Vitamin E therapy beyond cancer: Tocopherol versus tocotrienol

    Vitamin E therapy beyond cancer: tocopherol versus tocotrienol Hong Yong Peh, W.S. Daniel Tan, Wupeng Liao, W.S. Fred Wong PII: DOI: Reference:

S0163-7258(15)00229-6 doi: 10.1016/j.pharmthera.2015.12.003 JPT 6842

To appear in:

Pharmacology and Therapeutics

Please cite this article as: Peh, H.Y., Daniel Tan, W.S., Liao, W. & Fred Wong, W.S., Vitamin E therapy beyond cancer: tocopherol versus tocotrienol, Pharmacology and Therapeutics (2015), doi: 10.1016/j.pharmthera.2015.12.003

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ACCEPTED MANUSCRIPT P&T 22896

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Vitamin E therapy beyond cancer: tocopherol versus tocotrienol

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Hong Yong Peh a, W.S. Daniel Tan a, Wupeng Liao a, W.S. Fred Wong a,b,*

Department of Pharmacology, Yong Loo Lin School of Medicine, National University

Health System, Singapore

Immunology Program, Life Science Institute, National University of Singapore, Singapore

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b

*Corresponding Author and Reprint Request:

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W.S. Fred Wong, PhD

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Department of Pharmacology, Yong Loo Lin School of Medicine 16 Medical Drive, Block MD3, #04-01

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National University of Singapore, Singapore 117600

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Tel: 65-6516-3266; Fax: 65-6776-6872; E-mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT (250 / 250 words) The discovery of vitamin E (α-tocopherol) began in 1922 as a vital component required in

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reproduction. Today, there are eight naturally occurring vitamin E isoforms, namely α-, β-, γ-

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and δ-tocopherol and α-, β-, γ- and δ-tocotrienol. Vitamin E are potent antioxidants, capable

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of neutralizing free radicals directly by donating hydrogen from its chromanol ring. αTocopherol is regarded the dominant form in vitamin E as the α-tocopherol transfer protein in the liver binds mainly α-tocopherol, thus preventing its degradation. That contributed to the

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oversight of tocotrienols and resulted in less than 3% of all vitamin E publications studying

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tocotrienols. Nevertheless, tocotrienols have been shown to possess superior antioxidant and anti-inflammatory properties over α-tocopherol. In particular, inhibition of 3-hydroxy-3methylglutaryl-coenzyme A reductase to lower cholesterol, attenuating inflammation via

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downregulation of transcription factor NF-κB activation, and potent radioprotectant against radiation damage are some properties unique to tocotrienols, not tocopherols. Aside from

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cancer, vitamin E has also been shown protective in bone, cardiovascular, eye, nephrological and neurological diseases. In light of the different pharmacological properties of tocopherols

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and tocotrienols, it becomes critical to specify which vitamin E isoform(s) are being studied in any future vitamin E publications. This review provides an update on vitamin E therapeutic potentials, protective effects and modes of action beyond cancer, with comparison of tocopherols against tocotrienols. With the concerted efforts in synthesizing novel vitamin E analogues and clinical pharmacology of vitamin E, it is likely that certain vitamin E isoform(s) will be therapeutic agents against human diseases besides cancer.

Keywords: antioxidant; cardiovascular; cholesterol; inflammation; pharmacokinetics; radioprotectant

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ACCEPTED MANUSCRIPT Contents 1. Introduction History

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Biochemical and physical properties of Vitamin E isoforms

1.3

Sources of vitamin E

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1.1

2. Bioavailability and pharmacokinetics of vitamin E 3. Vitamin E as an antioxidant

4.2

Cardiovascular diseases

4.3

Diabetes

4.4

Eye disorders

4.5

Inflammatory diseases

4.6

Lipid disorder

4.7

Nephropathy

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Neurological diseases

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Radiation damage

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Bone diseases

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4.1

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4. Vitamin E therapy beyond cancer

5. Current limitations and developments of vitamin E 5.1

Limitations

5.2

Current developments

6. Summary and future outlook Conflict of interest statement Acknowledgement References

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ACCEPTED MANUSCRIPT Abbreviations α-TS, α-tocopherol succinate; AKT, protein kinase B; αTTP, α-tocopherol transport protein;

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Bcl-2, B-cell lymphoma 2; C/EBP, CCAAT-enhancer binding protein; CAT, catalase; CNS,

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central nervous system; COX, cyclooxygenase; ERK, extracellular-signal-regulated kinase;

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G-CSF, granulocyte-colony stimulating factor; GPx, glutathione peroxidase; GSH, glutathione; HDL, high-density lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutarylcoenzyme A; IL, interleukin; JNK, c-Jun N-terminal kinase; LDL, low-density lipoprotein;

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LOX, lipoxygenase; LT, leukotriene; MAPK, mitogen-activated protein kinase; MDA,

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malondialdehyde; MPP+, 1-methyl-4-phenylpyridinium ion; NF-κB, nuclear factor κ-lightchain-enhancer of activated B cells; NO, nitric oxide; NO2, nitrogen dioxide; NOS, nitric

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oxide synthase; Nrf-2, nuclear factor (erythroid-derived 2)-like 2; ONOO-, peroxynitrite;

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PI3K, phosphatidylinositide 3-kinase; PG, prostaglandin; PKC, protein kinase C; PPAR, peroxisome proliferator-activated receptor; RNS, reactive nitrogen species; ROS, reactive

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oxygen species; SOD, superoxide dismutase; STAT6, signal transducer and activator of transcription 6; T3, tocotrienol; TBARS, thiobarbituric reactive substances; TGF-β1,

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transforming growth factor-β1; TNF-α, tumor necrosis factor-α; TP, tocopherol; TRF, tocotrienol rich fraction; WMLs, white matter lesions.

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ACCEPTED MANUSCRIPT 1. Introduction 1.1 History

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Vitamin E is one of the most widely consumed vitamins known for its antioxidant capacity

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and multiple health benefits. In 1912, Casimir Funk coined the term ―vitamine‖ (obtained

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from vital and amine, meaning amine of life) as it was hypothesized that vitamine was chemical amines present in micronutrient food factors which prevented beriberi and other dietary-deficiency diseases (Funk, 1912). It was validated subsequently that not all vitamine,

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such as vitamin C and vitamin E, contained amine group, thus it was shortened to ―vitamin‖

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in English. The discovery of vitamin E began in 1922 by Herbert Evans and Katherine Bishop when they isolated an uncharacterized fat-soluble compound from green leafy

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vegetables which is required for reproduction (Evans & Bishop, 1922). Upon identifying the

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compound in 1924, it was termed as tocopherol (obtained from Greek words tokos and phero, meaning to bear children), also known as α-tocopherol presently. The search beyond α-

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tocopherol isoform started in 1947 by Stern et al, and it was in 1964 when tocotrienols were first discovered (Sen et al., 2007). Despite its discovery in 1964, it was only until 1980s

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where tocotrienol research took off in various diseases, which triggered the debate of supremacy between tocopherols and tocotrienols. α-Tocopherol was the main focus of vitamin E research and regarded as the major isoform with the most potent antioxidant and biological activity. However, recent studies have shown otherwise, where tocotrienols may triumph with its superior anti-cholesterolemic (unique to tocotrienols only), antioxidant, anticancer, anti-inflammatory, cardioprotective and neuroprotective properties. Despite the steady growth in publications of tocotrienols since 1966, it comprises merely 3% of all vitamin E research papers listed in PubMed (Fig. 1). Currently, there are eight naturally occurring isoforms in the vitamin E family.

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ACCEPTED MANUSCRIPT 1.2 Biochemical and physical properties of vitamin E isoforms Vitamin E can be classified into tocopherols and tocotrienols, resulting in a total of eight

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isoforms: namely α-, β-, γ-, and δ-tocopherol, and α-, β-, γ-, and δ-tocotrienol (Fig. 2).

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Vitamin E in its pure form is a yellow viscous oil/liquid that oxidizes readily when exposed

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to light, oxygen and transition metal ions. They are not water-soluble but dissolved in alcohol, organic solvents and vegetable oils (Bramley et al., 2000). They have the same basic chemical structure where a C16 isoprenoid side chain is attached at the C-2 of a chromane ring.

