SUPPLEMENTARY MATERIAL Identification and in vitro antioxidant activities of phenolic compounds isolated from Cynoglossum cheirifolium L. Bensaid Ilhem 1, Atik Bekkara Fawzia 1, El Haci Imad Abdelhamid 2, Belarbi Karima 1, Beddou Fawzia 3, Bekhechi Chahrazed 1 1 Laboratoire des Produits naturels, Département de Biologie, Faculté SNV-STU, Université Aboubekr BELKAÏD Tlemcen, B.P. 119, Imama 13 000, Algérie 2 Centre de Recherche scientifique et technique en Analyses Physico-chimiques (CRAPC), BP 384, Bou-Ismail 42004, Tipaza, Algérie 3 Université des Sciences et de la Technologie Houari Boumediene USTHB, Faculté des Sciences Biologiques. BP 32 EL ALIA 16111 Bab-Ezzouar Alger, Algérie Correspondence details: PhD BENSAID Ilhem Snvstu325@gmail.com +213 779 125 346
Abstract In an extensive search for bioactive compounds from plant sources, the quantitative and qualitative characterisation of the compounds present in Cynoglossum cheirifolium extracts was studied. Total phenolic and flavonoid contents were determined by spectrophotometric techniques. In vitro antioxidant and radical scavenging profiling was determined through DPPH scavenging activity and Ferric reducing antioxidant power. Our study showed that leaves produce more phenolic compounds, followed by flowering aerial part. The butanolic fraction obtained from leaves extract exhibited the highest total phenolics (78.65±3.58 mg GAE/g DW) and flavonoids (22.15±4.66 mg CE/g DW). In contrast, this fraction displayed the highest DPPH scavenging ability with IC 50 values of 0.06±0.003 mg/ml. The RP-HPLC-PDA analysis of phenolic compounds from leaves of C. cheirifolium lets to identify: rosmarinic acid, ferulic acid, caffeic acid, p-coumaric acid, syringic acid, sinapic acid and rutin. The obtained results indicate that this plant represent a valuable source of natural antioxidants. Keywords: Cynoglossum cheirifolium L., RP-HPLC-PDA, Antioxidant activity, phenolics, Flavonoids.
Experimental Plant materials Cynoglossum cheirifolium L. samples were collected from Aïn Youcef region (Tlemcen, Algeria) and identified by Dr. Stambouli from Department of Ecology and Environment, University of Tlemcen, Algeria. A voucher specimen (N B-2238) was deposited at the laboratory of Natural Products. Extraction of phenolic compounds Crude extracts were obtained by magnetic stirring for 24 h at room temperature of a dry organ powder with aqueous methanol (70% v/v). The resultant extracts were filtered and evaporated to dryness under vacuum at 60 C. Flavonoids were isolated by dissolving the dry residues obtained by the above procedure with hot water, followed successively by extraction with ethyl acetate and butanol (Bekkara et al. 1998). The obtained fractions were evaporated, weighed and dissolved in 3 ml of methanol. Quantification of phytochemicals Determination of total phenolic contents The total phenolic contents (TPC) of C. cheirifolium were determined using Folin-Ciocalteu reagent as described by Awah et al. (2012). The Gallic acid was used as a standard for the calibration curve. The results were expressed as mg Gallic acid equivalents (GAE) per g of dry weight (DW). Determination of total flavonoid contents Total flavonoid contents (TFC) were determined according to the method described by Barros et al. (2011). Catechin was used as a standard for the calibration curve. The results were expressed as mg Catechin equivalents (CE) per g of dry weight (DW). Assessment of the antioxidant activities 1,1-diphenyl-2-picrylhydrazyl assay
The hydrogen donating or radical scavenging ability of C. cheirifolium extracts was measured using the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) method according to Barros et al. (2011). The radical scavenging activity (RSA) was calculated as a percentage of DPPH discoloration using the equation: % RSA = [(A DPPH A s ) / A DPPH )] x 100 Where A S is the absorbance of the solution when the sample extract has been added at a particular level and A DPPH is the absorbance of the DPPH solution. The extract concentration providing 50% of radical-scavenging activity (IC 50 ) was calculated from the graph of RSA percentage against extract concentration. Vitamin E, Butylated hydroxytoluene (BHT) and Butylated hydroxyanisole (BHA) were used as standards. Ferric reducing antioxidant power (FRAP) assay The ability of the extracts to reduce Fe 3+ was assayed by the method of Oyaizu (1986). Vitamin E, BHT and BHA were used as standards and EC 50 value (effective concentration of the extract which corresponds to 0.5 of absorbance) was obtained from linear regression analysis. RP-HPLC-PDA identification of phenolic compounds RP-HPLC analysis was performed on a Perkin Elmar Flexar system equipped with a binary pump delivery system, an internal degasser, photo diode array detector (PDA) and an Eclipse ODS Hypersil C18 column (150 mm 4.6 µm) was used. The mobile phase consisted in solvent A- Water/Acetic acid (98/2) and B- Methanol. The gradient elution system was: 5 min with 5 % of B; 15 min with 10 % of B and 26 min of linear
gradient from 10 % to 100% of B, after that, 20 min were consisted for equilibration. Flow rate was 0.8 ml/min. The chromatograms were monitored at 280 and 340 nm. The compounds identification and peak assignments were done based on their retention times, UV-VIS spectra and also comparing with standards. Statistical analysis All results were carried out in triplicate and expressed as mean ± standard derivation (SD). References Awah FM. Uzoegwu PN. Ifeonu P. Oyugi JO. Rutherford J. Yao X. Fehmann F. Fowke KR. Eze MO. 2012. Free radical scavenging activity, phenolic contents and cytotoxicity of selected Nigerian medicinal plants. Food Chem. 131: 1279 1286. Barros L. Cabrita L. Boas MV. Carvalho AM. Ferreira ICFR. 2011. Chemical, biochemical and electrochemical assays to evaluate phytochemicals and antioxidant activity of wild plants. Food Chem. 127: 1600 1608. Bekkara F. Jay M. Viricel MR. Rome S. 1998. Distribution of phenolic compounds within seed and seedlings of two Vicia faba cvs differing in their seed tannin content, and study of their seed and root phenolic exudations. Plant Soil. 203: 27 36. Oyaizu M. 1986. Studies on Products of Browning Reactions: Antioxidative Activities of Products of Browning Reaction Prepared from Glucosamine. Jpn J Nutr Diet. 44: 307 315.
Figure S1. RP-HPLC chromatogram of ethyl acetate fraction of C.cheirifolium L. (leaves part) obtained at 280 nm