Echinophora tenuifolia L. branches phytochemical profile and antiproliferative activity on human cancer cell lines

: The methanolic extract of Echinophora tenuifolia L. branches and its fractions were evaluated for their in vitro cell growth inhibitory activity on different human cancer cell lines (C32, LoVo and SKBr3) and the normal BJ fibroblasts. All tested samples were effective against the melanoma cell line C32, with IC 50 values ranging from 22.8 ± 0.8 to 78.7 ± 1.2 μg/mL, the antiproliferative activity of the dichloromethane fraction being significantly higher. This fraction was also effective against the LoVo adenocarcinoma cell line, with an IC 50 value of 53.0 ± 2.1 μg/mL. The ethyl acetate and dichloromethane fractions showed the highest lipid peroxidation inhibitory activity, verified by means of the β-carotene bleaching test. The phytochemical profiles of E. tenuifolia branches extract were established by means of GC-MS and HPTLC. Overall, branches of E. tenuifolia L. could represent a rich source of bioactive compounds, potentially useful in the pharmaceutical field.


GC-MS analysis
The apolar volatile constituents of the n-hexane and dichloromethane fractions of E. tenuifolia L. branches were identified by means of GC-MS. The phytochemical profile was acquired on a Hewlett-Packard 6890 gas chromatograph equipped with an SE-30 capillary column (100 % dimethylpolysiloxane, 30 m length, 0.25 mm in diameter, 0.25 µm film thickness) and a selective mass detector Hewlett Packard 5973. Analyses were conducted using a programmed temperature from 60 to 280 °C (16 °C/min) with helium as carrier gas (linear velocity, 0.00167 cm/sec) (Conforti et al. 2012). The comparison of GC retention factors with those of standards, and the comparison of mass spectra with those present in the Wiley 138 library allowed the identification of compounds.

Evaluation of total phenolic and total flavonoid contents
The total phenolics and total flavonoids contents of the E. tenuifolia branches methanolic extract were assessed by means of spectrophotometric methods. Values were calculated from calibration curves based on the standard chlorogenic acid (analysis of phenolics) or quercetin (analysis of flavonoids); final results were expressed as mg of chlorogenic acid or quercetin equivalent per g of dry plant material, respectively. The Folin-Ciocalteau reagent was utilized to evaluate total phenolics (Menichini et al. 2013) with absorbance measurements at 726 nm. The flavonoids content was estimated using a colorimetric method based on the formation of a flavonoid-aluminum complex (Marrelli et al. 2015) with absorbance measurements at 430 nm.
Analyses were run in triplicate.

HPTLC analysis
Qualitative and quantitative analyses of polar constituents of the AcOEt fraction of E. tenuifolia L. were carried out by means of High-Performance Thin Layer Chromatography (HPTLC).
Operating conditions were the same as previously described (Marrelli et al. 2014). Plates were developed using a mixture ethyl acetate/dichloromethane/acetic acid/formic acid/water (100:25:10:10: For post-chromatographic derivatization, the plates were treated with NPR reagent (1 g diphenylborinic acid aminoethylester in 200 mL of ethyl acetate) or anisaldehyde reagent (1.5 mL p-anisaldehyde, 2.5 mL H 2 SO 4 ,1 mL AcOH in 37 mL EtOH; heating at 105°C for 3 min). The plates were examined under a UV light at 254 or 366 nm and under white light upper and lower (WRT) before and after derivatization.
Calibration curves were prepared using absolute amount (μg/band) as independent variable (X) and the peak area of standards as dependent variable (Y). Quantification of compounds was performed using regression equations (correlation coefficients R 2 , typically > 0.98). All determinations were carried out in triplicate (three spots on the same plate).

Free radical scavenging activity (FRSA) assay
The free radical scavenging activity (FRSA) was assayed using a test based on the reduction of a purple methanolic solution of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). 200 µl of samples solutions at different concentrations (5-1000 µg/mL) were added to 800 µl of a 10 -4 M methanol solution of DPPH.
After 30 min in the dark, absorbances were measured at 517 nm. Ascorbic acid was used as positive control and all experiments were run in triplicate (Conforti et al. 2006).

β-Carotene bleaching-linoleic acid assay
The antioxidant activity was determined using the β-carotene bleaching test as previously reported (Conforti et al. 2010). Briefly, 1 mL of a β-carotene solution (0.5 mg/mL in CHCl 3 ) was added to 0.02 mL of linoleic acid and 0.2 mL of 100% Tween 20. An emulsion was prepared by evaporation of chloroform and dilution with 100 mL of water. 0.2 mL of different samples solutions (1-100 μg/mL) were added to 5 mL of the prepared emulsion that was placed in a water bath at 45 °C; absorbances were measured at 470 nm at initial time and after 30 and 60 minutes. The antioxidant activity was measured in terms of successful prevention of β-carotene bleaching. Propyl gallate was used as positive control and all experiments were run in triplicate. Dichloromethane fraction α-Phellandrene epoxide 11.708 0.1 Carvacrol 12.445 5.0 a Compounds listed in order of elution from SE30 MS column. b Retention time (as minutes). c Relative area percentage (peak area relative to total TIC peak area %).