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Tocotrienols differ from tocopherols with an unsaturated farnesyl isoprenoid side chain at C-

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3’, C-7’ and C-11’ over a saturated phytyl isoprenoid side chain (Fig. 2). The greek letter prefixes of both tocopherols and tocotrienols depend on the number and position of methyl groups on the chromanol ring, whereby α-isoform is 2,5,7,8-tetramethyl; β-isoform is 2,5,8-

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trimethyl; γ-isoform is 2,7,8-trimethyl; and δ-isoform is 2,8-dimethyl. Therefore, using the αisoform, the structural terminology for α-tocopherol is 2,5,7,8-tetramethyl-2-(4,8,12-

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trimethyltridecyl)-6-chromanol

while

α-tocotrienol

is

2,5,7,8-tetramethyl-2-(4,8,12-

trimethyldeca-3,7,11-trienyl)-6-chromanol. Tocochromanols are amphipathic molecules with

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a polar chromanol ring and lipophilic isoprenoid side chain. The unsaturated isoprenoid side chain of tocotrienols potentially enhances its penetration into fatty tissues, such as brain and liver, and better distribution on the cell membranes (Suzuki et al., 1993). Vitamin E in nature consists of the dextrorotatory enantiomers only with a single stereoisomer. Tocopherols contain three chiral stereocenters at C-2, C-4’ and C-8’ (e.g.: d-RRR-α-tocopherol), while tocotrienols contain only one chiral stereocenter at C-2 as the other two chiral stereocenters are not possible with C=C unsaturation in the isoprenoid tail (Colombo, 2010).

1.3 Sources of vitamin E

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ACCEPTED MANUSCRIPT In nature, vitamin E can be found in vegetables, plants and plant oils. The distribution of tocopherols in the plant kingdom is primarily α-tocopherol in green leafy plants and γ-

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tocopherol in non-green plant parts such as fruits and seeds (Zielinski, 2008). Common food

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sources of α-tocopherol can be found in almonds, avocados, hazelnuts, peanuts and sunflower

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seeds; β-tocopherol in oregano and poppy seeds; γ-tocopherol in pecans, pistachios, sesame seeds and walnuts; δ-tocopherol in edamame and raspberries (obtained from United States Department of Agriculture nutrient database, http://ndb.nal.usda.gov/). Common food oils

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including corn, peanut and soybean oil contains largely α-tocopherol and γ-tocopherol. In

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contrast, tocotrienols are relatively rarer in food sources and plant kingdom. It was suggested that tocotrienols are bioconverted to tocopherols and may function as intermediates to

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biosynthesize α-tocopherol in plants (Pennock, 1983). In plants, tocotrienols can be found in

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the seeds of monocotyledonous plants, fruits of dicotyledonous plants [list of plants can be found in (Horvath et al., 2006; Zielinski, 2008)] and latex of rubber trees (Hevea brasiliensis)

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(Horvath et al., 2006). Tocotrienols levels thrive in abundance in plant foods, namely rice bran oil, palm oil and annatto seeds (ratio of tocopherol to tocotrienol in plant oils – 45:55,

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30:70 and 0:100 respectively). The extraction of crude palm oil from oil palm (Elaeis guineensis) fruits can reach up to 800 mg/kg of tocotrienols, consisting mainly of αtocotrienol and γ-tocotrienol (Theriault et al., 1999). Annatto seeds are unique as unlike rice bran and palm oil, it contain only tocotrienol (mostly δ-tocotrienol), but no tocopherols (Frega et al., 1998). Tocotrienols can also be acquired in cereal grains like barley, oat, rice, rye and wheat. They are characterized with higher content of tocotrienols to tocopherols within the kernels (ratio of tocopherol to tocotrienol – 21:79, 23:77, 33:67, 49:51 and 45:55 respectively). Table 1 lists the various sources of vitamin E and its respective components of tocopherols and tocotrienols.

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ACCEPTED MANUSCRIPT Synthetic forms of vitamin E consist mainly of α-tocopherol, and it was first synthesized in 1938 (Karrer et al., 1938). Unlike naturally occurring d-RRR-α-tocopherol, synthesized α-

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tocopherol consists of a racemic mixture of eight stereoisomers [all-rac-α-tocopherol (RRR-,

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RRS-, RSS-, RSR-, SRS-, SSR-, SRR- and SSS-α-tocopherol)] (Kiyose et al., 1997). Various

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attempts to synthesize tocotrienols were not as successful with tocopherols, as it only produced low to moderate yields and purity (Netscher, 2007).

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2. Bioavailability and pharmacokinetics of vitamin E

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Studies in the past two decades have shown that both α-tocopherol transport protein (αTTP) and ω-hydroxylase are vital components for the absorption of vitamin E, α-tocopherol in

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particular (Hosomi et al., 1997; Sontag & Parker, 2002). αTTP in the liver facilitates the

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packing of vitamin E into lipoproteins and transportation to other tissues via the bloodstream. While αTTP has a high affinity to α-tocopherol (100%), it has lower affinity for other vitamin

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E isoforms: approximately 50% for β-tocopherol, 10-30% for γ-tocopherol and 1% for δtocopherol (Brigelius-Flohe & Traber, 1999; Traber, 2007). A study has also shown that

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αTTP has 8.5 fold lower affinity to bind to α-tocotrienol than α-tocopherol (Hosomi et al., 1997). In the liver, unbounded isoforms of vitamin E to αTTP will be susceptible to catabolization via cytochrome P450 (CYP4F2)-initiated ω-hydroxylation and oxidation by ωhydroxylase (Jiang, 2014). Due to these two antagonizing interactions, α-tocopherol is regarded as the predominant isoform to be accumulated in tissues where other vitamin E isoforms

are

metabolized

to

carboxychromanols,

hydroxycarboxychromanol

and

carboxyethyl-hydroxychroman derivatives (for tocotrienols) (Birringer et al., 2002; Lodge et al., 2001). With that in mind, it led to a huge debate for a decade if orally administered tocotrienols can reach vital organs in the body, which dampened the research on tocotrienols in the 1990s. Despite the fact that αTTP has a lower affinity towards tocotrienols, it is not

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ACCEPTED MANUSCRIPT clear, or to what extent, the transport of orally administered tocotrienols to vital organs depends on αTTP. In one study, female mice were rendered infertile when αTTP is knockout,

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where they suffered from α-tocopherol deficiency in spite of dietary α-tocopherol

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supplementation (Jishage et al., 2001). Interestingly, oral supplementation of α-tocotrienol to

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female mice restored fertility in these αTTP knockout mice under α-tocopherol deficiency (Khanna et al., 2005a). This suggests other transport machinery or mechanisms for the

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absorption and transport of tocotrienols beyond the αTTP.

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Following up on the debate of tocotrienol viability, pharmacokinetics studies on tocotrienols were performed. A study in 2003 determined the pharmacokinetics of α-, γ-, and δ-tocotrienol

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via intramuscular, intraperitoneal, intravenous and oral routes in rats (Yap et al., 2003). The

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absorption of tocotrienols administered via intramuscular and intraperitoneal routes was negligible and should be avoided. Tocotrienols have incomplete absorption and limited

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bioavailability in rats, where the bioavailability of α-tocotrienol was approximately 28%, and 9% for both γ- and δ-tocotrienols (Yap et al., 2003). The time required to reach peak plasma

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concentration for α-, γ-, and δ-tocotrienol were 3.3, 3.0 and 2.8 hours respectively, while the half-life was approximately 3 hours for α-tocotrienol and 2 hours for γ- and δ-tocotrienols in rats (Yap et al., 2003). In humans, the half-life of α-, γ-, and δ-tocotrienol in plasma was estimated to be 2.3, 4.4 and 4.3 hours, respectively (Yap et al., 2001), while the half-life of αtocopherol and γ-tocopherol were 57 and 13 hours, respectively (Leonard et al., 2005). In a separate double-blind placebo-controlled study, human subjects took tocotrienol supplements at a dose of 250 mg/day for 8 weeks, where the mean plasma levels of α-, γ-, and δtocotrienol were 0.8 µM, 0.54 µM and 0.09 µM (O’Byrne et al., 2000). As tocotrienols are oil-based compounds, it faces limited bioavailability. As emulsions are known to increase absorption of fat-soluble compounds, based on self-emulsifying drug delivery systems

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ACCEPTED MANUSCRIPT technology (Gao & Morozowich, 2005), self-emulsifying formulations of tocotrienols were produced, such as Tocovid™ SupraBio™ and Naturale³. The supplementation of Tocovid

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SupraBio™ elevated peak plasma concentration of α-tocotrienol in humans to approximately

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3 µM, a threefold increase as compared to 0.8 µM in previous study without self-

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emulsification (Khosla et al., 2006).

