Garry Clarke 1, Kang Nee Ting 2, Ch

Garry Clarke 1, Kang Nee Ting 2, Christophe Wiart 2 and Jeffrey Fry 1,*
1 School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2UH, UK;
E-Mail: garry.clarke@nottingham.ac.uk
2 School of Biomedical Sciences, University of Nottingham, Jalan Broga, 43500, Semenyih, Selangor,
Malaysia; E-Mails: Kang-Nee.Ting@nottingham.edu.my (K.N.T.);
Christophe.Wiart@nottingham.edu.my (C.W.)
* Author to whom correspondence should be addressed; E-Mail: jeff.fry@nottingham.ac.uk;
Tel.: +44-115-8230160; Fax: +44-115-8230142.
Received: 24 October 2012; in revised form: 29 November 2012 / Accepted: 20 December 2012 /
Published: 4 January 2013
Abstract: Extracts of plants from the Malaysian rainforest and other fragile habitats are
being researched intensively for identification of beneficial biological actions, with
assessment of antioxidant behavior being a common component of such assessments. A
number of tests for antioxidant behavior are used, with the 2,2-diphenyl-1-picrylhydrazyl
(DPPH) and ferric reduction activity potential (FRAP) assays often being used in parallel,
and also with measurement of total phenolics content (TPC) as a surrogate marker for
antioxidant capacity. The present study investigated the possible redundancy in using all
three assays to determine antioxidant capacity in 92 extracts obtained from 27 plants from
the Malaysian rainforest. The results demonstrated that the assays displayed a high
(R ≥ 0.82) and significant (P < 0.0001) correlation with one another, indicating a high level
of redundancy if all three assays are used in parallel. This appears to be a waste of
potentially valuable plant extracts. Because of problems with the FRAP assay relating to
color interference and variable rates of reaction point, the DPPH assay is the preferred
assay in preliminary screening of extracts of plants from the Malaysian rainforest.
OPEN ACCESS
$50406
Antioxidants 2013, 2 2
Keywords: antioxidant; plant extracts; DPPH; FRAP; phenolics; correlation
1. Introduction
There is considerable interest in charting the biological activities of plants from geographically
remote and fragile habitats such as the primary rainforests of Malaysia and other locations in
South-East Asia, in the hope of identifying novel compounds of potential therapeutic value [1].
Oxidative stress is believed to be a major contributor to the pathogenesis of a number of chronic
diseases [2], and it is for this reason that antioxidant behavior is one of the most commonly determined
biological activities in extracts of plants [3]. A wide variety of antioxidant assays are used when
determining the antioxidant activity of plant extracts, two common ones being based on the scavenging
of the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical (DPPH assay) and ferric reduction activity potential
(FRAP assay). Many utilize both the DPPH and the FRAP assays in their plant activity screening
programs, presumably on the assumption that a combination of the data would provide a better
description of antioxidant activity than obtained from a single assay (for recent examples see [4–7]).
However, should this assumption not be correct, use of these two assays will use up potentially
valuable plant material without providing additional information.
As part of a program mapping the biological activities of plants of the Malaysian rainforest, a total
of 96 extracts isolated from 27 plants have been tested using both the DPPH and FRAP assays, and an
assessment made of the correlation between the two assays. These findings are presented in this paper,
together with data on the total phenolics content (TPC) of the extracts, which is often taken as an
indirect measure of antioxidant capacity [8].
2. Experimental Section
2.1. Collection of Plants and Preparation of Extracts
Plant extracts were collected from various sites in the Malaysian primary rainforest, with relevant
authority as required under the framework of the United Nations Convention on Biodiversity, and water
(W), ethanol (EtOH), ethyl acetate (EtAc) and hexane (Hex) fractions were prepared from leaf, bark or
the whole aerial parts of the plant using standard techniques [9]. A full listing of the plants and extracts
is given in Table 1.
Table 1. Plant extracts used in this study and their antioxidant activity as determined by
results of the 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reduction activity potential
(FRAP) and total phenolics content (TPC) assays.