Determination of antioxidant effect of polyols in a cell-free environment

Introduction

A free radical can be defined as any molecular species (capable of independent existence) that contains an unpaired electron in an atomic orbital. These are highly reactive structures, damaging by lipid peroxidation (both in nucleus and in cell membrane) biologically relevant molecules such as DNA, proteins, carbohydrates and lipids. The most important oxygen-containing free radicals are the hydroxyl radical, the superoxide anion radical, hydrogen peroxide and the nitric oxide radical. These free radicals are involved in oxidative stress mechanism, contributing significantly to many diseases. The human body is in constant battle to control the free radicals steady state. 
Polyols are widely used by the food and pharmaceutical industries. They serve, e.g., as carrier in pharmaceutical preparations. Mannitol is considered as excipient of choice for instable active pharmaceutical ingredients (API). In vitro, mannitol has free radical scavenging properties. It can protect active substances like hyaluronic acid against radicalic degradation, especially by oxygen-derived free radicals.¹ Moreover, it hinders the release of reactive oxygen species (ROS) by thermal- or photodecomposition of tetracyclines.² A total oxy-radical scavenging capacity assay3 demonstrated that xylitol, sorbitol and mannitol inhibit oxidation reactions. 
This study evaluates an antioxidant effect of four selected polyols, using a cell free environment in order to measure its scavenging effects from different reactive oxygen species families. It consists of three single assays to evaluate possible antioxidant effects: superoxide radical scavenging activity (NBT assay), hydroxyl radical scavenging activity (HRS assay) and a lipid peroxidation inhibition assay (MDA).

Materials and methods

Materials

All tested polyol samples (mannitol, maltitol, sorbitol and xylitol) came from Roquette Frères, Lestrem, France, with pharmaceutical excipient quality. Each substance was dissolved in demineralized water to obtain a master solution with a final concentration of 50 mg/ml in dry substance. The finally tested dilutions were 5µg/ml, 2.5 mg/ml and 5 mg/ml for all assays. Three well-known antioxidant references served as positive controls at the following concentrations: 
(+)-Catechin : from 10 nM to 10 mM (0.0029 to 2.9 mg/ml), Ascorbic acid from 20 nM to 20 mM (0.00376 to 3.76 mg/ml) and Quercetin from 5 nM to 5 mM (0.00151 to 1.51 μg/ml). 

Scavenging activity trials

 The measurement of superoxide radical-scavenging activity (NBT–assay) was carried out as described in^4,5: 
20 μl of 15mM Na₂EDTA in buffer (50 mM KH₂PO₄/KOH, pH 7.4), 50 μl of 0.6 mM nitro blue tetrazolium chloride (NBT) in buffer, 30 μl of 3 mM hypoxanthine in 50 mM KOH, 5 μl of the test substance in DMSO, and 145 μl of buffer were mixed in 96-well microplates. The reaction was initiated by adding to each mixture 50 μl of xanthine oxidase buffer solution (1 unit in 10 ml buffer). The final preparations were incubated at room temperature while the absorbance at 570 nm was measured every min up to 8 min using a spectrophotometric plate reader.The measurement of the hydroxyl radical scavenging (HRS assay) effect by polyols was done using iron-EDTA solution (solution: ferrous ammonium sulfate/ EDTA/ DMSO/ ascorbic acid). The mixture was incubated in a hot water bath at 80 to 90°C for 15 min. After incubation, ice-cold trichloroacetic acid and Nash’s reagent were added to each sample and incubated at room temperature for 15 min. The absorbance at 412 nm was measured with a spectrophotometric plate reader.Lipid peroxidation inhibition in egg yolk extract (10% final v/v) was performed in presence of 3.5 mM FeSO₄ after 60 minutes pre-incubation at 37 °C. After the incubation time (60 minutes at 95 °C), malondialdehyde (MDA) was dosed in each sample and compared to MDA standard curve⁶ using a commercial assay kit and following the manufacturer instructions. All chemicals came from Sigma Aldrich, USA.

Results 

The superoxide and hydroxyl radicals scavenging activities are expressed as a percentage of inhibition (see figures 1 and 2). The inhibition is calculated based on the background sample vs. concentration tested for all samples.

