
2,3,6-Trimethylbenzaldehyde O Compound 1 was isolated as a white amorphous powder. Positive DCI-HRMS of this substance gave pseudo-molecular ion peaks [M+H] + at m/z 289.0916, which is consistent with the molecular formula C12H16O8 and five degrees of unsaturation. The IR spectrum displayed intense absorption bands at 3341 (OH), 1606 (C=C).The 1 H and 13 C-NMR spectral data of compound 1 were assigned in Table 2.  [14,15]. The presence of the disubstituted γ-methylene-γ-lactone moiety was confirmed by the 13   White X-ray quality crystals of compound 2 was obtained by crystallization in CHCl3/MeOH (95:5). The X-ray diffraction analysis of 2 was carried out on a single crystal ( Figure 2). This study confirmed the structure of compound 2 and clearly established that the absolute configuration of the double bond is (E). From these data, the structure of 2 was identified as (E)-4-hydroxy-3-methylbut-2-enoic acid, indicated for the first time as a natural compound.

Antioxidant Activity
In this study, DPPH radical scavenging activity and ABTS radical cation activity were measured to assess the antioxidant activity of compounds from F. lutea flowers. The results of the antioxidant activity are presented in Table 3. Only the phenolic compounds were tested of the antioxidant activity.  Among the compounds from F. lutea flowers 7, 6 and 5 exhibited the most potent DPPH free radical scavenging activity, with IC50 values of 18.0 ± 0.5 µmol/L, 26.6 ± 1.3 µmol/L and 39.2 ± 0.5 µmol/L, respectively. Compound 4 showed a moderate activity, with an IC50 value of 127.4 ± 1.2 µmol/L, compared to the positive control, ascorbic acid (vitamin C) (IC50 = 25.0 ± 0.1 µmol/L). The ABTS +. scavenging activity of compounds from F. lutea flowers was similar to the DPPH free radical scavenging activity. Although the compound 8 is phenolic, does not have interesting antioxidant activity.
The antioxidant activity of compound 7 is explained by the presence of the caffeoyl moities at C-3 and C-5 of the quinic acid, each having two phenol groups in the ortho position. The effective contribution of the two caffeoyl systems in compound 7 and even in its analogue 6 is confirmed by the considerable loss in the activity of compound 4 which contains only one in its structure.
The relative loss in antioxidant activity of compound 6 is certainly due to methylation of the carboxylic acid function which seems to contribute to this activity through its hydroxyl group. The significant antioxidant activity of compounds 6 and 7 may partly explain the use of this plant in traditional medicine against convulsions.

Cytotoxicity Evaluation
The cytotoxic activity of compounds 1-11 isolated from F. lutea flowers against the human colon carcinoma cell line HCT-116 and ovary cells lines IGROV-1 and OVCAR-3 was assessed using MTT assay, which is reliable to detect proliferation of cells. The activities of compounds were presented in Table 3. It is important to mention that none of these products have ever been previously tested for their cytotoxic activity against the human cell lines HCT-116, IGROV-1 and OVCAR-3.
Compounds 5 and 9 presented the best cytotoxic effect against the human colon carcinoma cell line HCT-116, with IC50 values of 22.5 ± 2.4 and 50.0 ± 4.9 µmol/L, respectively. The other compounds did not exhibit activities up to 100 µmol/L against the same cell line.
Only compound 5 exhibited the cytotoxic effects against the ovary cell lines IGROV-1 and OVCAR-3, with IC50 values of 17.8 ± 1.1 and 25 ± 1.1 µmol/L, respectively. The other compounds did not exhibit any activity up to 100 µmol/L against the IGROV-1 and OVCAR-3 cell lines.
Compound 5 showed higher toxicity against a colon cancer cell line (LoVo) and is less toxic against another type of colon cancer (HCT-116) and two ovary cancer cell lines (IGROV-1 and OVAR-3). Compound 6 showed a good activity against the liver cell line Hep-G2 but it showed no activity against HCT-116, OVCAR-3 and IGROV-1. This difference is due to the selectivity of the product towards the cancer cell lines.
The significant activity of caffeoyl acid methyl ester (5) towards the three cancer cell lines compared with that of its analogue where in caffeic acid is esterified with one of the alcohol functions of the quinic acid showed a considerable effect of the latter in reducing the activity of compound 5. This conclusion is confirmed by the inactivity of compounds 6 and 7, which both contain in their structures quinic acid and despite the presence of two caffeoyl moities.
The high activity of compound 9, only against the HCT-116 cell line, might be explained by a selectivity phenomenon. The literature reports that narcissin 8 showed a low cytotoxic activity against the human chronic myelogenous leukemia cell line K562 with an IC50 value of 800 µmol/L [31]. parameters. The structure solution was obtained by direct methods (SHELXS-97) and was refined with anisotropic thermal parameters using full-matrix least squares procedures on F 2 to give R = 0.0751 using SHELXL-97 [32]. Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1030232. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44(0)-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).

Collection of Plant Material
Ferula lutea flowers were collected in the region of Béja (Tunisia), on April, 2010 and identified by Professor Féthia Harzallah Skhiri, in the Laboratory of Genetic, Biodiversity and Valorization of Bioresources, Higher Institute of Biotechnology of Monastir, University of Monastir, Tunisia. A voucher specimen was deposited in the same laboratory (F.L.F-10).

