The Neuroprotective Properties of Rosmarinic Acid

Rosmarinic acid has been shown to be a neuroprotective agent in both in vitro in vivo experiments. Today we will be reviewing the mechanisms by which rosmarinic acid induces neuroprotection and looking at a number of supporting studies.

Data for the bioavailability of rosmarinic acid is limited, however, some researchers have shown that rosmarinic acid is metabolized in mammals, including in humans. A study by Al-Sereti et al. showed that rosmarinic acid is absorbed by both and skin and gastrointestinal tract (1). Another study by Ritschel et al. explored the absorption properties of rosmarinic acid and showed that the absolute bioavailability was 60% after topical application on the skin of rats (2). In addition to this impressive finding, rosmarinic acid was also found to be present in the lung, brain, liver, heart, spleen and bone tissue of the rats after intravenous administration by the same research team.

In humans, Baba et al showed that rosmarinic acid and its metabolites are excreted in the urine within six hours of consumption (3). The dosage was 200 mg via oral consumption in this experiment. This means that rosmarinic acid is able to reach brain cells and elicit several effects via a number of different mechanisms which we will discuss in the following article.

In vitro studies

A study by Lee et al. showed that rosmarinic acid in the range of 14-56 µM 30 minutes before being exposed to hydrogen peroxide exhibited a cytoprotective effect on SH-SY5Y neuroblastoma cells (4). At 56 µM, rosmarinic acid blocked caspase-3 activation and cell apoptosis in the SH-SY5Y neuroblastoma cells. With a dosage of 28 and 56 µM but not 14 µM, rosmarinic acid prevented the increase of ROS production caused by exposure to the hydrogen peroxide.

Some of this antioxidant effect was caused by the induction of Heme oxygenase 1 (HO-1) expression, the researchers confirmed this by using zinc protoporphyrin an inhibitor of HO-1 which prevented the antioxidant effects of rosmarinic acid. The induction of HO-1 was via a mechanism associated with the activation of protein kinase A (PKA) and phosphoinositide-3-kinase (PI3K).

This was confirmed by the research team by treating the cells with PKI, a compound that inhibits PKA or LY294002, an inhibitor of P13K. Using either of these inhibitors suppressed the effect of rosmarinic acid on the induction of HO-1. Indeed the study authors observed that there was an increased level of apoptosis in the cells treated with these protein kinases inhibitors.

In a study by Du et al. the research team demonstrated that pretreatment with rosmarinic acid at a dose of 10-9 mol/L for 30 min, MES23.5 protected dopaminergic cells against toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposure (5).

Rosmarinic acid prevented the loss of cell viability and the release of lactate dehydrogenase (LDH) and also prevented the decrease in dopamine production in MES23.5 cells exposed to MPTP. Rosmarinic acid created an antioxidant effect by decreasing the level of ROS production generated by exposure to MPTP. Rosmarinic acid was also able to attenuate the loss of mitochondrial membrane potential caused by MPTP exposure.

Another study by Dashti et al, found that rosmarinic acid at 30-100 µM over 24 hours protected mouse cerebellar granule cells against toxicity induced by potassium dichromate (K2 Cr2 O7) exposure (6). Co-treatment of rosmarinic acid and K2 Cr2 O7 counteracted loss of cell viability in mature cerebellar granule cells, although it did not protect immature neurons. Rosmarinic acid was also shown to suppress the increased production of reactive oxygen species caused by exposure to K2 Cr2 O7 toxicity. However, rosmarinic acid at 3 and 10 µM however, did not show any beneficial effects.

In 2014 a study by Ghaffari et al.showed that rosmarinic acid counteracted the pro-oxidant and cytotoxic effects of Hydrogen peroxide (H2 O2) exposure (7). Rosmarinic acid co-treatment inhibited the loss of cell viability caused by the H2 O2) in a dose dependent manner in the range of 1 – 50 µM. Not only that but rosmarinic acid was also shown to be effective at preventing the release of lactate dehydrogenase (LDH) in cells exposed to H2 O2.

