PALM OIL TOCOTRIENOLS AND ITS POTENTIAL AGAINST ALZHEIMER’S DISEASE AND BRAIN ISCHEMIA

Many neurodegenerative disorders and syndromes are associated with an excessive generation of reactive oxygen species (ROS) and oxidative stress. In the past few years, the pathophysiological role of ROS has been intensively studied in in-vitro and in vivo models of chronic neurodegenerative disorders such as Alzheimer’s disease (AD) and stroke. In AD, oxidative neuronal cell dysfunction and cell death caused by protofibrils and aggregates of the AD-associated amyloid β protein (Aβ) may causally contribute to pathogenesis and progression. ROS and reactive nitrogen species also take part in the complex cascade of events and the detrimental effects occuring during ischemia and reperfusion in stroke. Lately tocotrienols have gained increasing attention as its therapeutic potential against neurodegenerative disorders was investigated. Compared to tocopherols, recent evidences have shown that tocotrienols are more potent antioxidant. In this review, the potential of tocotrienols to provide protection against selected neurodegenerative disorders is discussed.


Introduction
A lot of research efforts into the elucidation of the mechanisms of neurodegeneration in neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and stroke has led to an increased understanding of the basic mechanisms of nerve cell death. In general, cell death can follow two basic pathways; apoptosis and necrosis. Apoptosis which is also known as programmed cell death, is an active form of cell degeneration and is executed by enzymes such as caspases. On the other hand, necrosis is frequently occurs during acute insults and is characterized by rapid cell lysis. Both process of cellular degeneration are frequently associated with an excessive generation of free radicals and other cellular events which later lead to oxidative stress 1,2,3 . By definition, the term oxidative stress refers to the excessive oxidation of cellular biomolecules that leads to cellular damage, which is carried out by reactive oxygen species (ROS) including superoxide radicals (O 2 . -), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH.), nitric oxide (NO.), and metabolites thereof 4 . Numerous types of radicals with diverse lifetimes, reactivities, and oxidative potencies exist, but some of the most biologically detrimental ones are formed, for instance, during the reduction of molecular oxygen to water in mitochondria.
The high reactivity of certain ROS causes oxidative damage. Oxidation may affect the DNA, which may lead to nucleotide dimerization and ultimately to errors during the replication process. Lastly, they frequently affect membrane lipids, or more specifically, the unsaturated carbohydrate side chains of phospholipids that are preferred sites for oxidation, leading to membrane dysfunction and cell lysis 5 . ROS are regular byproducts of cellular physiology. When generated in excess, for example in the mitochondria or after peractivation of intracellular oxidases, ROS can have destructive effects. In general, however, many ROS also exert vital functions that range from host defense to neuronal signal transduction. In order to maintain the balance between ROS generation and detoxification, as well as to prevent ROS from accumulates, several cellular defense systems exist. These comprise of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT)and glutathione peroxidase (GPx), and nonenzymatically actine compounds, for example; ascorbate, vitamin C, glutathione (GSH) and vitamin E. Mitochondrial dysfunction, various endogenous oxidase systems (eg. Cyclooxygenases) and other enzymes activated by exogenous triggers, which include neurotoxins, may cause overproduction and IJBAR (2012) 03 (09) www.ssjournals.com accumulation of ROS which may lead to oxidative stress 6 . The evidence that suggest oxidative stress is tightly associated with the neurodegenerative disorders is increasing. These disorders include Alzheimer's disease (AD), Parkinson's disease (PD), stroke, traumatic brain injury (TBI) and amyotrophic lateral sclerosis (ALS). Although the specific sources of the accumulating oxidants and the affected target structures may differ between the particular pathologies in the nervous system, general features can be defined that include: 1. Increased levels of oxidatively altered metabolites are found in the post mortem tissue in neurodegenerative disease, 2. Compensatory defense reactions (oxidative stress response) can be seen in the affected nerve cells, 3. Disturbances of mitochondrial metabolism are observed which may account for an increased leakage of ROS originating from the reactions of the respiratory chain. The brain appears to be particularly vulnerable to oxidations. Neuronal brain tissue is characterized by: 1. High energy requirements and high oxygen consumption, 2. An enrichment in peroxidizable fatty acids, 3. High levels of transition metals, 4. A relative deficit of antioxidant defenses compared with other tissues.

