The Polar Paradox Theory: Revisited Shreeya Ravisankar

Outline Introduction Polar Paradox Theory Mechanisms Challenges Conclusions

Introduction Lipid oxidation is a complex process: Initiation, propagation and termination (heat, light, oxygen, enzymes, transition metals, metalloproteins and/or micro- organisms). Preventing lipid oxidation: Antioxidant – most effective, convenient and economical strategy. Effectiveness depends on: chemical structure, temperature, type of oxidation substrate, physical state of system media and presence of antagonists/synergists. Antioxidants can prevent or delay oxidation by scavenging free radicals, quenching singlet oxygen, inactivating peroxides and other reactive oxygen species (ROS), chelating pro-oxidant metal ions, quenching secondary oxidation products, and inhibiting pro-oxidative enzymes, among others.3 The

Polar Paradox Theory Polar antioxidants more effective in less polar media (bulk oils) Non-polar antioxidants more effective in more polar media (oil-in-water emulsions, liposomes, biological membranes and whole tissues) Early evidence that supported this hypothesis included studies on trolox, ascorbic, gallic, caffeic, and ferulic acids, among others, which exhibited higher antioxidant efficacies in bulk oil and lower efficacies in emulsions than their correspondent nonpolar alkyl esters.7-11 In addition, synthetic lipophilic antioxidants, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), were found to be more active in emulsions than in dry lard or vegetable oil; R-, β-, γ-, and δ-tocopherols showed opposite trends in efficacy in liposomes and bulk oil.

Mechanism In bulk oil: Initial theory: Polar antioxidant oriented themselves in the oil-air interface- where surface oxidation occurs. But air is more non-polar than oil!! Association colloids hypothesis: Bulk oil contains small amounts of water, mono and diacylglycerols, phospholipids, sterol and free fatty acids- they are surface active by forming reverse micelles and lamellar structures creating oil-water interfaces. Early studies on oxidation in bulk oils were based on the assumption that oxidation occurs in a homogeneous medium. The oil-air interface was considered to be the site where oxidation was initiated and propagated to the inner parts of the oil. However, the distribution of polar antioxidants at the oil-air interface was questioned because air is even less polar than oil. In bulk oils, reverse micelles are typically formed by surfactants Figure 3 Simplified view of a spherical reverse micelle. with low hydrophilic-lipophilic balance (HLB) values such as free fatty acids (∼1), diacylglycerol (∼1.8), and monoacylglycerol (∼3.4–3.8) (24). Monoacylglycerols are known to form reverse micelles in triacylglycerol oils (Gulik-Krzywicki and Larsson, 1984). Phospholipids have intermediate HLB value (∼8), which may explain why they form lamellar structures as well as reverse micelles (Gupta et al., 2001). Increased effectiveness of the hydrophilic antioxidants would occur if the association colloids were a major site of oxidation due to their ability to concentrate transition metals and surface active lipid hydroperoxidesTherefore, oxidation is postulated to be likely to take place at the interfaces of these associationTherefore, oxidation is postulated to be likely to take place at the interfaces of these association colloids, rather than at the air-oil interface HLB (Hydrophile-Lipophile Balance) is an empirical expression for the relationship of the hydrophilic ("water-loving") and hydrophobic ("water-hating") groups of a surfactant. The table below lists HLB values along with typical performance properties. The higher the HLB value, the more water-soluble the surfactant. Thus higher HLB value surfactants form lamellar structures (phospholipids) whereas lower ones form reverse micelles (mono and diacylglycerols)

Mechanisms In oil-in-water emulsions According to polar paradox theory, non-polar antioxidants show better affinity to the oil, forming a protective membrane around the lipid droplet. Various studies have shown these trends: In bulk oil : Eriodictyol, Caffeic acid, Hydroxyltyrosol > BHT/ α-tocopherol1 In oil-in-water emulsions: BHA, BHT, α-tocopherol > 17 phenolic antioxidants2 1 Mateos, R.; Trujillo, M.; Pereira-Caro, G.; Madrona, A.; Cert, A.; Espartero, J. L. New lipophilic tyrosyl esters. Comparative antioxidant evaluation with hydroxytyrosyl esters. J. Agric. Food Chem. 2008, 56, 10960–10966. 2 Cuvelier, M. E.; Bondet, V.; Berset, C. Behavior of phenolic antioxidants in a partitioned medium: Structure-activity relationship. J. Am. Oil Chem. Soc. 2000, 77, 819–824.

