Volume 21, Issue 2, April 2013, Pages 143–152 A good number of abstracts and research articles (in total 74) published, so far, for evaluating antioxidant activity of various samples of research interest were gone through where 407 methods were come across, which were repeated from 29 different methods. These were classified as in vitro and in vivo methods. And those are described and discussed below in this review article. In the later part of this review article, frequency of in vitro as well as in vivo methods is analyzed with a bar diagram. Solvents are important for extracting antioxidants from natural sources. Frequency of solvents used for extraction is also portrayed and the results are discussed in this article. As per this review there are 19 in vitro methods and 10 in vivo methods that are being used for the evaluation of antioxidant activity of the sample of interest. DPPH method was found to be used mostly for the in vitro antioxidant activity evaluation purpose while LPO was found as mostly used in vivo antioxidant assay.
Ethanol was with the highest frequency as solvent for extraction purpose.The human body has a complex system of natural enzymatic and non-enzymatic antioxidant defenses which counteract the harmful effects of free radicals and other oxidants. Free radicals are responsible for causing a large number of diseases including cancer (Kinnula and Crapo, 2004), cardiovascular disease (Singh and Jialal, 2006), neural disorders (Sas et al., 2007), Alzheimer’s disease (Smith et al., 2000), mild cognitive impairment (Guidi et al., 2006), Parkinson’s disease (Bolton et al., 2000), alcohol induced liver disease (Arteel, 2003), ulcerative colitis (Ramakrishna et al., 1997), aging (Hyun et al., 2006) and atherosclerosis (Upston et al., 2003). Protection against free radicals can be enhanced by ample intake of dietary antioxidants. Substantial evidence indicates that foods containing antioxidants and possibly in particular the antioxidant nutrients may be of major importance in disease prevention. There is, however, a growing consensus among scientists that a combination of antioxidants, rather than single entities, may be more effective over the long term.
Antioxidants may be of great benefit in improving the quality of life by preventing or postponing the onset of degenerative diseases. In addition, they have a potential for substantial savings in the cost of health care delivery.Various methods are used to investigate the antioxidant property of samples (diets, plant extracts, commercial antioxidants etc.). The objective of this review article is to accumulate all probable methods that are used to evaluate the antioxidant property of various samples. A compiled description of all available in vitro and in vivo antioxidant models would provide prolific advantages to the researchers of this arena by reducing their time for literature review and method development. Two review articles have been published earlier ( Chanda and Dave, 2009 and Badarinath et al., 2010) on in vitro evaluation of antioxidant activity. In this article, attempts have been taken to include in vivo too and to analyze the frequency of the use of different methods.Internet browsing from Google Scholar database was used to identify and to download abstracts and research papers related to antioxidant activity study using suitable keywords (antioxidant + plant extract + in vitro + in vivo) in the month of August 2011.
In the first thirty-four pages, a total of three hundred and forty articles appeared and those were subjected to preliminary screening. The basis of the selection of the articles was (i) antioxidant activity of plant extracts and (ii) description of antioxidant test procedures. A total of seventy-four papers and abstracts were identified and reviewed for in vivo and in vitro methods related to antioxidant evaluation. Solvents used for extraction purpose are also reviewed from the downloaded scientific records.Antioxidant activity should not be concluded based on a single antioxidant test model. And in practice several in vitro test procedures are carried out for evaluating antioxidant activities with the samples of interest. Another aspect is that antioxidant test models vary in different respects. Therefore, it is difficult to compare fully one method to other one. To some extent comparison among different in vitro methods has been done by Badarinath et al. (2010), while we discussed the methods in terms of grouping in the present manuscript.
Researcher has to critically verify methods of analysis before adopting that one for his/her research purpose. Generally in vitro antioxidant tests using free radical traps are relatively straightforward to perform. Among free radical scavenging methods, DPPH method is furthermore rapid, simple (i.e. not involved with many steps and reagents) and inexpensive in comparison to other test models. On the other hand ABTS decolorization assay is applicable for both hydrophilic and lipophilic antioxidants. In this article all in vitro methods are described and it is important to note that one may optimize logically the respective method to serve his/her experimental objective as no one method is absolute in nature rather than an example.The molecule 1, 1-diphenyl-2-picrylhydrazyl (α,α-diphenyl-β-picrylhydrazyl; DPPH) is characterized as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, so that the molecule does not dimerize, as would be the case with most other free radicals.
The delocalization of electron also gives rise to the deep violet color, characterized by an absorption band in ethanol solution centered at about 517 nm. When a solution of DPPH is mixed with that of a substrate (AH) that can donate a hydrogen atom, then this gives rise to the reduced form with the loss of this violet color.In order to evaluate the antioxidant potential through free radical scavenging by the test samples, the change in optical density of DPPH radicals is monitored. According to Manzocco et al., 1998 the sample extract (0.2 mL) is diluted with methanol and 2 mL of DPPH solution (0.5 mM) is added. After 30 min, the absorbance is measured at 517 nm. The percentage of the DPPH radical scavenging is calculated using the equation as given below:where A is the absorbance before reaction and A is the absorbance after reaction has taken place.Human beings are exposed to H2O2 indirectly via the environment nearly about 0.28 mg/kg/day with intake mostly from leaf crops. Hydrogen peroxide may enter into the human body through inhalation of vapor or mist and through eye or skin contact.