Vitamin E family: Tocopherol and
Tocotrienol: In 1922, Herbert Evans and Katherine Bishop, two prominent researchers from Berkeley, first isolated fat-soluble vitamin E from green leafy vegetables and described it as a fertility factor. Deficiency of this vitamin is now known to cause severe degenerative diseases such as ataxia, Duchenne muscular dystrophy-like muscle degeneration, and infertility 7 . Vitamin E is present in most edible oils to various extents, including those extracted from wheat germ oil, wheat, rice bran, barley, oats, coconut and palm. While alpha-tocopherol was the first vitamin E analogue to be recognized, eight chemically distinct analogues are now known, consisting of alpha (α), beta (β), gamma (γ) and delta (δ)-tocopherols (TP), and α-, β-, γ-, and δ-tocotrienols (T3). Tocopherols are the saturated forms of vitamin E, whereas the tocotrienols are unsaturated and possess as isoprenoid side chain ( Figure 1).  Palm tocotrienol-rich fraction (TRF) is an extract of palm oil and consists of 25% αtocopherol and 75% tocotrienols. TRF has been shown to possess potent antioxidant 9 , 10 , antiinflammatory 11 , anticancer 12, 13,14 , neuroprotection 15,16 and cholesterollowering 17,18,19 activities. Previously, most studies have been focused on the antioxidant property of tocopherol. However, its passage through the blood brain barrier (BBB) is limited. Compared to tocotrienol, its unsaturated side chain allows for more efficient penetration into tissues that have saturated fatty layers such as the brain and liver 20 . In this review, the potential of tocotrienol to provide protection against selected neurodegenerative disorders is discussed.

Tocotrienol and Alzheimer's disease (AD)
AD is characterized symptomatically by progressive cognitive and memory decline, speech loss and personality changes. It is histopathologically characterized by: 1. Synaptic alterations, 2. Nerve cell loss in certain brain regions, www.ssjournals.com 3. Formation of protofibrils of amyloid β protein (Aβ) and extracellular deposition of amorphous Aβ, 4. The intracellular aggregation of hyperphosphorylated tau protein A commonly held hypothesis regarding the development of AD is that it is due to altered processing of the amyloid precursor protein (APP), which leads to excessive β-amyloid (Aβ) formation or aggregation 21 . Several genes are known to cause AD in a small percentage of patients. All these genes, for example, APP gene on chromosome 21, presenilin 1 gene on chromosome 14, and presenilin 2 gene on chromosome 1, are associated with an increase in β-amyloid formation 22 . The apolipoprotein (apo) E-4 genotype is a significant risk factor for AD. Similar to the other genes that cause AD, the APOE*E4 allele is associated with increased amyloid deposition. Aβ aggregates then trigger the activation of microglia, inducing the production of oxidants, cytokines and prostanoids, that work in various ways to: (1) boost the production of Aβ by neurons and astrocytes; (2) kill neurons, either directly or by increasing their sensitivity to excitotoxicity; (3) disrupt neuron structure and function by promoting excessive phosphorylation of tau, giving rise to the characteristic neurofibrillary tangles; (4) disrupt the protective function of astrocytes 23 . It has been demonstrated that treatment of neuronal cells with TRF is able to reduce neurotoxicity caused by the exposure to Aβ protein 24 . In this study, neuronal cells were divided into 4 groups; untreated cells served as control, treated with 10 µM Aβ peptide for 24 hours, 5 µg/ml TRF for 24 hours followed by 10 µM Aβ peptide for 24 hours (pre-treatment) or 10 µM Aβ peptide for 24 hours followed by 5µg/ml TRF for 24 hours (post-treatment). From this study, results showed that when neurons were exposed to 10 µM Aβ peptide, percentage of DNA damage is significantly higher compared to control. Treatment with TRF significantly lowers the DNA damage. Pre-and post-treatment with 5 µg/ml TRF also significantly increased the number of viable cells, which demonstrated that TRG was able to prevent cell death induced by the Aβ peptide. This observation is further supported through fluorescence staining, where cells that treated with TRF still able to retain their membrane integrity. Aβ can induce ROS production through multiple pathways. One way is by binding metal-ions (Fe 