Antioxidant testing: Combining assays points to ‘biological relevance’

By Stephen Daniells

- Last updated on GMT

Related tags: Antioxidant

Combining a series of chemical and cell-based antioxidant tests is the best way to understanding antioxidant effects and the biological relevance of the data, says a new study with acai.

Two cell-based assays and one chemical test were combined to show that polyphenols were the major source of antioxidant activity and that only three of the seven major flavonoids identified could actually penetrate the cell and “contribute to the reduction of oxidative damage within the cell”, according to findings published in Food Chemistry​.

“Due to the complexity of the anti-oxidant defense system in the body, a single anti-oxidant assay cannot reflect all aspects of activities of a given compound,” ​wrote scientists from the USDA Arkansas Children’s Nutrition Center at the University of Arkansas for Medical Sciences, the Shanghai Institute of Pharmaceutical Industry, NIS Labs, and AIBMR Life Science.

“In evaluating the anti-oxidant capacities of natural products, combining both chemical and cell-based assays will provide a useful approach towards understanding the anti-oxidant effects of natural compounds and their biological relevance to any health benefits observed ​in vivo.

“Such combined use of assays may prevent premature conclusions being made about which compounds are responsible for a foods biological effects, based on reliance of chemical assays,”​ they added.

The conclusions echo those of Dr Karen Schaich from Rutgers University, who told attendees at the NutraIngredients Antioxidants 2010 conference in Brussels last week that the best approach to understanding antioxidant activity was to combine results from various assays. Schaich also pointed to the potential of emerging tests such as Cuprac, which uses the copper(II)−neocuproine [Cu(II)-Nc] reagent as the chromogenic oxidizing agent (J. Agric. Food Chem., 2004, Vol. 52, pp. 7970-7981).

Understanding açai

The study also deepens our understanding of açai, with the presence of two flavonoids – vitexin and quercetin – reported for the time in açai pulp.

Açai berries (pronounced ah-sigh-ee) have long formed part of the staple diet of Indian tribes. With the appearance of a purple grape and taste of a tropical berry, it has been shown to have powerful antioxidant properties thanks to a high level of anthocyanins, pigments that are also present in red wine.

It is presently being sold in a number of countries, including New Zealand, Australia, South America, Japan, USA, and the Middle East.

Study details

The researchers performed a two-step study: The first used chromatographic methods to isolate and identify seven major flavonoids from freeze-dried açai pulp (Earth Fruits LLC, Brazil). Data showed the compounds to be chrysoeriol, dihydrokaempferol, homoorientin, luteolin, orientin, quercetin, and vitexin. They then turned their attention to measuring the antioxidant activity of these compounds.

“The vast majority of studies that assess anti-oxidant capacities of açai and other anti-oxidant rich fruits and berries have utilised chemical-based assays. Unfortunately, such assays do not reflect or correlate with the test item’s cellular response,”​ explained the researchers.

“The second objective of this study was to evaluate the anti-oxidant capacities of these flavonoids with one chemical-based assay and two cell-based assays: ORAC, CAP-e assay and ROS PMN assay. By doing these additional assays, we hope to understand not only their in vitro anti-oxidant capacities, but also their behaviours in living cells to reduce oxidative stress.”

Data from the ORAC test showed that flavonoids in the aglycone form had higher antioxidant activity than the glycoside forms. The CAP-e data showed that only luteolin, quercetin, and dihydrokaempferol could penetrate the cell and “contribute to the reduction of oxidative damage”​. Finally, the ROS PMN data showed that five of the flavonoids exhibited “exceptional effects by reducing ROS formation in PMN cells, which produced high amounts of ROS under oxidative stress”​.

“In evaluating the anti-oxidant capacity of natural products, combining both chemical and cell-based assays will provide more comprehensive understanding of anti-oxidant effects and potential biological relevance,”​ concluded the researchers.

Source: Food Chemistry
Volume 122, Pages 610–617
"Anti-oxidant capacities of flavonoid compounds isolated from acai pulp (Euterpe oleracea Mart.)"
Authors: J. Kang, Z. Li, T. Wu, G.S. Jensen, A.G. Schauss, X. Wu

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