In the range of 1-25 µM for 24 hours, cells exposed to rosmarinic acid were protected from increased ROS production caused by exposure to H2 O2 in N2A cells. Rosmarinic acid was also able to block genotoxicity induced by H2 O2 in the same study.

Interestingly in this same study a dosage of 25 µM for 24 hours of rosmarinic acid also increased the expression of tyrosine hydroxylase, an important regulator of the antioxidant response in cells.  Treatment also increased the level of brain-derived neurotrophic factor (BDNF) in N2A cells. In the brain, BDNF is released by nerve cells or by support cells, such as an astrocytes, and then binds to a receptor on a nearby nerve cell. This then prompts the increased production of proteins associated with nerve cell survival and function.

This shows that rosmarinic acid was effective at protecting N2A neuroblastoma cells against damage caused by exposure to prooxidant agents and was also able to modulate neurotransmission and neurotrophic signaling in a beneficial way. The study authors do not detail the exact mechanism by which rosmarinic acid is able to exert these effects, the data is useful nonetheless.

A study by Braidy et al. showed that rosmarinic acid had a neuroprotective effect on primary human neurons (8). The study authors found that pretreatment of rosmarinic acid in the range of 0.01 – 0.1 mg/mL protected primary neurons against damage from ciguatoxin (CTX). Ciguatoxin is a toxic molecule obtained from the dinoflagellate microalgae Gambierdiscus and causes neurological dysfunction in humans.

Rosmarinic acid once again as in previous studies, decreased LDH release from primary neurons and protected neuronal cells against nicotinamide adenine dinucleotide (NAD+) depletion caused by exposure to CTX. It is worth noting that NAD+ is a key player in DNA and mitochondrial DNA repair and its loss is thought to contribute significantly to the aging process (9-10). NAD+ facilitates DNA repair via the Poly (ADP-ribose) polymerase (PARP) enzyme (11-13).

Braidy et al demonstrate that rosmarinic acid prevented the DNA damage caused by CTX in their study thus helping to maintain genomic integrity. Genomic instability is thought to be one of the primary reasons we age therefore preventing it could be beneficial to health and avoiding various age-related diseases (14).

Additionally, NAD+ is linked to the function of the sirtuins, a family of genes linked to longevity and aging (15) and also the tumour suppressor protein, p53 (16).

In vivo studies

Rosmarinic acid has also been tested in in vivo experiments in regards to its ability to protect brain cells from chemically induced damage. A study in 2012 by Wang et al. showed that rosmarinic acid at a dose of 20 mg/kg via intragastric administration for 21 days protected rat striatum from exposure to 6-hydroxydopamine (6-OHDA) and subsequent damage (17).

Rosmarinic acid was able to prevent the decrease in dopamine and Tyrosine hydroxylase levels caused by exposure to the 6-OHDA. Thus, rosmarinic was able to prevent the loss of Tyrosine hydroxylase positive neurons in the striatum of the rats.

The method by which it protected striatal cells from the 6-OHDA was via increasing Bcl-2 (a gene family that allows the cell to resist apoptosis) and decreasing levels of the Bax proteins, a regulator of apoptosis. Treatment with rosmarinic acid allowed the cells to become more resistant to damage and subsequent cell death. Finally, rosmarinic acid was effective at reducing the level of iron-positive cells present in the striatum exposed to the 6-OHDA. Thus, rosmarinic acid was able to protect the cells against iron overload caused by 6-OHDA, this was very likely by decreasing the rate of reaction of iron with hydrogen peroxide via the Fenton reaction (18).

Mushtaq et al. observed an antioxidant effect from rosmarinic acid in the brains of rats exposed to streptozotocin (STZ). At a dose of 10 mg/kg via intragastric administration for 21 days, rosmarinic acid was able to suppress lipid peroxidation inducing by the STZ in the hippocampus, striatum and cerebral cortex (19). The rosmarinic acid also prevented an increase in acetylcholinesterase activity caused by exposure to the STZ in all the areas of the rat brain mentioned previously.