2+ , Cu 2+ ), which then can drive the production of OH . from peroxides through the Fenton reaction. Initial lysis of neurons occurring in AD tissue also can leads to the release of excitatory amino acids such as glutamate, which subsequently induces oxidative processes either though binding to NMDA receptor or through its competetition for the neuronal cystine antiporter system leading to a depletion of intracellular redox-shuttle glutathione, GSH 25,26 . Aβhas been reported to produce H 2 O 2 through the metal ion reduction pathway 27 . In the past few years, more studies have been conducted to observe the neuroprotective effects of tocotrienol against peroxides. Tocomin 50% which contains αtocopherol, and α-, γ-, and δ-tocotrienols significantly inhibited H 2 O 2 -induced neuronal death using primary cultures of rat striatal neurons, in contrast toα-tocopherol, it failed to reduce H 2 O 2 -induced neuronal death 28 . In this study, both the LDH release assay and MTT assay revaled that treatment of striatal cultures with various concentrations (10-100 µM) of H 2 O 2 for 24 hours resulted in a concentrationdependent reduction in cell viability. Then the cultures were exposed to 30 µM H 2 O 2 in the presence or absence of Tocomin 50% (0.1-10 µM) for 24 hours. As expected, H 2 O 2 caused neuronal death, however, Tocomin 50% produced a concentration-dependent suppression of H 2 O 2 -induced neurotoxocity, with significant protection at concentrations of 1 and 10 µM. This study also demonstrated that, in terms of neuroprotective efficacy in rat striatal cultures, vitamin E analogs ranked as follows: αtocotrienol > γ-tocotrienol > δ-tocotrienol. According to indices of protein oxidation and lipid peroxidation, however, γ-tocotrienol is most effective followed by α-tocotrienol, δtocotrienol and α-tocopherol 29,30 . The suggestion that tocotrienols can protect nerve cell from hydrogen peroxide toxicity is further supported by other study. In a recent study, the death of neurons that have been exposed to various concentrations of H 2 O 2 were prevented when treated with 5 µM of vitamin E isoforms 31 . The preventive effects of α-and γtocotrienols were significantly stronger than αtocopherol. To detect early events of cell degeneration, neurite formation was checked with phase-contrast microscopy. Treatment with H 2 O 2 induced abnormal morphology and significantly induced neurite beading, which was prevented by treatment with α-and γtocotrienols. This study also focused the effect of vitamin E treatment towards the changes of IJBAR (2012) 03(09) www.ssjournals.com collapsin response mediator proteins (CRMPs). These proteins belong to a family of cytoplasmic proteins in the brain and plays a crucial role in neurite polarity and axon guidance 32,33,34 . Changes in CRMP-2 level have been implicated in various neurodegenerative disorders, including AD, ischemia and Wallerian degeneration 35,36 . Administration of α-or γtocotrienol significantly attenuated the induction of unusual CRMP-2 protein in H 2 O 2 -treated neurons. The effects of H 2 O 2 toxicity towards viability and apoptosis of astrocytes also has been assessed by the MTS, LDH release assays (viability) and ssDNA assay (apoptosis) by using primary culture of astrocytes. Cell viability decreased by 50% when exposed to 100 µM of H 2 O 2 , and at more than 200 µM H 2 O 2 , almost all cells were dead. Pre-treatment with 1-10 µM γ-tocotrienol protected cells against H 2 O 2 -induced cell loss significantly 37 . α-Tocotrienol and γ-tocotrienol in µM concentration have also been reported to be more effective than α-tocopherol in protecting against glutamate-induced cell death in HT4 neuron cell cultures 20 . More interestingly, 100 nM of α-tocotrienol has been showed to be able to protect HT4 hippocampal neurons 16,38 and primary cortical neurons against glutamateinduced cell death. There are several other studies have verified that tocotrienols were more effective antioxidants than α-tocopherol. For example, α-tocotrienol exhibited a higher level of antioxidant activity against lipid oxidation in rat liver microsomal membranes than αtocopherol 10 , and greater peroxyl radicalscavenging activity in liposomes 20 . TRF has been reported to be more effective than αtocopherol in protecting rat brain mitochondria and rat liver microsomes against oxidative damage 29,30 .