 

 

Figure 1. Superoxide radical scavenging activity (NBT Assay) of polyols compared with standards

 

 

Figure 2. Hydroxyl radical scavenging activity (HRS) of polyols compared with standards

 

The three tested antioxidant references catechin, ascorbic acid and quercetin showed powerful effects on both particular ROS (superoxide and hydroxyl radicals). The results confirm the validity of the design of the NBT and HRS assays. 
Sorbitol is a slight scavenger of the superoxide radical (see figure 1), with a maximum inhibition of 28%. Under the test conditions, the other polyols did not display any significant antioxidant effect on superoxide radicals.
Considering hydroxyl radicals, mannitol shows an equivalent antioxidant effect compared with the reference ascorbic acid (figure 2): 77% inhibition at 2500 µg/ml. The other polyols samples maltitol, sorbitol and xylitol have a low antioxidant activity toward the hydroxyl radical.

 

Table 1. Inhibition of lipid peroxidation (MDA Assay)

The lipid oxidation was limited by adding the antioxidant controls catechin, ascorbic acid and quercetin. Mannitol has a moderate effect on the prevention of lipid oxidation, compared to the antioxidant references (see table 1). The efficiency of xylitol is high, whereas the other polyols sorbitol and maltitol did not display any significant effect on malondialdehyde production.

Conclusion

These results are in accordance with data from literature:  polyols show antioxidant activities. Especially mannitol showed scavenging effect toward hydroxyl free radicals.^1,2,3 Xylitol inhibits the lipid peroxidation with high efficiency. The antioxidant activity of these polyols is mainly due to hydroxyl groups, interacting with free radicals to inhibit the oxidation reaction.³ Therefore, it was expected that the scavenging capacity of polyols increase with the number of hydroxyl groups, especially when comparing xylitol (C5) with sorbitol and mannitol (C6) and with maltitol (C12). However, our results did not show a significant evolution in antioxidant effects considering the number of hydroxyl groups.  
Finally, these results underline the interest of using polyols, especially mannitol, to limit the effects of oxidative stress in pharmaceutical preparations. Its presence could reduce the degradation of actives substances and hence can improve the overall stability of these formulations.  

References

1. André P. and Villain F. Free radical scavenging properties of mannitol and its role as a constituent of hyaluronic acid fillers: a literature review, International Journal of Cosmetic Science. 39, 355-360 (2017).
https://doi.org/10.1111/ics.12386

2. Miyazaki T.; Yomota C. and Okada S. Hyaluronate depolymerization following thermal decomposition of oxytetracycline. Chem. Pharm. Bull. 49, 118–122 (2001).
https://doi.org/10.1248/cpb.49.118

3. Kang K.W.; Kwak S.H.; Yun S.Y. and Kim S.K Evaluation of Antioxidant Activity of Sugar Alcohols Using TOSC (Total Oxy-radical Scavenging Capacity) Assay, Toxicological Reasearch. 23, 143-150 (2007).
http://dx.doi.org/10.5487/TR.2007.23.2.143

4. Chang S.T.; Wu J.H.; Wang S.Y.; Kang P.L.; Yang N.S. and Shyur L.F. Antioxidant Activity of Extracts from Acacia confusa Bark and Heartwood. J. Agric. Food. Chem. 49, 3420-3424 (2001).
https://doi.org/10.1021/jf0100907

5. Tung Y.T.; Wu J.H.; Hsieh C.Y.; Chen P.S. and Chang S.T. Free radical-scavenging phytochemicals of hot water extracts of Acacia confusa leaves detected by an on-line screening method. Food Chem. 115, 1019-1024 (2009).
https://doi.org/10.1016/j.foodchem.2009.01.026

6. Halliwell B. and Gutteridge C. Formation of a thiobarbituric-acid-reactive substance from deoxyribose in the presence of iron salts. Febs Letters. 128, 347-351 (1981).
https://doi.org/10.1016/0014-5793(81)80114-7

 

Disclaimer

The information contained in this document is to the best of our knowledge true and accurate, but all instructions, recommendations or suggestions are made without guarantee. Since the conditions of use are beyond our control, we disclaim any liability for loss and/or damage suffered from use of these data or suggestions. Furthermore, no liability is accepted if use of any product in accordance with these data or suggestions infringes any patent. No part of this document may be reproduced by any process without our prior written permission