Alternative test-strategy

Project team HU

Dr. Robert Jan Veldman (WP1); Dr. Jean-Paul ten Klooster (WP2); Dr. Cristina Chamorro-Perez (WP3); Dr. Laura M’Rabet; Dr. Cyrille Krul; Tineke Malherbe-Dupont; Dr. Raymond Pieters (PL)


RAAK-PRO, Stichting Innovatie Alliantie (PRO-2-001) 

Key words

Natural compounds, in vitro strategy, inflammation, obesity 

Project summary

The interest for plant- and /or nutrition-derived natural health products is increasing. However, it remains unclear how the safety and efficacy of such products can be assessed. This has resulted in European legislation to ensure scientific validation of health claims (The EFSA Journal 2008, 644, 1-44). In these validation processes, mechanistic knowledge is of crucial importance. In addition, there is an urgent need to reduce and replace animal testing. The current project aims to meet both demands.The general aim of this project is to optimize and validate a translational animal-free strategy to assess safety and efficacy of natural health products.

The project activities are translated to 3 work packages:

  • WP1 - Fast and specific screenings-assays.
  • WP2 - Methods to assess inflammatory and other immune reactions, in relation to gut health.
  • WP3 - Innovative methods to determine the chemical composition of plant derived compounds in relation to biological effect parameters.


Drie mensen testen


WP1: Development of an in vitro test battery for testing of natural anti-oxidants

The anti-oxidative capacity of food-derived compounds is usually assessed by ‘classical’ methods such as the FDA-approved oxygen radical absorbance capacity (ORAC) assay. From a chemical point of view these assays tell whether a particular compound or extract possesses anti-oxidant properties or not. The biological relevance of its outcomes might however be questioned since delivery parameters such as intestinal absorption, biotransformation and cellular membrane passage of anti-oxidants are not taken into account. In collaboration with several project partners (ORAC Europe BV, Bio Detection Systems BV, Phytogenix BV and TNO) we aim to develop an array of complementary in vitro tests by which these issues can be addressed. Tests that are currently being implemented include (in addition to the ORAC assay): in silico redox analysis, FACS-based analysis of oxidant probe-loaded sheep erythrocytes and a human Nrf2-luciferase reporter cell line. For validation purposes these assays are currently challenged with a standard panel of (defined mixtures of) natural compounds and (fractions of) selected plant extracts. Future plans aim for combining these assays with in vitro metabolism by recombinant Cyp450 enzymes or rat liver microsomal fractions.

WP2: Methods to assess inflammatory and other immune reactions, in relation to gut health.

In WP2 we focus on two different organs of the human body, namely, the intestine and adipose tissue. Both organs have an intimate interplay with the immune system and currently, we are developing novel 2D and 3D in vitro systems for the intestine and adipose tissue, including specific immune cells. These tissue culture-based in vitro systems will subsequently be used for monitoring metabolism, toxicity and effectiveness of novel drugs, food supplements and phytochemicals in, for instance, immune enhancement and diseases such as type 2 diabetes. This research is performed in collaboration with Danone, Fytagoras, IRAS and the WUR.

WP3: Processing, extraction, analysis and isolation of active ingredient from daily food

Phytochemicals are secondary metabolites produced by plants which provide colour and flavour to fruit, vegetables and, herbs. Prevention of oxidative stress, antioxidant, antiiinflammatory, antiviral, and antimicrobial properties are some of the interesting biological activities attributed to phytochemicals ingested by human diet. Unfortunately, very little is known about qualitative and quantitative composition of phytochemicals and correlation of the composition with biological activity. In order to evaluate that, reliable data on phytochemical content is needed. Therefore, we are interested in the development and/or optimization of procedures which allow us efficient extraction and analysis of phytochemicals. Fruits, vegetables, herbs and other complex matrices will be extracted, analyzed and tested for antioxidant and anti-inflammatory activities. Furthermore, correlations of the phytochemical composition and biological activity will be established. In collaboration with Phytogenix, Fytagoras, TNO, and IRAS (UU), we have already extracted interesting matrices such us fennel, raspberries, white/black pepper and menopausal supplements. Extracts have been obtained by steam distillation, reflux, soxhlet extraction, accelerated solvent extraction, comprehensive fractionation and ultrasound as extraction methods. Moreover, TLC, GC, HPLC and/or LC-MS methods have been developed to analyze the chemical composition of the extracts. Finally, antioxidant activity of some of the extracts has been measured by DPPH, NP-PEG, and ORAC tests. Preliminary results showed differences in antioxidant activity (ORAC) of extracts depending on the solvent and the extraction method used.