Tocotrienols and Stroke (Brain ischemia)
Free radicals have also been linked to a variety of neurological disorders and brain injuries accompanied by acute nerve cell death 4,39 . In focal and global cerebral ischemia, cerebral blood and oxygen flow is reduced in brain regions that are normally supplied by the occluded vessels. Brain damage associated with stroke is dependent on the severity and the duration of the interrupted blood supply to the area. Superoxide, O 2 .and nitric oxide, NO. are generally believed to play major roles in the development of cerebral ischaemic damage 40 . Reperfusion leads to an abrupt supply of glucose and oxygen to previously deprived neurons, and this disrupts mitochondrial function by uncoupling oxidative phosphorylation and bringing about inhibition of complex I. These processes increase superoxide generation. In addition, disturbed intracellular homeostasis, including increased calcium levels, activates nitric oxide synthase, NOS to produce nitric oxide. The high concentration of superoxide and nitric oxide thus form peroxynitrie, ONOO-, which can directly induce damage or converted to other strong oxidants. In addition to the direct oxidative damage to cellular macromolecules, ROS may also induce secondary events that mediate ischemia-related cellular changes. As major intracellular oxidative stress-sensitive targets, mitochondria, intracellular signaling cascades and transcription factors have been identified. ROS generated inside the mitochondria during ischemia/reperfusion cause the release of cytochrome c, which then interacts with the factors Apaf-1 and caspase-9, ultimately leading to the activation of apoptosis-associated caspases 4 . Downstream activation of caspase 3 leads to DNA-fragmentation and apoptosis. Mitochondria, however, are well known to be involved in necrotic pathways, and there is strong evidence that it depends on the severity of the ischemic insult, the particular cell type affected, and the nature of the intracellular signaling status, whether necrosis, apoptosis or intermediate forms of cell death occur. It has been demonstrated that if mitochondrial ATP production is completely stopped during ischemia, necrosis occurs 41,42 . The pathogenetic role of the excitatory amino acid L-glutamate in ischemic damage is of particular interest. Glutamate in an excitatory neurotransmitter that is present throughout the central nervous system (CNS). Elevated levels of extracellular glutamate however, are neurotoxic 26 . Glutamate toxicity is a major contributor to pathological cell death within the nervous system. Two forms of glutamate toxicity are present; receptorinitiated excitotoxicity 43 and non-receptor mediated toxicity 44 . The physiological consequences of extracellular glutamate are mediated by three classes of membrane proteins within the CNS. These are ionotropic glutamate receptors, metabotropic glutamate receptors and the cystine/glutamate antiporter. Glutamate acts post-synaptically on three families of ionotropic receptors, named after their preferred agonists, N-methyl-D-aspartate (NMDA), α-amino-5methyl-4-isoxazolepropionic acid (AMPA) and IJBAR (2012) 03(09) www.ssjournals.com kainite. These receptors all incorporate ion channels that are permeable to cations, although the relative permeability to Na + and Ca 2+ varies according to the family and subunit composition of the receptor. In addition, glutamate can activates a family of G-protein receptors, referred as metabotropic glutamate receptors 45 . Both families of receptors are situated on various aspects of an excitatory synapse, including the pre-synaptic terminal, the postsynaptic terminal and astrocytes that sheath the synapse 46 (Engelman and MacDermott, 2004). Another target for extracellular glutamate in the central nervous system (CNS) is through the inhibition of the glutamate/cystine antiporter X c -, which is responsible for the transportation of cystine that is required for the synthesis of GSH. The major tocotrienol-sensitive signaling pathways which are known to be involved in glutamate-induced neurodegeneration include c-Src and 12-lipoxygenase 16,38,47,48 . The c-Src and its structurally related members of the Src family are non-receptor tyrosine kinases that reside within the cell associated with cell membranes and appear to transduce signals from transmembrane receptors to the cell interior. Treatment of HT4 neuronal cells with elevated levels of extracellular glutamate resulted in more than 95% loss of cell viability within a duration of 12 hours, and this glutamate-induced cell death was prevented when pre-treated with nanomolar concentration of α-tocotrienol 16 . In this study, it was shown that, on a concentration basis, α-tocotrienol was more effective than αtocopherol in protecting HT4 cells against glutamate-induced cytotoxicity. A dosedependent study of α-tocotrienol and αtocopherol showed