Although tocotrienols are not well-absorbed in the liver, they appear to be similarly absorbed

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as tocopherols with dietary fat and are secreted into chylomicron particles. The chylomicron-

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bound vitamin E isoforms were reported to be transferred to peripheral tissues such as adipose tissues, bones, brain, lung, muscle and skin, via the lymphatic system (Ikeda et al., 2001; Uchida et al., 2012). This might explain the accumulation of non-α-tocopherol

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isoforms of vitamin E in peripheral tissues, despite the fact of low uptake in the liver αTTP. A study involving Caco2 human epithelial colorectal cells elucidated that absorption of γ-

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tocotrienol supersedes that of α-tocopherol (Tsuzuki et al., 2007). The rapid epithelial transport of tocotrienols over tocopherols in Caco2 cells were attributed to the difference in

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saturation of the isoprenoid side chains, where unsaturated tocotrienols were more lipophilic. The toxicological aspect of tocotrienol is relatively safe, where human subjects fed with 250 mg/day for 8 weeks (O’Byrne et al., 2000), and female rats fed with 130 mg/kg daily for 13 weeks to one year displayed no side effects (Nakamura et al., 2001; Tasaki et al., 2008).

3. Vitamin E as an antioxidant Vitamin E is widely accepted as one of the most potent antioxidant. The antioxidant property is attributed to the hydroxyl group from the aromatic ring of tocochromanols, which donates hydrogen to neutralize free radicals or reactive oxygen species (ROS). In homogenous solutions and in vitro assays, the reaction rates between the vitamin E isoforms largely

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ACCEPTED MANUSCRIPT depends on the number of methyl groups on the chromanol ring. The antioxidant activity of α-, β- and γ-isoforms of both tocopherol and tocotrienol are similar, except the δ-isoform has

weaker

activity,

when

tested

in

pyrogallolsulfonphthalein

and

2,7-

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dichlorodihydrofluorescein diacetate assays (Peh et al., 2015; Yoshida et al., 2007).

However, the distinction of antioxidant activities in biological systems between tocopherols and tocotrienols is clear. Free radicals attack on cell membrane results in peroxyl radicals and

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lipid peroxidation which is responsible for hypercholesterolemia and cardiovascular diseases.

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Studies have displayed the superiority of α-tocotrienol over α-tocopherol in neutralizing the peroxyl radicals and lipid peroxidation in rat liver and liposomal membranes (Ghafoorunissa et al., 2004; Serbinova et al., 1991; Suzuki et al., 1993). In HUVEC (human umbilical vein

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endothelial cell) cells, palm tocotrienol rich fraction (TRF) was more efficient than αtocopherol in attenuating thiobarbituric reactive substances (TBARS) (Ghafoorunissa et al.,

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2004). In rat brain mitochondria, γ-tocotrienol elicited stronger protective effect against oxidative damage (Kamat & Devasagayam, 1995). Interestingly in Caenorhabditis elegans,

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administration of tocotrienols abated protein carbonylation and extended mean life span, where α-tocopherol supplementation had no effect (Adachi & Ishii, 2000). This could be explained with a few possible mechanisms. Firstly, tocotrienols are more uniformly distributed in the cell membrane bilayer (Palozza et al., 2006). Secondly, tocotrienols have a stronger disordering effect on phospholipids due to its unsaturated isoprenoid side chain which results in its ―arc‖ conformation over the chromanol ring, thus rendering a more effective interaction to lipid radicals (Serbinova et al., 1991; Simone et al., 2008). Lastly, tocotrienols have a higher recycling efficiency from its chromanoxyl radicals over tocopherols (Packer et al., 2001; Theriault et al., 1999).

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ACCEPTED MANUSCRIPT The antioxidant efficacy of vitamins E on reactive nitrogen species (RNS) is gaining more attention recently. RNS includes nitric oxide (NO), nitrogen dioxide (NO2) and peroxynitrite

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(ONOO-). The reaction of α-tocopherol with NO2 yields a nitrosating agent, while γ-

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tocopherol reduces NO2 to NO without generating nitrosating species (Cooney et al., 1995). It

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was hypothesized that possessing an unsubstituted 5-position on the chromanol ring, γ- and δisoforms, but not α- and β-isoforms of vitamin E, renders the ability to neutralize RNS (Christen et al., 1997). The scavenging of NO2 and ONOO- by γ-tocopherol is superior to that

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of α-tocopherol, which resulted in the formation of 5-nitro-γ-tocopherol. This was proven in a

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study where 5-nitro-γ-tocopherol was detected in the plasma of zymosan-induced peritonitis in rats (Christen et al., 2002). Similarly in radiological threat, nitric oxide synthase (NOS) is

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stimulated and produced RNS, where only prophylactic administration of γ-tocotrienol

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Ghosh et al., 2009).

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among the vitamin E isoforms protected mice from radiation damage (Berbée et al., 2009;

4. Vitamin E therapy beyond cancer

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4.1 Bone diseases

Osteoporosis is a metabolic bone disease, which is manifested with the degeneration of bone density and microarchitecture, leading to bone fragility and fracture. The causes of osteoporosis include estrogen deficiency due to menopause in women, testosterone deficiency in men, prolonged use of glucocorticoid, chronic smoking, alcohol abuse, inflammatory bowel syndrome, and so on (Iniguez-Ariza & Clarke, 2015). Both oxidative stress and inflammation are implicated in the pathogenesis of osteoporosis. As anti-oxidative and anti-inflammatory agents, the effects of vitamin E isoforms have been widely explored in a variety of experimental osteoporosis models.

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ACCEPTED MANUSCRIPT The effects of tocopherol supplementation on bone are inconsistent and somehow contradictory (Chin & Ima-Nirwana, 2014a). High-dose -tocopherol was shown to exert

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negative effect on bone in normal animals but was protective in stressed animals (Arjmandi et

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al., 2002; Hampson et al., 2015; Smith et al., 2005). A recent study showed that there was

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increased bone resorption and decreased bone mass in mice fed with high-dose -tocopherol, probably due to increased osteoclast fusion and differentiation (Fujita et al., 2012), which was however, not supported by another report (Iwaniec et al., 2013). Some studies exhibited

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beneficial effects of -tocopherol on bone loss while others did not but instead showing

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improved bone quality (Chai et al., 2008; Feresin et al., 2013). When compared with tocotrienols, tocopherols revealed either comparable or less effective in protecting bone in

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animals (Hermizi et al., 2009; Maniam et al., 2008; Mehat et al., 2010; Muhammad et al.,

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2012).

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The bone-protective effects of tocotrienols have been demonstrated in various animal models which were subjected to estrogen deficiency, testosterone deficiency, glucocorticoid, nicotine

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and oxidizing reagents. In general, the studies have shown that tocotrienols increased osteoblast number, mineral deposition, and bone formation activity and decreased osteoclast number, erosion on bone, and bone resorption activity, thus preventing the degeneration of bone mineral density and bone microarchitecture in osteopenic animals. These effects could be attributed to various activities exerted by tocotrienols.

It is well-established that oxidative stress is implicated in the development of osteoporosis (Ibanez et al., 2014). Supplementation of tocotrienols reduced oxidative stress product malondialdehyde (MDA), preserved and increased antioxidant enzyme activities in vivo (Abd

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ACCEPTED MANUSCRIPT Manan et al., 2012; Maniam et al., 2008). In an in vitro study, it was shown that -tocotrienol

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homologue decreased oxidative damage on primary osteoblast culture (Nizar et al., 2011).

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Tocotrienols exerted its biological actions not only by anti-oxidant property, but by

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suppressing mevalonate pathway which promotes bone loss by regulating osteoblastogenesis and osteoclastogenesis through activation of GTPase (Mo et al., 2012). A recent study found that mice supplemented with emulsified -tocotrienol via subcutaneous injection were

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significantly protected from ovariectomy-induced bone loss as evaluated by various bone

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structural parameters, bone metabolic gene expression levels and serum levels of biochemical markers for bone resorption and bone formation (Deng et al., 2014). The effect of tocotrienol

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was achieved by downregulating the activity of 3-hydroxy-3-methylglutaryl-coenzyme A

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(HMG-CoA) reductase.

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Other mechanisms by which tocotrienol executes its bone-protective activity include preventing proinflammatory cytokines such as IL-1, IL-6 and TNF-, and enhancing genes

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related to bone formation and osteoblast activity as such alkaline phosphatase, beta-catenin, collagen type I  1, and osteopontin (Chin & Ima-Nirwana, 2014b; Norazlina et al., 2010; Norazlina et al., 2007).