A 2010 study by Shimojo et al. showed that rosmarinic acid was effective at protecting mouse brains in an experimental model of familial amyotrophic lateral sclerosis (FALS) using human SOD1 G93A transgenic mice (20). In humans with FALS there is impairment of SOD1 function and also an increase in microglial activity suggesting neuroinflammation plays a role in the disease (21-23). Therefore, a compound that has antioxidant and anti-inflammatory effects is a potential treatment for FALS.

In the study the authors observed that FALS model mice given rosmarinic acid at a dose of 013 mg/kg via intraperitoneal injection twice a week over a period of 14 days, showed a significant increase in survival. They also showed improvement in a number of cognitive tasks.

In addition to this, neurons in the anterior horn of the lumbar spinal cord appeared to have augmented size in comparison to the control mice. Thus, rosmarinic acid treatment reduced the degeneration of motor neurons leading to a reduction of impairment and an improvement of behaviour in the mice.

In 2013 Luan et al. reported that rosmarinic acid had anti-inflammatory effects in vitro on SH-SY5Y neuroblastoma cells and in vivo in a rat model of diabetes (24). Rosmarinic acid was shown to be effective at decreasing the loss of cell viability caused by exposure to oxygen-glucose deprivation (OGD) in the SH-SY5Y neuroblastoma cells. The study authors also observed that rosmarinic acid induced cytoprotective effects by particularly inhibiting lactate dehydrogenase leakage.

Rosmarinic acid also inhibited cell apoptosis and the tumor necrosis factor-α (TNF-α)-induced activation of NF-κB, as well as partially blocking NF-κB binding activity. NF-κB is a protein complex and a master regulator of the inflammatory process and is generally considered to be central to the aging process.

A dosage of 25-200 mg/kg of rosmarinic acid via intravenous administration 30 minutes after inducing cerebral ischemia/reperfusion injury decreased the infarct size and brain water content in the test animals. Indeed, rosmarinic acid at 50 mg/kg via intravenous administration given at 1, 3, 5 and even 7 hours after injury had very similar effects.

The authors also described that rosmarinic acid at 50 mg/kg via intravenous administration decreased Evans blue extravasation (a measure of brain blood barrier integrity) and myeloperoxidase (MPO) activity (a measure of inflammation) in the mice. The authors also note that at this dosage rosmarinic acid also suppressed activation of the NF-κB protein complex thus showing anti-inflammatory effects via the modulation of the inflammatory response pathway.

Confirming this data an earlier study by Swarup et al.observed that rosmarinic acid at 25 mg/kg twice a day via intraperitoneal injection exerted an anti-inflammatory effect in mice exposed to Japanese encephalitis virus (25).

Rosmarinic acid decreased the number of activated microglia and downregulated the expression of proinflammatory cytokines, including the NF-κB protein complex, the master regulator of inflammatory response. Rosmarinic acid was effective at reducing the replication of the virus in the brain and the subsequent inflammation it causes.

Conclusion

Rosmarinic acid displays antioxidant and anti-inflammatory action in brain cells as well as cytoprotective action. However the mechanisms of action are not yet fully understood and therefore further research is needed ascertain all the mechanisms are involved.

It should be noted that the dosages used in these in vitro experiments are not usually utilized in in vivo testing, so the concentrations should be considered very high compared to those present in living animals, this is demonstrated in the bioavailability studies for example.

Given this context, the protective effects observed in in vitro studies may not translate to humans, therefore further study is needed. Toxicological analyses are needed due to the risk of rosmarinic acid intoxication and to establish a therapeutic dose for disease treatment. Studies to explore ways to improve the bioavailability would also be very useful as well as research focused on ways to deliver rosmarinic acid to target cells and tissues.

 

References

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