SUPPLEMENTARY MATERIAL Antioxidant and antibacterial activities and polyphenolic constituents of Helianthemum sessiliflorum Pers

In this study the various extracts of aerial parts of Helianthemum sessiliflorum Pers. were examined in vitro for possible source of antioxidants and for antibacterial activity. The antioxidant activity was performed by DPPH radical scavenging method which showed that ethyl acetate extract possessed the best antioxidant potential (IC50 = 32.75 ± 2.07 μg/ml). The significant linear correlation was realized between the values of the total phenolic/flavonoid content and antioxidant activity of plant extracts. The ethyl acetate and n-butanol extracts showed moderate antibacterial activity. In addition, the phytochemical study of n-butanol extract afforded nine known phenolic compounds (1-9). This is the first report of six of them (1, 3, 5-8) in Cistaceae family. The structural identification of the isolated compounds was achieved using several spectroscopic methods.


General experimental procedures
The separation and purification of n-butanol extract were realised using column chromatography (SiO 2 : 320-400 mesh, Merck, Polyamide: SC-6 and Sephadex . The TLC and preparative TLC used in this study were Silica gel (Kieselgel 60 F 254 , Merck) and RP-18 reversed-phase, detection at 254 and 366 nm. The structures of isolated compounds were established using several spectroscopic methods including NMR experiments (Bruker-Avance-600 Spectrometer; δ in ppm rel. to Me 4 Si as internal standard, J in Hz), UV spectra (Beckman DU-600 spectrophotometer; λ max in nm), optical rotation measurement (Perkin-Elmer 241 polarimeter) and MS spectra were obtained by (ESI-MS ion trap Bruker Esquire, EI-MS Bruker Micromass Q-TOF).
Spectrophotometer used in biological study was a Perkin-Elmer (Lambda 950).

Preparation of plant extracts
The aerial parts of H. sessiliflorum were collected on May 2011 in Biskra (south of Algeria) and identified by Prof. Bachir Oudjhih of Agronomic Institute of Batna-1 University (Algeria) where a voucher specimen number (664/LCCE) was deposited.
Air-dried and powdered aerial parts of H. sessiliflorum (1 kg) were extracted three times with 70% ethanol (10 L each) for 24 h at room temperature. The ethanol extract was suspended in water and then successively partitioned with cyclohexane (3 × 150 ml), ethyl acetate (3 × 150 ml) and n-butanol (3 × 150 ml).

Total phenol content (TPC)
The concentration of phenolic compounds in plant extracts was determined using Folin-Ciocalteu method (Singleton et al. 1999). 0.5 ml of methanolic solution of various extracts were mixed with Folin Ciocalteu reagent (2.5 ml, 1:10 diluted with distilled water) for 5 min and aqueous Na 2 CO 3 (2.5 ml, 7.5 %) were then added. The mixture was allowed to stand for 45 min in thermostat at 45 °C and the phenols were determined by colorimetric method at 765 nm. The standard curve was prepared using different concentrations of gallic acid in methanol (20-200 µg/ml) and calibration line was constructed (r = 0.988). Total phenolic content was expressed in terms of micrograms of gallic acid equivalents per milligrams of dry extract.

Total flavonoid content (TFC)
Total flavonoid content in H. sessiliflorum extracts was measured by the aluminium chloride spectrophotometric assay (Zhishen et al. 1999). The sample contained 2 ml of methanol solution of the extract in the concentration of 2 mg/ml and 2 ml of 4% AlCl 3 solution dissolved in methanol. The samples were incubated for an hour at room temperature. The standard curve was prepared using the standard solution of quercetin in methanol (0.5-5 mg) and the absorbance was determined using spectrophotometer at 415 nm for constructing a calibration line (r = 0.989). Total flavonoid content of the different extracts was expressed in micrograms of quercetin equivalents per milligrams of extract.

Evaluation of antioxidant activity
The ability of the plant extracts to scavenge in vitro 1,1-diphenyl-2-picryl-hydrazyl (DPPH)-free radicals was assessed by the standard method, adopted with suitable modifications (Kumarasamy et al. 2007). The solution of extracts was prepared in methanol to achieve the concentration of 2 mg/ml. Diluted solutions were mixed with 2 ml of methanolic solution of DPPH in concentration of 2 mg/ml. These solution mixtures were kept in dark for 30 min at room temperature and optical density was measured at 517 nm. The same procedure was repeated for the standard solution of quercetin. Percentage of inhibition was calculated using equation:

% inhibition = [1-(Abs sample/Abs control)]×100
Linear graph of concentration versus percentage inhibition was prepared and IC 50 values (micromolar concentration required to inhibit DPPH radical formation by 50%) were calculated. The antioxidant activity of each sample was expressed in terms of IC 50 .

Evaluation of antibacterial activity
In vitro antibacterial activity of different extracts was investigated by the disk diffusion method (Bauer et al. 1996) (Tortora et al. 2004) collected from antibacterial laboratory of hospital public institution of Djelfa, Algeria. Different concentrations of plant extracts (50,100,200,300,400,500, 600, 700 mg/ml) were prepared using DMSO as solvent. The sterile filter paper discs (6 mm diameter) were saturated by 20 µl of different concentrations of each extract and then were placed on lawn cultures prepared. Similarly, each plate carried a blank disk by adding DMSO alone to serve as a negative control, and antibiotic disk (6 mm diameter) was also used as positive control. The Petri dishes were subsequently incubated at 37 °C for 24 h and the inhibition zone around each disc was measured in mm.

Statistical analysis
Data are expressed as mean ± standard derivation from three separate observations. For antioxidant assays, one way ANOVA test (p<0.05) was used to analyze the differences among IC 50 of various extracts. The correlation and linear regression analysis were performed using Microsoft Office Excel 2007.