Extraction and Isolation
The fresh flowers (5.8 kg) were macerated at room temperature with methanol/water (7/3, 20 L) for 7 days. The corresponding aqueous residue obtained after filtration and evaporation of the organic solvent (MeOH) under reduced pressure was partitioned successively with ethyl acetate and n-butanol yielding after evaporation of the solvents the corresponding ethyl acetate (56 g) and n-butanol (220 g) extracts. We used a CombiFlash ® Rf 200 flash chromatography system. This liquid chromatography apparatus allows us to split and purify several grams of an extract or a complex fraction with high throughput. It is performed using two pumps (5-200 mL/min; 0-200 psi), provided with packed columns (4 g to 330 g), a precolumn using RediSep Rf silica gel normal phase and a fraction collector with a diode array detector (200-780 nm).

Biological Activity
The absorptance of the extract, at the wavelength of each test and with each concentration, was removed to eliminate the effect of colouring.

Free Radical Scavenging Activity DPPH Test
The antioxidant activity of compounds was assessed according to their DPPH scavenging ability as described by Khlifi et al., [33] with some modifications. Reaction mixtures, containing 0.1 mL of the relevant compounds solution in methanol, with concentrations ranging 3 mg/mL and 0.1 mL of a 0.2 mM DPPH methanolic solution, were added to 96-well microtiter plates and incubated at 25 °C for 30 min. Absorbances were measured at 520 nm, the wavelength of maximum absorbance of DPPH, were recorded as A(sample). A blank experiment was also carried out applying the same procedure to a solution without the test material and the absorbance was recorded as A(blank). The free radical-scavenging activity of each solution was then calculated as percent inhibition according to the following equation: Antioxidant activity extracts was expressed as IC50, defined as the concentration of the test material required to cause a 50% decrease in initial DPPH concentration. Ascorbic acid was used as a standard. All measurements were performed in triplicate.

ABTS Radical-Scavenging Test
The radical scavenging capacity of the samples for the ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonate) radical cation was determined as described by Khlifi et al., [33] with some modifications. ABTS was generated by mixing a 7 mM of ABTS at pH 7.4 (5 mM NaH2PO4, 5 mM Na2HPO4 and 154 mM NaCl) with 2.5 mM potassium persulfate (final concentration) followed by storage in the dark at room temperature for 16 h before use. The mixture was diluted with ethanol to give an absorbance of 0.70 ± 0.02 units at 734 nm using spectrophotometer. For each sample, diluted solution (20 μL) was allowed to react with fresh ABTS solution (180 μL), and then the absorbance was measured 6 min after initial mixing. Ascorbic acid was used as a standard and the capacity of free radical scavenging was expressed by IC50 (µmol/L) values calculated denote the concentration required to scavenge 50% of ABTS radicals. The capacity of free radical scavenging IC50 was determined using the same previously used equation for the DPPH method. All measurements were performed in triplicate.

5-Lipoxygenase Inhibitory
The anti-inflammatory activity activities of the isolates was determined on Soybean lipoxygenase as described by Khlifi et al. [33] with some modifications. 20 µL of each compound was mixed individually with 150 µL sodium phosphate buffer (pH 7.4) containing 20 µL of 5-lipoxygenase and 60 µL of linoleic acid (3.5 mM), yielding a final volume of 250 µL. However, the blank does not contain the substrate, but will be added 30 µL of buffer solution. All compounds were re-suspended in the DMSO followed by dilution in the buffer so that the DMSO does not exceed 1%. The mixture was incubated at 25 °C for 10 min, and the absorbance was determined at 234 nm. The absorption change with the conversion of linoleic acid to 13-hydroperoxyoctadeca-9,11-dienoate (characterized by the appearance of the conjugated diene at 234 nm) was followed for 10 min at 25 °C. Nordihydroguaiaretic acid (NDGA) was used as positive control. The percentages of enzyme activity were obtained at 200 µmol/L. All measurements were performed in triplicate.

Cytotoxicity Evaluation
The human cancer cell line was used for cytotoxic assay: (HCT-116, IGROV-1 and OVCAR-3). The cells were grown in RPMI-1640 medium supplemented with 10% faetal calf serum (Gibco, Langley, OK, USA), air and 5% CO2. The isolate was added to a medium containing 1 × 10 6 cells/mL, 2 mM L-glutamine and 50 μg/mL gentamycin, and kept at 37 °C in a fully humidified atmosphere. After 18 h of incubation at 37 °C in 5% CO2 incubator, the tubes were centrifuged at 8000 g for 10 min. The supernatant was decanted, and the pellets taken and washed with 20 mM of phosphate buffered saline solution. Each pellet was dissolved in 100 μL (2 mg/mL) MTT solution in a tube, incubated at 22 °C for 4 h and centrifuged at 8000 g for 10 min. All the pellets were dissolved in 500 μL DMSO and read spectrophotometrically at 500 nm. IC50 was calculated by nonlinear regression analysis. Doxorubicin (HCT116) and tamoxifen (IGROV-1 and OVCAR-3) were used as positive control.