SUPPLEMENTARY MATERIAL Antioxidative activities and phenolic compounds of pumpkin (Cucurbita pepo) seeds and amaranth (Amaranthus caudatus) grain extracts Pier Giorgio Peiretti a, Giorgia Meineri b, Francesco Gai a, Erica Longato b*, Ryszard Amarowicz c a Institute of Sciences of Food Production, National Research Council, Grugliasco, Italy; b Department of Veterinary Science, University of Torino, Grugliasco, Italy; c Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland. * Corresponding author: Erica Longato, Postal address: Department of Veterinary Science, University of Torino, Largo Paolo Braccini 2, 10095 Grugliasco, Italy. Tel.: +390116709305 E-mail address: erica.longato@unito.it Abstract Phenolic compounds were extracted from pumpkin (Cucurbita pepo) seed and amaranth (Amaranthus caudatus) grain into 80% (v/v) methanol. The extracts obtained were characterized by the contents of total phenolic compounds (TPC), trolox equivalent antioxidant capacity (TEAC), ferric-reducing antioxidant power (FRAP) and antiradical activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH ) radical. The content of individual phenolic compounds was determined by HPLC-DAD method. Pumpkin seeds showed the higher content of TPC than that from amaranth. The TEAC values of both extracts were similar each other. The lower value of FRAP was observed for pumpkin seed. Phenolic compound present in amaranth grain exhibited strongest antiradical properties against DPPH radical. Several peaks were present on the HPLC chromatograms of two extracts. The UV-DAD spectra confirmed the presence of vanillic acid derivatives in the amaranth grain. The three main phenolic compound present in pumpkin seed were characterized by UV- DAD spectra with maximum at 258, 266, and 278 nm. Key words: pumpkin, amaranth, phenolic compounds, antioxidant activity, FRAP, ABTS, DPPH.
1. Experimental 1.1 Plant material C. pepo seeds were purchased from Tavi S.p.A. (Genova, Italy), while A. caudatus grain were furnished by Pedon S.p.A. (Molvena, Italy). Both seeds were authenticated by Prof. Achille Schiavone and the voucher specimen bearing number of each (151 and 152, respectively) was deposited in Department of Veterinary Science, University of Torino. Each sample was stored at 25 C until the analysis. 1.2 Preparation of methanolic extracts Seeds and grains were milled with a laboratory mill and then defatted with hexane. Phenolic compounds were extracted from so obtained material with 80% methanol for 15 min, at 75 C, at solid material to solvent of 1:10 (w/v) (Karamać et al. 2015). The extractions were repeated three times, filtrates were combined and methanol was evaporated under vacuum using a rotary evaporator (Büchi, Flawil, Switzerland). The remaining aqueous solution was freeze-dried. 1.3 Total phenolic contents TPC was determined using Folin & Ciocalteu s phenol (FPC) reagent as described by Amarowicz et al. (1996). Briefly, solutions of extracts (0.25 ml) were mixed with 0.25 ml of the FCP reagent, 0.5 ml of a aqueous saturated solution of Na2CO3 and 4 ml of distilled water. After 20 min standing in dark, the samples were centrifuged for 5 min at 3000 g. Absorbance of supernatants was read at 725 nm with the Beckman DU 7500 spectrophotometer (Beckman Instruments Inc., Fullerton, USA). The results were expressed as mg catechin equivalents per g of extract. 1.4 Trolox Equivalent Antioxidant Capacity The TEAC was determined by using a method of Re et al. (1999). For this assay, 2,2 -azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) cation radical (ABTS +) solution was prepared by dissolving 96 mg of ABTS in 2.45 mm Na2S2O8. This mixture was shaken for 16 h at room temperature in the dark until reaching a stable oxidative state. Before analysis, the ABTS + stock solution was diluted with methanol to an absorbance of 0.70±0.02 at 734 nm. For the spectrophotometric assay, 2 ml of the ABTS + solution and 20 μl of extracts were mixed and the absorbance was recorded at 734 nm with the spectrophotometer after samples incubation at 30 C for 6 min (TH-24 block heater, Meditherm, Poland). The calibration curve was plotted by using 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) as a standard. The results were expressed as mmol Trolox equivalents per g of extract.