4.2 Cardiovascular diseases Atherosclerosis is a chronic inflammatory disorder that occurs as a result of lymphocyte infiltration to the arterial wall, smooth muscle cell proliferation and damage in the arterial wall caused by extracellular matrix accumulation. The effects of TRF on the microscopic development of atherosclerosis and lipid peroxidation in the aorta of rabbits have been investigated (Nafeeza et al., 2001). After 10 weeks of treatment with TRF, cholesterol-fed 14

ACCEPTED MANUSCRIPT rabbits had lower aortic contents of MDA, less intimal thickening and greater preservation of the internal elastic lamina, consistent with reduced lipid peroxidation as compared to the

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rabbits fed with normal diet. Another study showed that tocotrienol-enriched palm oil

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prevented atherosclerosis through modulating the activities of peroxisome proliferator-

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activated receptors (PPAR) (Li et al., 2010). Recently, a clinical trial was conducted to compare TRF (180 mg tocotrienols, 40 mg tocopherols) with placebo (0.48 mg tocotrienols, 0.88 mg tocopherols) in patients undergoing chronic hemodialysis, in which accelerated

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atherosclerosis might be contributed by dyslipidemia, inflammation and an impaired

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antioxidant system (Daud et al., 2013). TRF supplementation showed improvement in lipid profiles in terms of plasma total cholesterol, triglycerides, and high-density lipoprotein

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(HDL), when compared with placebo. The changes in the TRF group were associated with

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higher plasma apolipoprotein A1 level and lower cholesteryl-ester transfer protein activity. These studies suggest TRF supplementation improved lipid profiles to prevent atherosclerosis.

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It is rather controversial for the effects of tocopherol isoforms in atherosclerosis models. In a study of chronic ethanol consumption-induced atherosclerosis in animals, elevation of

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inflammatory cells, disorganization of endothelium and increase in pro-inflammatory mediators were observed in the ethanol group. Significant amelioration of endothelial wall changes, along with the restoration of elevated mediators were found in -tocopherol-treated animals (Shirpoor et al., 2013). However, quite on the contrary, dietary supplementation with -tocopherol (400 IU/d) oxidized HDL-2 and HDL-3, surprisingly showing a proatherogenic effect (Wade et al., 2013). Data on the superior effect of another tocopherol isoform tocopherol over -tocopherol were described in terms of preventing oxidative stress, where tocopherol but not -tocopherol supplementation reduced biomarkers of oxidative stress in patients with metabolic syndrome (Devaraj et al., 2008). In a clinical study conducted in healthy men, the supplementation of -tocopherol-rich mixture of tocopherols attenuated 15

ACCEPTED MANUSCRIPT postprandial hyperglycemia-induced MDA level, oxidative stress and vascular endothelial

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dysfunction, independent of inflammation (Mah et al., 2013).

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Myocardial ischemia reperfusion injury resulted from severe impairment of coronary blood

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supply produces a spectrum of clinical syndromes. Although all of the tocotrienol isoforms have shown some degree of cardioprotection, -tocotrienol was found to be the most potent in boosting pro-survival genes in myocardial ischemic injury model (Das et al., 2008). The

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differential interaction of mitogen-activated protein kinase (MAPK) with caveolin 1/3 in

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conjunction with proteasome stabilization was proposed to play a unique role in tocotrienolsmediated cardioprotection. Another study investigated the cardioprotective property of -

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tocotrienol and/or resveratrol. Both agents showed the protection alone, and furthermore

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acted synergistically, providing a greater degree of cardioprotection than either alone, in an ischemia reperfusion model (Lekli et al., 2010). The protective effect was proposed to be

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their ability to induce autophagy as well as generate Akt-Bcl-2 survival pathway which might help to recover the cells from injury. Myocardial ischemia reperfusion injury induced by

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global ischemia and cold-induced functional hyperthyroidism was also investigated by tocopherol treatment (Mukherjee et al., 2008; Venditti et al., 2011). It was observed that tocopheryl phosphate exhibited cardioprotection in a rat global ischemia model by enhancing anti-apoptotic signals such as p42/44 ERK, p38β MAPK, Bcl-2 and Akt, and reducing proapoptotic signals like p38α MAPK, JNK and c-Src (Mukherjee et al., 2008). In addition, tocopherol was also found to attenuate harmful effects of the myocardiac response to coldlinked oxidative stress, probably through restoration of superoxide dismutase (SOD) and catalase (CAT) activities, and recovery of mitochondrial function (Venditti et al., 2011).

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ACCEPTED MANUSCRIPT Thromboembolic events are the likely cause of various clinical pathologies including renal and splenic microinfarctions, stroke and heart failure. The effects of -tocopherol, -

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tocotrienol, or TRF on in vivo platelet thrombosis and ex vivo platelet aggregation were

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compared (Qureshi et al., 2011). After intravenous injection in anesthetized dogs, tocotrienols

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were significantly better than tocopherols in inhibiting cyclic flow reductions, a measure of the acute platelet-mediated thrombus formation, and collagen-induced platelet aggregation. These data suggest tocotrienols provide a better therapeutic benefit in conditions like platelet

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thrombosis, stroke and myocardial infarction, as compared to tocopherols.

4.3 Diabetes

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Diabetes mellitus often results in various complications due to nitrosative and oxidative stress

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induced by high levels of glucose in the blood. A 2004 prospective cohort study demonstrated that the intake of vitamin E reduce the risk of onset of type 2 diabetes after a

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23-year follow-up (Montonen et al., 2004). TRF reduces total cholesterol, low-density lipoprotein (LDL) and total lipid in diabetic patients (Baliarsingh et al., 2005). Patients given

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tocotrienol-enriched canola oil (200 mg/day tocotrienols) also showed significant reduction in serum C-reactive protein (CRP) and urine microalbumin (Haghighat et al., 2014). This could be due to the increase in PPAR target gene expression when Db/Db mice were fed with TRF, increasing insulin sensitivity (Fang et al., 2010). It was further shown in vitro that α-, γ-, and δ-tocotrienols but not α-tocopherol, could bind PPARα and increase its interaction with LXXLL motif of PGC-1α peptide (Fang et al., 2010). Using Wistar rats placed on a diet high in fructose, it was demonstrated that combination treatment with α-lipoic acid and either αtocopherol alone or TRF can both prevent and reversed metabolic and cardiovascular changes in fructose-fed rats (Patel et al., 2011). Wound healing was accelerated by α-tocopherol,

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ACCEPTED MANUSCRIPT together with reduced plasma MDA level and increased glutathione peroxidase activities in

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diabetic rats (Musalmah et al., 2002).

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Impaired endothelial function in streptozotocin-induced diabetic rats was improved with both

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TRF and α-tocopherol that were isolated from palm oil (Muharis et al., 2010). Another study also demonstrated that TRF can reduce erythrocyte membrane MDA levels and leukocyte DNA damage together with increased SOD and glutathione (GSH) in erythrocytes (Matough

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et al., 2014). However, TRF was unable to reduced serum advanced glycation end products in

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diabetic rats, only lowering blood glucose and glycated hemoglobin levels (Wan Nazaimoon & Khalid, 2002). TRF also resulted in reduced serum glucose, glycated hemoglobin

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concentrations, low-density lipoprotein cholesterol, plasma total cholesterol and triglyceride

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levels while having higher SOD activity (Budin et al., 2009). TRF was also shown to have a protective effect on blood vessel walls, lowering levels of MDA and 4-hydroxynonenal

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(Budin et al., 2009).

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In studying diabetes-associated cognitive decline, streptozotocin-induced diabetic mice had dose-dependent reduction of inflammation, NF-κB translocation and cell death through the combined treatment of insulin and tocotrienol (Kuhad et al., 2009). Diabetic neuropathic pain was also shown to be alleviated by chronic treatment of insulin and tocotrienol in a rat model, whilst reducing plasma TNF-α, TGF-β, IL-1β and caspase-3 (Kuhad & Chopra, 2009b). The combined treatment of TRF and α-tocopherol also ameliorated glutamate cytotoxicity in astrocytes (Selvaraju et al., 2014).

4.4 Eye disorders

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ACCEPTED MANUSCRIPT Cataractogenesis, a form of eye disorder characterized by the progression of lenticular opacities, has been shown to be driven by nitrosative and oxidative stress in the lenticular

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cells. Using a galactose-induced experimental cataracts in rats, it was observed that topical

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tocotrienol in the range of 0.01-0.05% reduced nitrosative and oxidative stress, delaying the

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onset and progression of cataract (Abdul Nasir et al., 2014). However, 0.2% or higher concentration of topical tocotrienol led to increased lens oxidative stress and aggravates cataractogenesis (Abdul Nasir et al., 2014). In studying the distribution of vitamin E in ocular

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tissue from topical administration, the level of α-tocotrienol was significantly higher than α-

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tocopherol in administered tissues (Tanito et al., 2004). However, the intraocular penetration of γ-tocotrienol and γ-tocopherol did not differ significantly (Tanito et al., 2004).

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Using Tenon’s capsule fibroblast cultures, indicative of fibrosis from post-glaucoma filtration surgery with excessive fibroblast proliferation and extracellular matrix production, it was

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shown that only α-tocotrienol significantly inhibited growth of fibroblasts, but not other isoforms of vitamin E such as α-tocopherol (Meyenberg et al., 2005). On the other hand, α-

al., 2001).

