Throughout its history, synthetic feed-grade Vitamin E acetate has been known for its (high) price volatility combined with a global supply chain prone to disruptions. Polyphenol-rich natural extracts, on the other hand, have a rather high stability in cost. However, not all types of polyphenolic antioxidants are readily bio-available to the animal and thus suited to partially substitute Vitamin E for feed cost reduction. Luckily, reliable assays become increasingly disposable to evaluate a product’s true antioxidant capacity in animal cells.

Global Product Manager Antioxidants
Impextraco – Belgium

Vitamin E (VE) is applied in feed for a dual purpose. Firstly, for its unique bio-essential functions for the animal regarding immunity, reproduction, preservation of cell membranes, etc. Secondly, as a standard antioxidant with a high biological activity in the animal, thanks to its phenolic structure (Figure 1) and beneficial reduction potential. The latter implies it’s an effective scavenger of oxidising agents (i.e. free radicals) in the animal’s body. The most widely used type of VE in premixes and compound feed is the synthetic form all-rac-DL-α-tocopheryl acetate 50% Adsorbate. This is an ester of α-tocopherol and acetic acid, being stable during storage and handling. Within the animal’s body, it is converted into highly bio-available free tocopherol via a necessary hydrolysis step.
BIOACTIVE ANTIOXIDANT ALTERNATIVES TO VITAMIN E
Vitamin E acetate is known for its typical price volatility and riskful global supply chain that can lead to supply disruptions. Consequently, more and more formulators are replacing the antioxidant part of synthetic VE’s dosage in the premix or compound feed by natural polyphenols. It is crucial to stress that, in the animal, polyphenols can only substitute the antioxidant properties of dietary VE and not its essential vitamin functions. Polyphenols are a diverse group of more than 8,000 phytobiotic compounds, occurring in almost every plant. Plants like tea, grapes, berries and nuts are rich in polyphenols. Thanks to their phenolic chemical nature like VE, polyphenols serve as good antioxidants able to capture unstable free radicals (like Reactive Oxygen Species). However, not all phenolic compounds have the same scavenging capacity (i.e. free radical affinity) due to intrinsic differences in their chemical structure. Next to capturing radicals, some polyphenols can also work as effective recyclers of used up antioxidants like oxidised VE (Figure 2). This thanks to their chemical redox potential value, which can be even favourably lower than the one of the latter. As such, specific types of polyphenols can also prolong the effect of VE via a synergistic interaction.

Even more important is that not all polyphenols are strong antioxidants under biological conditions. How can an antioxidant be claimed to replace the (systemic) antioxidant properties of for instance VE in the animal, when it doesn’t make its way to the blood and the cells of the body tissues? That is why high values for antioxidants in classical in vitro methods (e.g. ORAC, TEAC, DPPH, FRAC) do not necessarily mean a high intra-cellular antioxidant capacity. These conventional tests typically comprise a direct oxidative challenge, induced in a test tube containing nothing but a synthetic radical solution. Needless to say, such a concept will not take into account the intestinal stability, bio-availability or intra-cellular antioxidant activity of a compound or product.
HOW TO EVALUATE AN ANTIOXIDANT’S BIOLOGICAL ACTIVITY?
In vitro methods with their typical shortcomings were mentioned before. So how to test a product’s antioxidant capacity in a biologically relevant way for the animal? It is important to note that live animal trials with a (small) partial replacement of VE by an antioxidant are not always ideal. This is because, within such an application, zootechnical performance is anyhow not expected to show a significant drop, as most diets are overdosed in VE.

A more direct and representative way is to measure the level of Intra-Cellular Oxidative Stress (ICOS), preferably within an oxidative challenge model. This records accurately the protective effect of an antioxidant on radical-challenged animal cells (Figure 3), in terms of intra-cellular accumulation of Reactive Oxygen Species (ROS).
The University of Antwerp in Belgium, for instance, has established its own validated ICOS model via fluorescence-based measurements in animal gut epithelial cells (i.e. the IPEC-J2 porcine type). Such an assay creates the following benefits for an antioxidant compound or -product compared to other evaluation methods:
1. assessing its bio-availability or uptake by gut epithelial cells (its systemic, whole-body effect)
2. simulating its protective effects on gut integrity (vs. ROS-induced inflammation) and thus also disease susceptibility
3. measuring its antioxidant effect inside the cell (where most oxidative stress is typically generated)
4. allowing a head-to-head comparison with Vitamin E (its water-soluble analogue Trolox®)
INTRA-CELLULAR OXIDATIVE STRESS TEST
The aim of this ex vivo test is to measure the integrity of IPEC-J2 gut epithelial cells, after an induced oxidative challenge by either hydrogen peroxide (H2O2) or menadione, which are strong ROS generators. This by measuring the oxidation across the cell membrane via fluorescence, with the use of an indicator for ROS radicals. This indicator diffuses into the animal cell and if ROS are present therein, it becomes oxidised. When this occurs, the molecule turns fluorescent and thus can be measured (Figure 4). Therefore, the higher the oxidation, the higher the fluorescence.

