Submitted: 13 May 2015
Accepted: 27 Aug 2015
ePublished: 08 Sep 2015
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Int J Phytocos Nat Ingred. 2015;2(1): 10.
doi: 10.15171/ijpni.2015.10
  Abstract View: 4688
  PDF Download: 2902

Original Research

N-methyl-2-phenylindole Colorimetric Method as Biomarker for Lipid Peroxidation in Pisum sativum

María Juliana Restrepo Vega 1, José Hipólito Isaza Martínez 1*, Ana Julia Colmenares Dulcey 1

1 Research Group on Natural Products and Food (GIPNA), Department of Chemistry, Faculty of Natural and Exact Sciences, Ed. 320, Of 2096., Ciudad Universitaria-Meléndez, Universidad del Valle, Cali, Colombia

Abstract

A novel and selective method to quantify lipid peroxidation in Pisum Sativum by measuring one of its secondary products, malondialdehyde, by its reaction with N-methyl-2-phenylindole was validated in this study.  It has been reported that indirect effect of heavy metals in plant macromolecules via reactive oxygen species (ROS) production is more toxic and fast than its direct effect. The OH• is produced by metal-catalized Fenton reaction, as a consequence, metal presence in soils may potentiate lipid peroxidation. Enzymes such as cyclooxygenase and lipoxygenase may initiate lipid peroxidation too, therefore, either ROS scavenging or LOX or COX inhibition is convenient routes to diminish lipid peroxidation occurrence.  Two way anova was performed to determine if interaction between time and treatment result in a significant difference between MDA levels in twelve plants. Bonferoni test revealed that treatment was not consistent with time. The day that resulted in significant difference of MDA was the eighth day. MDA to perform the reaction was extracted from groups of twelve 8-day plants by sonication in hydrochlorhydric acid. A calibration curve using 1,1,3,3, tetramethoxypropane as MDA standard, HCl 1 N in methanol, acetonitrile-methanol (3:1) as solvent was performed. Its linear range was 0,0078 μM to 1 μM with a 99,8% correlation. Sensibility of the method was enhanced by variation of time and temperature. Repeatability reported as variation coefficient yielded 7,8% for the standard curve and 19,6% for the samples, being these latter representative of combined biological and chemical variability. A natural flavonoid rich fraction extracted from Siparuna gigantotepala and a synthetic pyrimidine-type compound were evaluated as potential lipid peroxidation inhibitors. Flavonoid rich fraction inhibits the reaction by scavenging radicals while pyrimidine-type compound inhibits LOX and COX enzyme-catalized lipid peroxidation. Both potential inhibitors showed lipid peroxidation inhibition as evidenced by returning MDA concentration in stressed plants to that in control plants.
Keywords: Lipid peroxidation, Pisum sativum, N-methyl-2-phenylindol method, Malondialdehyde, Flavonoids, Oxidative Stress
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