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tocopherol was also shown to protect rat lens against ultraviolet-B photo damage (Reddy et

4.5 Inflammatory diseases In lipopolysaccharide-induced RAW264.7 macrophage and IL-1β-induced lung epithelial cell models, vitamin E isoforms could attenuate PGD2 and PGE2, respectively, with varying potencies: γ-tocotrienol ≈ δ-tocopherol > γ-tocopherol >> α-/β-tocopherol (Jiang et al., 2008). Likewise, the production of LTB4 and LTC4 in HL-60 cells and human neutrophils when stimulated with ionophore (A23187) was abrogated by γ-, δ-tocopherol and γ-tocotrienol. αTocopherol was much less potent in reducing leukotrienes with IC50 of 40-60 µM as

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ACCEPTED MANUSCRIPT compared to γ-tocotrienol with IC50 of approximately 5 µM (Jiang et al., 2008). It has been shown that γ-tocotrienol was more potent than all tocopherol isoforms in ameliorating

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lipopolysaccharide-induced RAW246.7 macrophage production of IL-6 and G-CSF, probably

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via the downregulation of NF-κB activation and suppression of CCAAT-enhancer binding

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protein (C/EBP) (Wang & Jiang, 2013).

In asthma, it was observed that patients had lower levels of antioxidants in the lungs, which

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sparks off the usage of α-tocopherol (regarded as the main vitamin E isoform back in 1990s)

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for asthma (Kelly et al., 1999). A study showed that oral γ-tocopherol in ovalbumin-induced allergic asthma and rhinitis in rats decreased eosinophilia in the lung, nose and sinus duct

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(Wagner et al., 2008). It also abrogated inflammatory cytokines (IL-4, IL-5 and IL-13) in the

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bronchoalveolar lavage fluid and mucus production in the airways. However, tocopherols in asthma yielded inconclusive outcomes in subsequent studies (Cook-Mills et al., 2013;

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McCary et al., 2011), thus the investigation was shifted towards using tocotrienols for asthma. The screening of different vitamin E isoforms in a house dust mite-induced asthma mouse

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model has identified γ-tocotrienol as the promising candidate in treating asthma (Peh et al., 2015). In that study, 30-250 mg/kg oral γ-tocotrienol attenuated inflammatory leukocytes infiltration into the airways, mucus hypersecretion in the bronchial epithelium, inflammatory cytokines in bronchoalveolar lavage fluid and house dust mite-specific IgE in the serum; and restored antioxidants capacity in the lungs. These were mediated by inhibition of NF-kB nuclear translocation and promoting nuclear Nrf2 level, whereby α-tocopherol failed to modulate NF-κB and Nrf-2 activation. The protective effects of γ-tocotrienol were comparable to those of prednisolone. Notably, γ-tocotrienol was able to decrease neutrophil infiltration into the airways and neutrophils-related cytokines (e.g.: G-CSF), which is not achievable by corticosteroids (Ito et al., 2008). In another study, γ-tocotrienol was also shown

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ACCEPTED MANUSCRIPT to be superior among vitamin E isoforms in suppressing IL-13-induced production of eotaxin3 by inhibiting STAT6 in human lung epithelial A549 cells (Wang et al., 2012). Taken

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together, γ-tocotrienol may have therapeutic potential in treating asthma, and it may be

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considered as an add-on therapy to corticosteroid for optimal asthma control.

As for allergic dermatitis, in a randomized, double-blind, placebo-controlled trial, patients were fed 400 mg/day α-tocopherol for 60 days, and beneficial effects were observed

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(Javanbakht et al., 2010; Kosari et al., 2010). In a murine picryl chloride-induced dermatitis

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model, mice developed scratching behavior, dermal thickening, mast cell degranulation and heightened histamine levels in serum (Tsuduki et al., 2013). Oral 1 mg/day of rice bran

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tocotrienol (97.5% of tocotrienol – 3.5% α-tocotrienol, 89.9% γ-tocotrienol and 4.1% δ-

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tocotrienol) reversed the disease outcome via the inhibition of protein kinase C (PKC)

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activity.

Patients suffering from inflammatory bowel diseases, such as Crohn’s disease and ulcerative

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colitis, were found to have lower levels of vitamin E than normal healthy subjects (Bousvaros et al., 1998). In experimental colitis induced by acetic acid in rats, oxidative damage and both macroscopic and microscopic structural damages in colon were observed. Oral administration of 100 mg/kg/day of α-tocopherol abated oxidative and structural damage on the acetic acidchallenged colon (Bitiren et al., 2010). In a randomized controlled trial of stable but oxidatively stressed Crohn’s disease patients, supplementation of 533 mg/day α-tocopherol for four weeks significantly reduced oxidative stress (Aghdassi et al., 2003). The development of bowel stenosis in Crohn’s disease involves extracellular matrix deposition. Human intestinal fibroblasts isolated from colonic or ileal tissue of Crohn’s disease and ulcerative colitis, when treated with TRF, reduced cell proliferation and enhanced

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ACCEPTED MANUSCRIPT programmed cell death via apoptosis and autophagy of the intestinal fibroblasts (Luna et al., 2011b). In a follow-up study, it was observed that TRF-mediated decrease in extracellular

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matrix production and fibrogenesis of human intestinal fibroblasts was due to inhibition of

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TGF-β1 (Luna et al., 2011a).

In a transgenic KRN/NOD mouse model of human rheumatoid arthritis, treatment with 0.134 mg/day α-tocopherol for six weeks decreased IL-1β, reduced white blood cell counts to those

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of the control, abated oxidative stress and prevented joint destruction. However, it was unable

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to modify the date of onset of disease or alter the disease intensity (Bandt et al., 2002). In a carrageenan-induced air pouch inflammation model, combined γ-tocopherol and aspirin

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provided better anti-inflammatory action than combined α-tocopherol and aspirin or aspirin

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alone. In a collagen-induced rheumatoid arthritis rat model, oral treatment with 5 mg/kg/day γ-tocotrienol for 25 days significantly attenuated synovial fibrosis, congestion and

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hyperplasia in histopathological analysis of joint morphology (Radhakrishnan et al., 2014). In another similar study, 10 mg/kg δ-tocotrienol given for 25 days was found to reduce paw

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edema, restore bodyweight, and suppress collagen-specific T-cells and CRP level (Haleagrahara et al., 2014). These findings suggest that tocotrienols may be a better vitamin E isoform than tocopherols as anti-inflammatory agents.

4.6 Lipid disorder Lipid disorders are a group of conditions that involve high levels of fatty molecules, such as cholesterol and triglycerides in the blood. Tocotrienols research gained traction with the discovery of its cholesterol-lowering properties in the 1980s (Qureshi et al., 1986), via the inhibition of HMG-CoA reductase, a mechanism which is not prevalent in tocopherols (Pearce et al., 1992; Qureshi et al., 2002). Tocotrienols were observed to inhibit HMG-CoA

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ACCEPTED MANUSCRIPT reductase by post-transcriptional suppression of the enzyme itself (Parker et al., 1993). Interestingly, γ-tocotrienol has a 30-fold increase over α-tocotrienol in inhibiting HMG-CoA

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reductase (Teoh et al., 1994). A double-blind 8 weeks pilot study in hypercholesterolemic

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human subjects revealed that γ-tocotrienol may be the most potent cholesterol inhibitor

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among the vitamin E isoforms, where it reduced serum levels of total cholesterol, LDL, apolipoprotein B, thromboxane, platelet factor 4 and glucose (Qureshi et al., 1991). In a 2002 study, it was shown that the American Heart Association’s Step-1 diet and TRF25 (25-200

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mg/day) from rice bran reduced serum total cholesterol, LDL, triglycerides and

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apolipoprotein B in hypercholesterolemic patients (Qureshi et al., 2002). The combination of 270 mg citrus flavonoids and 30 mg tocotrienols in hypercholesterolemic patients also

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reduced serum total cholesterol, LDL, triglycerides and apolipoprotein B (Roza et al., 2007).

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Mixed tocotrienols treatment on hypercholesterolemic patients with non-alcoholic fatty liver disease resulted in a higher percentage having normal hepatic echogenic response (Magosso

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et al., 2013). TRF (180 mg tocotrienols and 40 mg tocopherols) also found beneficial to patients undergoing chronic hemodialysis, decreasing serum total cholesterol, triglycerides,

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LDL and cholesteryl-ester transfer protein activity, whilst increasing serum HDL and apolipoprotein A1 (Daud et al., 2013). However, a 1997 study using palm TRF (40 mg γ- and α-tocotrienols and 16 mg α-tocopherol) only reduced serum TBARS, but serum total cholesterol, LDL-cholesterol and triglyceride levels remained unaffected (Kooyenga et al., 1997). Another similar study in 1999 revealed that oral palm TRF (35 mg tocotrienols and 20 mg α-tocopherol) had no effect on serum lipid level, lipoprotein level or platelet function (Mensink et al., 1999). Both of these earlier studies using TRF preparations with relatively higher proportion of -tocopherol demonstrated no effects on hypercholesterolemia, which might be due to the opposing action by tocopherols on HMG-CoA reductase, as it has been

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ACCEPTED MANUSCRIPT shown that α-tocopherol increased HMG-CoA reductase activity in avian (Qureshi et al.,

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2002).

