Leaf Disc Assays for Rapid Measurement of Antioxidant Activity

Deepak M. Kasote,Bhimanagouda S. Patil,Guddadarangavvanahally K. Jayaprakasha

doi:10.1038/s41598-018-38036-x

Abstract

Antioxidant levels are key parameters for studies of food quality, stress responses, and plant health. Herein, we have demonstrated that excised leaf disc has both radical scavenging activity and reducing power, and used this concept to develop 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and potassium permanganate reduction (PPR) leaf disc assays. Reaction time and reagent concentration for these assays were optimized using leaves from spinach, kale, collards, mustard, and watermelon. Further, these assays were validated for linearity and intra-assay precision. Ultra-high performance liquid chromatography coupled to an electrospray quadrupole time-of-flight mass spectrometer (UPLC/ESI-HR-QTOFMS) was used for phytochemical profiling and studying relative abundances of certain phenolic compounds in various leaf discs suspended and cell-free extracts. The mass spectral analysis showed that leaf disc suspended methanolic extracts had almost same phytochemical profiles to those of cell-free extracts. The DPPH leaf disc assay demonstrated better radical scavenging potential than the conventional cell-free extract method. By contrast, the observed antioxidant activity values in ABTS and PPR leaf disc assays were lower than those of conventional cell-free extract-based methods. In conclusion, the developed leaf disc assays are simple and rapid for the qualitative and comparative assessment of the antioxidant potential of leaf samples, as well as can be a good alternative to conventional cell-free extract based methods.

Highlights

The antioxidant potential of the plant samples is routinely assessed by three approaches such as (a) direct measurement of antioxidant enzymes activity, (b) in vitro radical scavenging and reducing power assessment and (c) measuring the protective response of plant samples against chemical-induced oxidative stress
To give a broader window of detection, the dilution ratio of 1:40 of ABTS reagent was confirmed as an optimal concentration for leaf disc assays for the assessment of linearity
The ABTS assay better estimates the antioxidant capacity of foods, fruits, vegetables, and beverages compared to the DPPH method[32,33]

Summary

Introduction

The antioxidant potential of the plant samples is routinely assessed by three approaches such as (a) direct measurement of antioxidant enzymes activity, (b) in vitro radical scavenging and reducing power assessment and (c) measuring the protective response of plant samples against chemical-induced oxidative stress. Each of these approaches has its own limitations about applicability[3]. Methyl viologen or paraquat-induced oxidative stress leaf disc assay is simple and does not require any sample preparation[12] This assay provides limited information about physiological role, response and mechanism of action of phyto-antioxidants. The fundamental principle behind each assay was that the antioxidants oozes out from the edges of the excised leaf disc into the reaction medium and scavanges free DPPH and ABTS+ radicals and reduces KMnO4 to manganese dioxide (MnO2)

Results

Discussion

Conclusion