An effective defence against oxidative stress

Preventing peroxidation of lipids

Oxidative stress is one of the driving forces of the aging process (1) and is associated with a number of pathologies including atherosclerosis, cancer and neurodegenerative diseases such as Alzheimer’s and Parkinson’s 9 (2). Oxidative stress is mainly associated with the generation of reactive oxygen species and to a lesser extent, reactive nitrogen species. Both of these types of free radicals damage the components of the cell including DNA, proteins and membrane lipids. We are going to focus on membrane lipids in this article and look at how oxidative stress damages them leading to diseases.

A membrane lipid is a compound that is structurally similar to fats and oils and forms the double-layered surface of all cells (the lipid bilayer). The three major types of membrane lipids are phospholipids, glycolipids, and cholesterol. Free radicals generated by oxidative stress responses can strike these lipids and modify them, this can then interfere with various cellular processes including endocytosis (the taking in of matter by a living cell), exocytosis (the movement of matter from a living cell), regulation of membrane enzymes, transport of metabolites and more. The resulting impaired membrane functions have led to a number of research studies into lipid peroxidation (the oxidative degradation of lipids) and the role of antioxidants present in membranes.

There are a variety of molecules both endogenous (originating from within an organism) and exogenous (originating from outside an organism) that can protect against lipid peroxidation. Vitamin E, plasmalogens and polyphenols can all offer protection from oxidative stress and free radical damage (3-7). Polyphenols including rosmarinic acid, isoquercetin, quercetin, pterostilbene, berberine and many others naturally occur as dietary sources and are increasingly recommended for supplements to prevent oxidative stress. There are over 8000 different polyphenols produced by plants with more being discovered as research continues (8).

Polyphenols disrupt free radical production

Polyphenols have the ability to penetrate into the lipid bilayers which is a crucial step in them being able to protect the cell from oxidative stress and free radical damage. Polyphenols can inhibit lipid oxidation via two mechanisms: interception of intermembrane free radicals and also by increasing membrane fluidity, which disrupts lipid chains hindering the creation of free radicals (9). A study showed that polyphenols from citrus and rosemary were able to disrupt lipid chains in the lipid membrane in this exact manner (10).

The researchers in this study concluded that this fluidizing effect may favor the antioxidant capability and free radical scavenging characteristics of these polyphenols by enhancing the interaction of antioxidant molecules with lipid radicals. A number of experiments with multiple flavonoids and different lipids, suggests that their interaction with membranes relies on establishing a hydrogen-bond among the hydroxyl groups of the flavonoid and the polar headgroups of phospholipids (11).

Putting rosmarinic acid to the test

A 2011 study decided to put rosmarinic acid to the test and see if it was useful against lipid peroxidation as well as investigating its interaction with lipid membranes (12). The interaction of rosmarinic acid with lipid membranes had at this point never been fully explored nor had that interaction been linked to the antioxidant properties of rosmarinic acid.

The research team used liposomes of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) to investigate the ability of rosmarinic acid to inhibit lipid peroxidation and also to measure the amount of polyphenols that interacted with the lipid membrane. To determine the antioxidant ability of rosmarinic acid the team prepared small unilamellar vesicles containing phospholipids with polyunsaturated chains, DPLC. They then mixed these preformed vesicles with varying dosages of rosmarinic acid then incubated the samples. The researchers then measured the lipid activity at various time points to assess lipid peroxidation.

The results

Dosages are indicated as follows in the charts: 0.25 μM (open circle); 1 μM (closed circle); 1.5 μM (open square); 1.7 μM (closed square); 2 μM (open triangle); 7.5 μM (closed triangle) and 60 μM (closed diamond).

(A). Results (from three independent experiments) were expressed as percentages of peroxidation and plotted versus time. The percentage of peroxidation for each concentration of rosmarinic acid was deduced from the control experiment and was plotted versus time.

(B). The percentage of peroxidation obtained at 60 min was plotted versus the concentration of rosmarinic acid. The percentage of peroxidation for each concentration of rosmarinic acid was deduced from the control experiment and was plotted versus time.