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Using a hypercholesterolemia and atheroma model of rabbits on atherogenic diet, treatment

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with 50 mg/day TRF (72% γ-tocotrienol, 18% α-, β-tocotrienols, and 10% α-tocopherol) led to a larger reduction in serum total cholesterol, lipid peroxide and LDL as compared to treatment with 50 mg/day α-tocopherol, but their effects were similar for serum triglyceride

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and HDL (Teoh et al., 1994). Another study also demonstrated that TRF reduced aortic

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content of MDA and degree of intimal thickening in treated cholesterol-fed rabbit model of atherosclerosis, preserving the continuity of the internal elastic lamina (Nafeeza et al., 2001).

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Hyperlipidemia induced chronic renal dysfunction was also studied in rats on an atherogenic

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diet, where TRF (100 mg/kg) reduced dyslipidemia and development of chronic renal dysfunction (Rashid Khan et al., 2015). Using human monocyte-derived macrophages to

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study atherogenesis, α-tocotrienol or FeAOX-6 (a novel compound that combines antioxidant structural features of both tocopherols and carotenoids) was shown to reduce cholesterol

2007).

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accumulation in the cells, with α-tocotrienol reducing to a larger extent (Napolitano et al.,

4.7 Nephropathy Nephropathy occurs as a result of damage or disease to the kidney and often requires dialysis as a treatment if a transplant is not possible. There has been development of a vitamin Ecoated membrane which serves to reduce circulating lipid peroxidation during dialysis (Cruz et al., 2008). Membrane-bounded α-tocopherol could effectively reduce 8-hydroxy-2deoxyguanosine (8-OHdG) level in leukocyte DNA of chronic hemodialysis patients (Tarng et al., 2000). It has been demonstrated that membrane coated with α-tocopherol was able to

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ACCEPTED MANUSCRIPT increase red blood cell survivability and decrease serum free 4-hydroxynonenal during

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dialysis (Odetti et al., 2006; Usberti et al., 2002).

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In a model of diabetic nephropathy using streptozotocin-induced diabetic rats, tocotrienol was

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found to be more effective than α-tocopherol in restoring renal function. When combined with insulin, tocotrienol drastically suppressed TNF-α-induced NF-κB and caspase 3 activation (Kuhad & Chopra, 2009a). Another study comparing the effects of palm oil-TRF

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(PO-TRF) and rice bran oil-TRF (RBO-TRF), revealed that PO-TRF was a more effective

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nephroprotective and hypoglycemic preparation for diabetic nephropathy (Siddiqui et al., 2010). This difference could be due to the relatively higher concentration of tocotrienol in

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PO-TRF as compared to RBO-TRF (Siddiqui et al., 2010). Using a model of lipid-induced

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diabetic nephropathy in rats, both PO-TRF and RBO-TRF were shown to improve renal

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function, serum lipid profile and glycemic status (Siddiqui et al., 2013).

Exposure to high levels of chromium compounds or high intake of monosodium glutamate

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can lead to nephrotoxicity. In a potassium dichromate (K2Cr2O7)-induced acute renal injury rat model, TRF (200 mg/kg) treatment preserved kidney proximal reabsorptive function, glomerular function and redox status (Khan et al., 2010). In a monosodium glutamateinduced nephrotoxicity model, α-tocopherol (100 and 200 mg) reduced oxidative stress and restored renal function in rats (Paul et al., 2012).

4.8 Neurological diseases Glutamate is the major mediator of excitatory signals in the mammalian central nervous system. Extreme amounts of glutamate in the extracellular spaces can lead to numerous neurodegenerative diseases. The prevention of glutamate-induced neural cytotoxicity and

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ACCEPTED MANUSCRIPT neurodegeneration by vitamin E has been extensively investigated (Khanna et al., 2010; Khanna et al., 2003; Khanna et al., 2005b; Rati Selvaraju et al., 2014; Selvaraju et al., 2014;

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Sen et al., 2000). Sen and co-workers identified nanomolar -tocotrienols were more

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effective than -tocopherol in preventing glutamate-induced injury and death by suppressing

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c-Src/ERK, 12-lipoxygenase phosphorylation and cytosolic phospholipase A2 (Khanna et al., 2010; Khanna et al., 2003; Khanna et al., 2005b; Sen et al., 2000). In contrast, other

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laboratories found that vitamin E isoforms, both TRF and -tocopherol could protect glutamate-injured neuronal cells and astrocytes through their anti-oxidative properties (Rati

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Selvaraju et al., 2014; Selvaraju et al., 2014).

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External oxidative and neurotoxic agents such as Fe2+ ions, 1-methyl-4-phenylpyridinium ion

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(MPP+) and high-fat diet induced substantial neural cytotoxicity and cause neurological disorders in animal models (Crouzin et al., 2010; Gutierres et al., 2012; Nakaso et al., 2014).

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It was identified that transient receptor potential cation channel V1 was involved in the Fe2+induced neuronal death and a negative modulation of this channel activity by -tocopherol

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might account, at least in part, for the long-lasting neuroprotection against oxidative stress (Crouzin et al., 2010). High-fat diets have an important role in neurodegenerative diseases and neurological disturbances. The effects of high-fat diets on ectonucleotidase activities in synaptosomes of cerebral cortex, hippocampus and striatum of rats were examined, where tocopherol was capable of modulating the adenine nucleotide hydrolysis in that experimental model (Gutierres et al., 2012). It was demonstrated that -/-tocotrienol exhibited not only antioxidant effects but also a receptor signal-mediated neuroprotective action (Nakaso et al., 2014). In a cellular Parkinson's disease model induced by MPP+, they found that PI3K/Akt pathway mediated the cytoprotective effects of tocotrienols via receptor-mediated caveola formation as early events of signal transduction. 26

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Ischemic stroke occurs as a result of an obstruction within a blood vessel supplying blood to

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the brain. -Tocopherol, -tocotrienol and -tocopherol significantly decreased the size of

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cerebral infarcts in the middle cerebral artery occlusion mouse model, while -tocotrienol, -

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tocopherol and -tocotrienol showed no effect (Mishima et al., 2003). Multidrug resistanceassociated protein 1 was further identified as a protective factor against stroke, which was

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regulated by -tocopherol through microRNA-199a-5p (Park et al., 2011).

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Multiple sclerosis is an inflammatory disease of the central nervous system (CNS) that is associated with demyelination and axonal loss, resulting in severe neurological handicap. In

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mouse models, -tocopherol and TFA-12 (a new synthetic compound belonging to

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tocopherol long-chain fatty alcohols) promoted oligodendrocyte regeneration and remyelination (Blanchard et al., 2013; Goudarzvand et al., 2010). Niemann-Pick C disease is

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a fatal neurodegenerative disorder characterized by the accumulation of free cholesterol in the lysosomes of CNS. In Niemann-Pick C mice, -tocopherol delayed loss of weight, improved

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coordination and locomotor function and increased the survival, probably via reduced astrogliosis, nitrotyrosine and phosphorylated p73 in cerebellum (Marin et al., 2014). These observations underscore the essential role of -tocopherol in maintaining CNS function.

Clinical studies were conducted to evaluate the neuroprotective effects of vitamin E supplementation in patients treated with cisplatin chemotherapy which is associated with severe peripheral neurotoxicity in up to 90% of patients (Pace et al., 2010). Oral -tocopherol (400 mg/day) or placebo started before chemotherapy and continued for 3 months after cisplatin treatment in a total of 108 patients. Both the incidence and the severity of neurotoxicity were significantly lower in -tocopherol supplemented group as compared to 27

ACCEPTED MANUSCRIPT the placebo. In a small cohort of multiple sclerosis patients, nine subjects treated for 30 months with -tocopherol failed to decrease disease annual relapse rate (Pantzaris et al.,

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2013). Another clinical study aimed to evaluate the protective activity of mixed tocotrienols

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in humans with white matter lesions (WMLs), which are regarded as manifestations of

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cerebral small vessel disease, reflecting varying degrees of neurodegeneration and tissue damage (Gopalan et al., 2014). A total of 121 volunteers aged ≥35 years with cardiovascular risk factors and magnetic resonance imaging-confirmed WMLs were randomized to receive

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to attenuate the progression of WMLs.