on intra-cellular ROS presence, after an oxidative challenge to animal gut epithelial cells.
ICOS & VITAMIN E EQUIVALENCE
In a peer-reviewed, independent study conducted by the University of Antwerp1, the above ICOS fluorescence test on animal cells was used to evaluate the biological efficacy of a dietary antioxidant (ELIFE®). The latter is a concentrated blend of different, EU-sourced botanical extracts rich in primarily short monomeric and oligomeric polyphenols (covering different subclasses of flavonoids and phenolic acids).
Within the setup of this ICOS test, the phytobiotic antioxidant blend was added to the IPEC-J2 cell culture at increasing dosages from 0 (control) up to 1,000 ppm. This was done both with and without an initial triggering of oxidation (generating of ROS). The fluorescence results of the ELIFE® treatments were compared to a treatment of Trolox® (VE analogue), which was added in an amount equating to 1,100 ppm of synthetic VE50 ADS. The intra-cellular ROS content by fluorescence measurement was expressed in arbitrary units.

* statistically significant differences (at p<0.05) in ROS content between non-stressed and stressed cells – (i.e. stressor effect)
# statistically significant decrease (at p<0.05) in ROS content after treating cells with Trolox® water-soluble Vit. E or with ELIFE®, when compared to untreated control cells (i.e. antioxidant effect)
The outcome of the test (Figure 5) proved that ELIFE®’s polyphenols, which are primarily of the short and water-soluble type, penetrated the gut epithelial cells and exerted a significant intra-cellular antioxidant activity. Such a biological action is key, as most ROS radicals are produced inside animal cells due to the high mitochondrial activity.
First of all, the test results showed a clear dose-response in the reduction of intra-cellular ROS content up to 1,000 ppm ELIFE®. Secondly, with regards to VE replacement, the decreased intra-cellular ROS presence was similar between the Trolox® treatment (equal to 1,100 ppm synthetic VE50 ADS.) and the 500 ppm ELIFE® group. As a result, a dosage ratio of at least 1:2 between the antioxidant blend and VE50 ADS. could be concluded. Such an antioxidant equivalence would signify a matrix value of min. 1,000,000 IU/kg product for partial VE replacement (1 mg ELIFE® antioxidant blend = 2 mg VE50 ADS. = 1 IU).
PRACTICAL EXAMPLES OF PROFITABLE & SAFE REPLACEMENT
In monogastric diets, no replacement is allowed by the antioxidant blend at VE levels up to 20 IU/kg feed (i.e. minimum nutritional requirement + safety margin). In subspecies with a higher VE need, like sows or breeder chickens, this lower limit lies at a higher level. Above this threshold, the replacement rate of VE50 ADS. by the antioxidant blend is only gradually increased with dietary VE level. Such safe substitution advice is key, so that the min. physiological requirement for VE, as set by independent animal research institutes (like NRC, INRA or FEDNA), is never compromised. Below Table 1 shows some replacement examples and their indicative feed cost savings with ELIFE® in different animal applications (calculated with an end user price of 7.5 USD/kg for VE50 ADS.).
CONCLUSIONS
Due to their chemical nature, polyphenols are more versatile antioxidant molecules compared to Vitamin E (or even C). However, not all polyphenols are equally bio-available nor evenly bio-effective inside animal body cells. ELIFE®, a blend of selected botanical extracts rich in short-type polyphenols, is a potent antioxidant equivalent to synthetic Vitamin E in the animal. This claim has been demonstrated in multiple researches using bio-relevant lab models, such as direct oxidative challenges to animal gut epithelial cells. Thanks to its high antioxidant capacity in such biological matrices, ELIFE® allows a reliable replacement of Vitamin E acetate 50% ADS. This translates into an efficient 1:2 dosage ratio and a safe substitution rate (depending on the initial Vitamin E level in the diet). In combination with its low cost per kg of VE50 ADS. equivalence, the product has proven to create interesting financial savings for formulators.
Sources
1Ayuso et al. (2020). A Medium-Throughput System for In Vitro Oxidative Stress Assessment in IPEC-J2 Cells. Int. J. Mol. Sci. 2020, 21, 7263.
Further references are available upon request via [email protected]
About Steven Beckers
Steven Beckers is a monogastric nutritionist with a Master’s degree in Applied Bio-Sciences Engineering from the University of Leuven (Belgium). Since 2019, Beckers has been a Global Product Manager of multiple specialty feed additives at Impextraco. Here he provides technical support to customers and end users, to help establish innovative concepts in different agricultural markets. He is devoted to maximizing animal health and performance, by correctly advising and optimizing the application of value-added products.