As you can see from the results, the peroxidation percentage increased over time for all the rosmarinic acid doses tested. Within an hour the DLPC had become fully peroxidized at 0.25 μM, however, at concentrations greater than 2 μM lipid peroxidation levels remained below 20% and the higher the dose the less peroxidation occurred in that time.

(C). These data points were fitted with a dose–response curve.

Finally the research team calculated the percentage of peroxidation at 60 minutes and plotted it against the concentration of rosmarinic acid. The resulting curve allowed them to work out that the concentration of rosmarinic acid needed to inhibit peroxidation by 50% is 1.51 μM.


These results confirm that rosmarinic acid is a highly efficient inhibitor of lipid peroxidation as well as being able to insert itself into lipid membranes. They went on to show that rosmarinic acid is able to reside in the membrane close to the headgroups of lipids, a region accessible to polar molecules such as hydrosoluble radicals from the bulk phase. They also discovered that the insertion of 1 mol% of rosmarinic acid was not sufficient to alter membrane structure or fluidity.

Most importantly, it showed for the first time that a small amount of rosmarinic acid is able to spontaneously insert itself inside the membrane, and is the reason why it is able to prevent lipid peroxidation so efficiently. This suggests that rosmarinic acid could help to protect the plasma membranes of cells effectively against oxidative stress and subsequent free radical damage.

Rosmarinic acid has long been used in traditional herbal medicines and the herbs and plants it comes from have been favorites in many cultures for centuries. As science is revealing even more of the secrets of polyphenols like this it gives even more more compelling reason to consider taking rosmarinic acid as part of your daily health regime.



(1) López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M., & Kroemer, G. (2013). The hallmarks of aging. Cell, 153(6), 1194-1217.
(2) Halliwell, B., & Gutteridge, J. M. (1990). [1] Role of free radicals and catalytic metal ions in human disease: An overview. Methods in enzymology, 186, 1-85.
(3) Kühn, H., & Borchert, A. (2002). Regulation of enzymatic lipid peroxidation: the interplay of peroxidizing and peroxide reducing enzymes. Free Radical Biology and Medicine, 33(2), 154-172.
(4) Wang, X., & Quinn, P. J. (1999). Vitamin E and its function in membranes. Progress in lipid research, 38(4), 309-336.
(5) Braverman, N. E., & Moser, A. B. (2012). Functions of plasmalogen lipids in health and disease. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1822(9), 1442-1452.
(6) Morandat, S., Bortolato, M., Anker, G., Doutheau, A., Lagarde, M., Chauvet, J. P., & Roux, B. (2003). Plasmalogens protect unsaturated lipids against UV-induced oxidation in monolayer. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1616(2), 137-146.
(7) Dragsted. (2003). Antioxidant actions of polyphenols in humans. International journal for vitamin and nutrition research, 73(2), 112-119.
(8) Fraga, C. G., Galleano, M., Verstraeten, S. V., & Oteiza, P. I. (2010). Basic biochemical mechanisms behind the health benefits of polyphenols. Molecular aspects of medicine, 31(6), 435-445.
(9) Oteiza, P. I., Erlejman, A. G., Verstraeten, S. V., Keen, C. L., & Fraga, C. G. (2005). Flavonoid-membrane interactions: a protective role of flavonoids at the membrane surface?. Journal of Immunology Research, 12(1), 19-25.
(10) Gutiérrez, M. E., Garcı́a, A. F., de Madariaga, M. A., Sagrista, M. L., Casadó, F. J., & Mora, M. (2003). Interaction of tocopherols and phenolic compounds with membrane lipid components: evaluation of their antioxidant activity in a liposomal model system. Life Sciences, 72(21), 2337-2360.
(11) Verstraeten, S. V., Keen, C. L., Schmitz, H. H., Fraga, C. G., & Oteiza, P. I. (2003). Flavan-3-ols and procyanidins protect liposomes against lipid oxidation and disruption of the bilayer structure. Free Radical Biology and Medicine, 34(1), 84-92.
(12) Fadel, O., El Kirat, K., & Morandat, S. (2011). The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1808(12), 2973-2980.