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200 mg mixed tocotrienols or placebo twice a day for 2 years. Mixed tocotrienols were found

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4.9 Radiation damages

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Radiation damage can be lethal at moderate to high doses. Despite its rare occurrence, there is an unmet medical need for humans exposed to radiation leakage. At lower dose of radiation,

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from 0.5-5.5 Gy, it results in hematopoietic syndrome, where blood cells, platelets and bone marrow are damaged, making victims prone to secondary infections and hemorrhage. Patients

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would survive at the lower range, but death prevails within six weeks for 50% of victims beyond 3.5 Gy dose. Moderate doses of radiation, from 5.5-10.0 Gy, affects the hematopoietic cells and gastrointestinal tract, where victims may develop sepsis, as bacteria can translocate through the defective intestinal mucosa barrier into the bloodstream. Death is probable within three weeks of radiation exposure. At high doses beyond 10.0 Gy, radiation damage reaches the central nervous system and causes cognitive dysfunction, cerebrovascular collapse, shock and pneumonitis, where death is certain within two weeks (Waselenko et al., 2004). Current radioprotector include amifostine which has significant side effects and a limited window of efficacy (Brizel et al., 2000; Rades et al., 2004). The transfer of energy from radiation to biomolecules has two actions: direct action – ionization of biomolecules

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ACCEPTED MANUSCRIPT from radiation, and indirect action – radiolysis of water to produce hydroxyl radicals to attack biomolecules (Michaels & Hunt, 1978). Thus, radiation also induces oxidative damage via

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the production of reactive oxygen/nitrogen species by activating xanthine oxidase and nitric

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oxide synthase enzymes (Deliconstantinos et al., 1996; Leach et al., 2001). As radiation

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damage at high doses is irreversible, the research focus shifted towards radiation countermeasure, to prophylactically prevent radiation damage to staff prone to radiation exposure. The radioprotector must have minimal/negligible side effects with antioxidant

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potential and can be consumed at a national level in the emergency like nuclear plant

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explosion or leakage. The use of α-tocopherol as a radioprotector has been found partially effective in mouse models since 1986 (Bichay & Roy, 1986). More recently, γ-tocotrienol

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was shown to be a better radioprotectant than α-tocopherol (Ghosh et al., 2009).

It is now widely accepted that both γ- and δ-tocotrienols are effective radioprotectants

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demonstrated in many pre-clinical studies (Kulkarni et al., 2010). γ-Tocotrienol is considered to be more efficacious than tocopherols against radiation damage due to its superior potency

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in inducing gene expression when tested in human endothelial cells (Berbée et al., 2012). The synergistic radioprotection of γ-tocotrienol (administered subcutaneously at 200 mg/kg dose) and pentoxifylline in mice exposed to 11.5 Gy total body irradiation, was shown to be due to phosphodiesterase inhibition (Kulkarni et al., 2013a). Radioprotectant γ-tocotrienol was also shown to up-regulate expression of G-CSF to promote the proliferation and survival of white blood cells such as neutrophils, thus reducing hematopoietic syndrome (Kulkarni et al., 2013b). This finding was further elaborated when G-CSF antibody co-administered with subcutaneous 200 mg/kg γ-tocotrienol prior to irradiation dampened the anticipated radioprotective effects in mice (Singh et al., 2014a). Gastrointestinal syndrome developed post-irradiation in mouse jejunum was also ameliorated when treated with 200 mg/kg of γ-

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ACCEPTED MANUSCRIPT tocotrienol subcutaneously, via the elevation of anti-apoptotic and down-regulation of proapoptotic factors (Suman et al., 2013). HMG-CoA reductase inhibition was also implicated as

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a key mechanism of γ-tocotrienol radioprophylactic properties as co-administration of

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mevalonate together with γ-tocotrienol conferred no radioprotection to irradiated mice

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(Berbée et al., 2009). Inability of tocopherols to inhibit HMG-CoA reductase may explain why tocotrienols are better radioprotectants (Pearce et al., 1992; Qureshi et al., 1996). δTocotrienol (subcutaneous 150-300 mg/kg) was also effective as a radioprotectant and acted

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through G-CSF as well (Satyamitra et al., 2011; Singh et al., 2014b). It attenuated

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gastrointestinal syndrome post-irradiation by decreasing IL-1β, IL-6 and protein kinase 6 (a

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stress-induced kinase which enhances apoptosis) in mouse intestines (Li et al., 2013).

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5. Current limitations and developments of vitamin E 5.1 Limitations

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One major reason which crippled tocotrienol research would be the inferior bioavailability of oral tocotrienols to that of α-tocopherol. The half-life of α-tocopherol is superior to γ-

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tocopherol or other tocopherol isoforms, and even more so against tocotrienols. This is mainly due to αTTP and ω-hydroxylase, which are responsible for the accumulation of αtocopherol in the body. Due to tocotrienols’ low bioavailability in the body and scarcity in nature, one must consume approximately 80-160 g of palm/rice bran oil, or 1.5-4 kg of wheatgerm/barley/oat to achieve published effective biological doses (Sen et al., 2007). As it is very costly to extract vitamin E from natural sources, particularly tocotrienols where 1 g of γ-tocotrienol cost over US$8,000 (Sigma-Aldrich, St. Louis, MO), chemically or biologically synthesized tocotrienols could bring down the economic concerns to be adopted as therapeutic drugs. α-Tocopherol was successfully synthesized as all-rac-α-tocopherol in 1938, but it is more challenging for tocotrienol synthesis with the presence of 3 C=C double bonds

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ACCEPTED MANUSCRIPT in the isoprenoid side chain. Nonetheless, nature has its way to challenge mankind where studies have shown that the human body is more selective towards the natural vitamin E (all-

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RRR) instead of its synthetic counterpart (all-rac) (Frega et al., 1998; Kosowski & Clouatre,

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2008). Further work on synthesizing tocotrienols as closely as possible to the naturally

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occurring forms is required. Although the acute exposure of tocotrienol is relatively safe, where human subjects were fed with 250 mg/day for 8 weeks (O’Byrne et al., 2000), the dose will escalate in more severe diseases like cancer and autoimmune diseases. The maximum

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tolerated dose of tocotrienol should be determined from more detailed toxicological studies.

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It is also essential to conduct chronic toxicity testing of tocotrienol in preparation for long-

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term usage in chronic diseases.

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5.2 Current developments

Chemical modification of tocopherol such as α-tocopherol succinate (α-TS) is produced by

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substituting the hydroxyl group on the chromanol ring with succinate (Fig. 3). This modification converts α-tocopherol into a radioprotector against radiation damage (11 Gy, 0.6

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Gy/min of 60Co γ-radiation) on the gastrointestinal tract, as determined by analysis of jejunum histopathology. The protective actions of α-TS was mediated via the upregulation of G-CSF and keratinocyte-derived chemokine (KC), where administration of G-CSF antibody abolished the protective effect (Singh et al., 2011). The follow-up study by the same team administered 400 mg/kg of α-TS subcutaneously 24 hours before a lethal dose of radiation observed reduction of DNA damage and apoptotic cells, while enhancing cell proliferation (Singh et al., 2013). α-TS was also shown to activate peroxidase activity of cytochrome c, which may explain its anti-cancer activity (Yanamala et al., 2014). The development of mitochondria-targeted vitamin E analog, Mito-chromanol (Fig. 3), from α-tocopherol depleted intracellular ATP and inhibited ATP-linked oxygen consumption in breast cancer

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ACCEPTED MANUSCRIPT cells, but sparing non-cancerous cells (Cheng et al., 2013). Mito-Chromanol selectively accumulated in tumor tissue of xenograft model of human breast cancer in mice, and

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exhibited both cytotoxicity and anti-proliferative effects of breast cancer cells, via the

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inhibition of glycolysis.

On the other hand, modifications to tocotrienols were performed as well. Tocotrienols which have lower bioavailability in the body and binding affinity to αTTP than tocopherol,

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prompted a research team to design tocoflexol (Fig. 3), where the trienyl chain of the farnesyl

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tail is converted to a dienyl chain (removal of the middle C=C double bond) to increase flexibility (Compadre et al., 2014). This resulted in extended half-life and enhanced bioavailability of δ-tocoflexol, and increased binding affinity to αTTP, while retaining its

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antioxidant potential. Vatiquinone (EPI-743), structurally similar to α-tocotrienol metabolite α-tocotrienol quinone, was developed for the treatment of Leigh syndrome and other

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inherited mitochondrial diseases by Edison Pharmaceuticals Inc. (Fig. 3). Leigh syndrome, a fatal neurodegenerative disorder which affects children mainly, is characterized with

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progressive loss of both mental and movement abilities, which results in death within couple of years (Rahman et al., 1996). A single arm clinical trial of 10 children suffering from Leigh syndrome was treated with EPI-743 daily for six months. All 10 subjects had reversal of disease progression regardless of genetic determinant defect or disease severity, with improvement on movement disorder (Martinelli et al., 2012). Leber hereditary optic neuropathy (LHON) is an inherited optic neuropathy resulting in isolated vision loss, but in some individuals, it extends to other neurological sequelae. The retinal ganglion cell is susceptible to disrupted ATP production and oxidative stress, the primary cell lost in LHON, via point mutations in mitochondrial DNA and mitochondrial dysfunction (Sadun et al., 2011). Five patients with genetically confirmed LHON suffered acute vision loss and were

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ACCEPTED MANUSCRIPT fed with EPI-743 thrice daily at 100-400 mg per dose. Four out of five patients regained vision, where two of them had total recovery and visual acuity, with no reported side effects

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in this study (Sadun et al., 2012). Currently, EPI-743 was approved by FDA (United States of

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and an ongoing Phase-IIb clinical trial for Friedreich’s ataxia.