Challenges to the Polar Paradox Theory The polar paradox theory may just be a particular case of a much wider global rule. The linear relationship between polarity and antioxidant effect is not always true. Why? Antioxidant effectiveness depends on too many factors: Presence of association colloids Phospholipids and water content Presence of transition metals Antioxidant stability and solubility Antioxidant molecular structure Presence of synergists/antagonists Concentration of antioxidants Broad array of pathways impacting oxidative reactions

In oil-in-water emulsion Non-linear hypothesis Cut-off effect Antioxidant activity increases as alkyl chain length increases until a threshold is reached, after which further chain length extension leads to drastic decrease in activity. Several studies are showing that medium chain lipophilic esters show improved efficiency as antioxidants in emulsions compared to long chain esters Conjugated autoxidisable triene Laguerre, M., L´opez Giraldo, L. J., Lecomte, J., Figueroa-Espinoza, M.-C., Bar´ea, B., Weiss, J., Decker, E. A. and Villeneuve, P. (2009). Chain length affects antioxidant properties of chlorogenate esters in emulsion: The cutoff theory behind the polar paradox. J. Agric. Food Chem. 57:11335– 11342. Laguerre, M., L´opez Giraldo, L. J., Lecomte, J., Figueroa-Espinoza, M.-C., Bar´ea, B., Weiss, J., Decker, E. A. and Villeneuve, P. (2010b). Relationship between hydrophobicity and antioxidant ability of phenolipids in emulsion: A parabolic effect of the chain length of rosmarinate esters. J. Agric. Food Chem. 58:2869–2876.

Cut-off effect In oil-in-water emulsions Shahidi, Fereidoon, and Ying Zhong. "Revisiting the polar paradox theory: a critical overview." Journal of agricultural and food chemistry 59.8 (2011): 3499-3504.

Cut-off effect - mechanisms The nonlinear behavior or cutoff effect of antioxidants in emulsified media may be explained by their partitioning, location, and mobility in the multiphase system, which are influenced by both polarity and their molecular size. As already mentioned, the partitioning properties of antioxidants in heterogeneous systems are crucial for their activities. It was found from partition analysis that the concentration of chlorogenates in the aqueous phase decreased with increased alkyl chain length, with the dodecyl ester presenting the lowest concentration, which correlated well with their antioxidant efficacies.32 Further extension in chain length (above 12 carbon atoms) resulted in an unexpected increase in partitioning in the aqueous phase. The authors suggested that this could be due to the micellization process of the long-chain chlrogenates, facilitating their existence as micelles or other aggregates in the water phase. The esters with long alkyl chains are generally amphiphilic and may aggregate readily in themedium rather than orienting themselves at the interfacial layer. Laguerre, Mickaël, et al. "What makes good antioxidants in lipid-based systems? The next theories beyond the polar paradox." Critical reviews in food science and nutrition 55.2 (2015): 183-201.

Cut-off effect - mechanisms Molecular size of the antioxidant: Increase in molecular size decreases mobility of the antioxidant : because of stearic hindrance which decreases diffusibility towards the reactive centers ie oxidizable substrates and free radicals. Increased hydrophobic interaction with environment (eg. Emulsifiers) decreases mobility.

In bulk oils Concentration of antioxidant The polar paradox appears to reflect only the situation when antioxidant concentration is above the critical concentration while overlooking its behavior at lower concentrations, that is, below the critical concentration. The bell-shaped curves indicate that the antioxidant activity increases with increasing concentration until a maximum activity is reached at the optimal concentration, after which the activity decreases with further concentration increase. As shown in Figure 1, below the critical concentration, the broken line is above the solid line, indicating higher antioxidant activity for nonpolar antioxidants than their polar counterparts. While above the critical concentration, the solid line is above the broken line, indicating an opposite trend. The polar paradox appears to reflect only the situation when antioxidant concentration is above the critical concentration while overlooking its behavior at lower concentrations, that is, below the critical concentration. Zhong, Ying, and Fereidoon Shahidi. "Antioxidant behavior in bulk oil: Limitations of polar paradox theory." Journal of agricultural and food chemistry 60.1 (2011): 4-6.