that at a concentration of 50 nM α-tocotrienol, but not α-tocopherol, partially protected the cells against glutamate-induced death. At 250 nM α-tocotrienol, but not αtocopherol, provided complete protection against loss of cell viability. Comparison of the two analogues of tocotrienol, α-and γ-, showed that α-tocotrienol was more effective than γtocotrienol in protecting against glutamate. Vitamin E and its analogs are known to be potent inhibitors of 5-LOX 49 , Vitamin E is also known to inhibit 15-LOX acitivity by specifically complexing with the enzyme protein 50 . A central role of inducible 12-LOX has been proposed in the execution of glutamateinduced neuronal death 57,58 . It has been reported that 12-LOX is subjected to rapid tyrosine phosphorylation in neuronal cells challenged with glutamate or GSH-lowering agents. Such phosphorylation is rapid and coincides with the c-Src activation. Previous studies have reported that rapid c-Src activation plays a central role in executing neurodegeneration 16,38 . It was also demonstrated in the other report that Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke 51 . It was also supported by other observation that Src family kinase-inhibitor PP2 reduces focal ischemic brain injury 52 . Further investigation has identified the 12-lipoxygenase (12-LOX) as a key tocotrienol-sensitive mediator of neurodegeneration 38 . This study showed that neurons isolated from 12-LOX-deficient mice are resistant to glutamate-induced cell death, which established that the 12-LOX indeed represents a critical checkpoint in glutamateinduced neurodegeneration. Further observation on in vivo model revealed that tocotrienolsupplemented rats showed more protection against stroke-induced injury compared to to controls 48 . Such protection was associated with lower c-Src activation and 12-LOX phosphorylation at the stroke site. Neurons and the brain are rich in arachidonic acid (AA). Massive amounts of AA are released from the membranes in response to brain ischemia or trauma 53 . Subsequent work has established that AA and its metabolites may be neurotoxic. There are three major pathways of AA metabolsim lipoxygenases, cyclooxygenases and cytochrome P450. In the lipoxygenase pathway, metabolites of 12-LOX seem to be the major metabolite of AA in the brain 54 as well as in cultured cortical neurons 55 (Ishizaki and Murota, 1991). Lipoxygenases, mainly 5-, 12-, and 15-LOX, are named after their ability to insert molecular oxygen at the 5, 12, or 15carbon atom of AA forming a distinct hydroperoxy-eicosatetraenoic (HPETE) acid 56 . 12-LOX produces 12(S)-HPETE which is further metabolized into 4 distinct products; an alcohol [12(S)-HETE], a ketone (12-ketoeicosatetraenoic acid), or two epoxy alcohols (hepoxilin A3 and B3). Using immature cortical neurons and HT cells, it has been shown that a decrease in intracellular concentration of GSH, [GSH]i triggers the activation of neuronal 12-LOX, which leads to the production of peroxides, the influx of Ca 2+ , and ultimately cell death 57,58 . The 12-LOX metabolite 12-HPETE proved to be capable of causing cell death 59 . Highly reactive oxygen radicals are produced during the conversion of HPETEs to HETEs 60 , contributing to the overall burden of oxidative stress following stroke. Under conditions of IJBAR (2012) 03(09) www.ssjournals.com GSH depletion, as in acute focal stroke, 12-LOX-derived 12-HPETE triggers nitric oxide (NO)-induced neural cell death 61 . 12-HETE has been shown to increase mitochondrial NO production, induce cytochrome c release, and subsequently cause mitochondrial dysfunction 62 . It has been proved that α-tocotrienol prevents glutamate-induced cell death in active c-Src overexpressing cells 16 (Sen et al., 2000). Furthermore, glutamate-induced c-Src activity is completely blocked by nanomolar amounts of αtocotrienol. Taken together, the powerful effects of palm oil-derived α-tocotrienol on c-Src and 12-LOX suggest that it is a potent inhibitor of 12-LOX-mediated AA metabolism and neurodegeneration.

Conclusion
Oxidative damage to cellular molecules and structures are related to the pathogeneses of neurodegenerative disorders. AD and stroke are among the most prominent examples. Previously, most works related to vitamin E are mainly focused on the antioxidative ability of αtocopherol. However, its passage through the blood brain barrier is limited. Compared to tocotrienol, its unsaturated side chain allows for more efficient penetration into tissues that have saturated fatty layers such as the brain and liver. Other than its neuroprotective property, tocotrienols has also demonstrated therapeutic potential in the treatment of cancer and hypercholesterolemia. Based on these increasing evidence, these isoforms of vitamin E remain as the most potent antioxidant available that could play a major role in the fight against neurodegenerative disorders.