1.5 Ferric-Reducing Antioxidant Power The FRAP assay was performed by using a colorimetric method (Benzie & Strain 1990). The sample solution analyzed was first properly diluted with deionized water to fit within the linearity range. The working FRAP reagent was prepared by mixing 10 volumes of 0.3 M acetate buffer (ph 3.6) with 1 volume of 10 mm 2,4,6-tris(2- pyridyl)-s-triazine in 40 mm HCl and with 1 volume of 20 mm FeCl3 x 6H2O. A volume of 2.25 ml of a working FRAP reagent was warmed to 37 C. Then, 75 μl of the sample and 225 μl of deionised water were added to the FRAP reagent and the absorbance was taken at 593 nm against reagent blank after 30 min of incubation. FRAP value was calculated and expressed as mmol Fe2+ equivalents per g of extract (using the calibration curve prepared for Fe2SO4). 1.6 Scavenging of the DPPH Radical The scavenging effect of phenolics from the extracts was monitored as described by Amarowicz et al. (2008). A 0.1 ml methanolic solution containing between 0.2-0.8 mg/ml of extract was mixed with 2 ml of deionized water and then added to a methanolic solution of DPPH (1 mm, 0.25 ml). The mixture was vortexed for 1 min, left to stand at room temperature for 20 min in the dark, and absorbance of the solution was then measured at 517 nm. 1.7 High performance liquid chromatography (HPLC) analysis Methanolic extract (20 mg) was dissolved in 2 ml of 80% methanol and filtered through a 0.45 μm cellulose acetate filter (Millipore Co. Bedford, Mass., USA). The phenolic compounds contained in sample were separated using a Shimadzu HPLC system (Shimadzu Corp., Kyoto, Japan) consisting of two LC-10AD pumps, a SCTL 10A system controller and a SPD-M 10A photodiode array detector. The chromatography was performed using a pre-packed Luna C18 column (4 x 250 mm, 5 µm; Phenomenex, Torrance, CA, USA) at room temperature. Elution proceeded for 50 min in a gradient system of 5-40% acetonitrile in water adjusted to ph 2.5 with trifluoroacetic acid; the detector was set at 320 and 350 nm, the injection volume was 20 µl and the flow rate was 1 ml/min (Cwalina-Ambroziak et al. 2014). 1.8 Statistical analysis All analytical determinations in this study were triplicated. Results are reported as mean value ± standard deviation. Mean values were compared by the Student s t-test using the MSTAT packaged program. Significant differences were considered at p<0.05.
Table S1: Characteristic of pumpkin seed and amaranth grain extracts. Assay Pumpkin Amaranth TPC (mg/g) 9.82 ± 0.51 a 4.35 ± 0.19 b TEAC (mmol Trolox/g) 0.074 ± 0.002 a 0.069 ± 0.002 a FRAP (mmol Fe 2+ /g) 0.054 ± 0.002 a 0.071 ± 0.002 b IC 50 (mg/ml) 11.9 ± 0.5 a 8.3 ± 0.4 b Mean values in the columns and denoted by the same letter are not significantly different at p < 0.05. Table S2: Content of main phenolic compounds in pumpkin seed and amaranth grain extracts. Plant Compound Content (mg/g) Pumpkin 1 a 4.25 ± 0.21 2 b 0.18 ± 0.01 3 b 0.54 ± 0.03 Amaranth 1 a 3.91 ± 0.15 2 a 2.29 ± 0.09 3 c 7.69 ± 0.32 a results expressed as gallic acid equivalents; b results expressed as vanillic acid equivalents; c results expressed as catechin equivalents.
Figure S1: Antiradical activity of the extracts of pumpkin seed [ ] and amaranth grain [ ] against DPPH radical. Figure S2: HPLC chromatogram of pumpkin seed extract and UV-DAD spectra of the three main compounds.
Figure S3: HPLC chromatogram of amaranth grain extract and UV-DAD spectra of the three main compounds. References Amarowicz R, Estrella I, Hernández T, Troszyńska A. 2008. Antioxidant activity of extract of adzuki bean and its fractions. J Food Lipids. 15:119-136. Amarowicz R, Karamać M, Kmita-Głażewska H, Troszyńska A, Kozłowska H. 1996. Antioxidant activity of phenolic fractions of everlasting pea, faba bean and broad bean. J Food Lipids. 3:199-211. Benzie IEF, Strain JJ. 1990. Ferric reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Method Enzymol. 299:15-27. Cwalina-Ambroziak B, Amarowicz R, Tyburski J, Janiak M, Nowak MK. 2014. Effect of farming systems on pathogen infections and content of phenolic compounds in carrot (Daucus carota L. Subsp Sativus Hoffm.) roots. J Anim Plant Sci. 24:1183-1189. Karamać M, Biskup I, Kulczyk A. 2015. Fractionation of buckwheat seed phenolics and analysis of their antioxidant activity. Polish J Food Nutr Sci. 65:243-249. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Bio Med. 26:1231-1237.