Zhong, Ying, and Fereidoon Shahidi Zhong, Ying, and Fereidoon Shahidi. "Antioxidant behavior in bulk oil: Limitations of polar paradox theory." Journal of agricultural and food chemistry 60.1 (2011): 4-6.

Zhong, Ying, and Fereidoon Shahidi Zhong, Ying, and Fereidoon Shahidi. "Antioxidant behavior in bulk oil: Limitations of polar paradox theory." Journal of agricultural and food chemistry 60.1 (2011): 4-6.

Other challenges Role of emulsifiers: Compete with antioxidants at the interfacial membrane thus leaving less interfacial area available for antioxidants At higher concentrations, emulsifiers may form micelles entrapping the antioxidants. Antioxidant mechanism of action: 2 ways of behavior: Prevent or retard initiation step by quenching initiators – polar antioxidants more better Breaking propagating chains – non-polar antioxidants more effective (in bulk oil) At higher concentrations, antioxidants can also act as pro-oxidants due to metal reduction or formation of phenoxy radicals that can initiate radical chain reactions. For initiator quenchers, the polar antioxidants can inhibit the initiation of oxidation at the interface (where most initiators are located) more effectively than nonpolar ones in the lipid phase, whereas the reverse is true for chain-breaking antioxidants. This is due to the fact that nonpolar chain-breaking antioxidants are more efficient in inhibiting the propagation reaction in the lipid phase, where the nonpolar oxygen molecules are preferentially dissolved.

Conclusion The polar paradox theory successfully explained behavior of antioxidants for last 2 decades However, in oil-in water emulsions the cut-off effect is showing contradictory results and in bulk oils, concentration of anti-oxidants is showing contradictory results. Thus, it is suggested that the polar paradox theory may be a particular case of a much wider global picture and more research and individual systems based approach should be undertaken.

References Mateos, R.; Trujillo, M.; Pereira-Caro, G.; Madrona, A.; Cert, A.; Espartero, J. L. New lipophilic tyrosyl esters. Comparative antioxidant evaluation with hydroxytyrosyl esters. J. Agric. Food Chem. 2008, 56, 10960–10966. Cuvelier, M. E.; Bondet, V.; Berset, C. Behavior of phenolic antioxidants in a partitioned medium: Structure-activity relationship. J. Am. Oil Chem. Soc. 2000, 77, 819–824. Laguerre, M., L´opez Giraldo, L. J., Lecomte, J., Figueroa-Espinoza, M.-C., Bar´ea, B., Weiss, J., Decker, E. A. and Villeneuve, P. (2009). Chain length affects antioxidant properties of chlorogenate esters in emulsion: The cutoff theory behind the polar paradox. J. Agric. Food Chem. 57:11335–11342. Laguerre, M., L´opez Giraldo, L. J., Lecomte, J., Figueroa-Espinoza, M.-C., Bar´ea, B., Weiss, J., Decker, E. A. and Villeneuve, P. (2010b). Relationship between hydrophobicity and antioxidant ability of phenolipids in emulsion: A parabolic effect of the chain length of rosmarinate esters. J. Agric. Food Chem. 58:2869–2876. Shahidi, Fereidoon, and Ying Zhong. "Revisiting the polar paradox theory: a critical overview." Journal of agricultural and food chemistry 59.8 (2011): 3499-3504. Laguerre, Mickaël, et al. "What makes good antioxidants in lipid-based systems? The next theories beyond the polar paradox." Critical reviews in food science and nutrition 55.2 (2015): 183-201. Zhong, Ying, and Fereidoon Shahidi. "Antioxidant behavior in bulk oil: Limitations of polar paradox theory." Journal of agricultural and food chemistry 60.1 (2011): 4-6.