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America Food and Drug Administration) in July 2014 for the treatment of Leigh syndrome

6. Summary and future outlook

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Vitamin E research is nearly a century long in a couple of years time. The masses know

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vitamin E (α-tocopherol) as a fertility factor and a key component in cosmetic products. However, the public is unaware that there are a total of 8 naturally occurring forms within the

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vitamin E family, and an additional seven relatively physiologically inactive forms created in

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synthetic dl-rac-α-tocopherol (Burton et al., 1998; Traber & Arai, 1999). Tocotrienols, consisting of one half of vitamin E family, has been the subject of approximately 3% of the

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studies published when obtained from PubMed. The scientific community recognizes that vitamin E is one of the most potent antioxidant in protecting biological systems from

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oxidative/nitrosative damage (Cooney et al., 1995; van Acker et al., 1993). In this review, we have presented biological activities of vitamin E beyond cancer in bone, cardiovascular, eye, inflammatory, allergic and neurological diseases, as well as diabetes, lipid disorder and potentially a radioprotectant against radiation damage (Fig. 4). In addition to vitamin E ability to neutralize free radicals as an antioxidant, it has also been shown to modulate signaling pathways including PPAR, C/EBP, STAT6, NF-κB and Nrf2, as well as signaling/inflammatory molecules such as apoptotic regulators (Bcl-2 and caspase-3), cytokines (IL-1β, IL-4, IL-5, IL-6, IL-13, TNF-α, TGF-β and G-CSF), kinases (c-Src, ERK, MAPK, PI3K, PK6 and PKC) and enzymes (AP, Cat, GPx, SOD, eNOS, GTPase, HMGCoA reductase, HO-1, COX-2, 5-LOX, 12-LOX and PLA2) (Table 2).

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α-Tocopherol had been regarded as the main isoform of vitamin E after the discovery of

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αTTP in the liver which selectively binds to α-tocopherol to prevent its degradation (Min et

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al., 2003), thus explaining its much longer half-life in the plasma over the other seven

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isoforms (Leonard et al., 2005). Despite that, γ-tocotrienol was observed to be absorbed significantly faster than α-tocopherol in the intestines (Tsuzuki et al., 2007). Aside from bioavailability issue, tocotrienols were shown to have comparable or superior efficacies in

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various diseases covered in this review (refer to Table 3 for tocopherol versus tocotrienol

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comparison in various diseases). In particular, tocopherols do not have cholesterol-lowering properties via the inhibition of HMG-CoA reductase as observed by tocotrienols (Qureshi et

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al., 2002). Supplementation of tocotrienols has potential to prevent atherosclerosis (Daud et

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al., 2013), and tocotrienols were shown to have better therapeutic benefits over tocopherols in platelet thrombosis (Qureshi et al., 2011). Tocotrienols anti-inflammatory actions are superior

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to α-tocopherol, possibly due to the ability to inhibit activation of STAT6 and NF-κB (Peh et al., 2015; Wang et al., 2012). Both γ-tocopherol and γ-tocotrienol, but not α-tocopherol,

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abrogate COX and 5-LOX-mediated eicosanoids in biological system to reduce inflammation (Jiang, 2014). The use of α-/γ-tocotrienol in neurological diseases was found to have better protective effects than tocopherols in pre-clinical models of glutamate-induced injury and Parkinson’s disease (Khanna et al., 2010; Nakaso et al., 2014). The use of vitamin E as radioprotectant against radiation damage has elucidated γ-tocotrienol as a better candidate over α-tocopherol when mice were exposed to whole body irradiation (Ghosh et al., 2009; Kulkarni et al., 2010). Nonetheless, despite vitamin E (tocopherols) has an established safety record, it is necessary to ascertain the safety of tocotrienols in chronic studies and determine the maximum tolerated dose since tocotrienols differ quite substantially from tocopherols. In

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ACCEPTED MANUSCRIPT the clinical trial registry portal by National Institute of Health, there are only 25 clinical trials

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on tocotrienols out of 927 trials on vitamin E (Table 4).

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In light of this, it is highly recommended that future research claims on vitamin E to specify

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the specific form employed to prevent any confusion. For instance, vitamin E was reported to perform below expectations in clinical trial in preventing cardiovascular diseases, and actually increase all-cause of mortality in test subjects (Miller et al., 2005). It is scientifically

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inaccurate to discount vitamin E on the whole when the study only adopted the use of α-

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tocopherol. In recent decade, vitamin E took quite a hit when clinical trials yielded disappointing or conflicting results (Cook-Mills et al., 2013; Greenberg, 2005; Lee et al., 2005; Lonn et al., 2005; Riccioni et al., 2006). The isoform of vitamin E applied in most

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clinical trials then was α-tocopherol. As shown in clinical trials registered and papers published, tocotrienols holds barely 3% foothold in the entire vitamin E collection. With

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tocotrienols success in various diseases in pre-clinical models, one frontier for tocotrienol research in the next decade shall be the translation from bench to bedside. It would be

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interesting to witness if tocotrienols would be the salvation for vitamin E’s fall from grace.

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ACCEPTED MANUSCRIPT Conflict of Interest Statement The authors declare that there are no conflicts of interest. The funders had no role in this

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review, including decision to publish and preparation of the manuscript.

Acknowledgement

Hong Yong Peh is a recipient of the NUS President Graduate Fellowship and NUS-Industry

Graduate

Fellowship.

This

work

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President

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Relevant Program (IRP) Research Scholarship. WS Daniel Tan is a recipient of the NUS was

supported

in

part

by

NMRC/CBRG/0027/2012 from the National Medical Research Council of Singapore and by

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NUHS Seed Fund R-184-000-238-112.

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Radioprotective properties of tocopherol succinate against ionizing radiation in mice. J Radiat Res 54, 210-220.

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radioprotector, preferentially upregulates expression of anti-apoptotic genes to promote

IP

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D

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MA

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ACCEPTED MANUSCRIPT Yoshida, Y., Saito, Y., Jones, L. S., & Shigeri, Y. (2007). Chemical reactivities and physical effects in comparison between tocopherols and tocotrienols: physiological significance

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and prospects as antioxidants. J Biosci Bioeng 104, 439-445.

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Zielinski, H. (2008). Tocotrienols: distribution and sources cereals – role in human health. In

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Tocotrienols: Vitamin E Beyond Tocopherols (Vol. 1, pp. 23-42): CRC Press, Boca Raton. Zielinski, H., Ciska, E., & Kozlowska, H. (2001). The cereal grains: focus on vitamin E.

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D

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Czech J Food Sci 19, 182-188.

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ACCEPTED MANUSCRIPT Figure Legends Fig. 1. Increasing trend of tocotrienol publications, although it comprises approximately 3%

T

of vitamin E research only. Number of tocopherols (white bars) and tocotrienols (black bars)

IP

publications per year since 1966 identified in PubMed were tabulated. The respective arrows

Information was accessed on 24th September 2015.

SC R

indicate the year where novel therapies beyond cancer by tocotrienols are discovered.

NU

Fig. 2. Chemical structures of tocopherol and tocotrienol. Numbering for the chromanol ring

MA

in green and numeration for the isoprenoid side chain in red. Blue R groups are listed for each isoform in the table with molecular weight. Tocopherol and tocotrienol are largely similar, with the exception of three C=C double bonds in the isoprenoid side chain for tocotrienol.

TE

D

Naturally occurring tocopherol chiral stereocenters are 2’R,4’R,8’R, while tocotrienol chiral

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stereocenter is 2’R only due to the C=C unsaturation in the isoprenoid tail.

Fig. 3. Chemical structures of modified tocopherols and tocotrienols. α-Tocopherol succinate

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and mito-chromanol are modified from tocopherols, while δ-tocoflexol and EPI-743 are modified from tocotrienols.

Fig. 4. Bubble map outlining vitamin E and their potential clinical applications in multiple diseases. Black bubbles indicate different class of human diseases, and bubbles with dotted lines indicate potential clinical applications in respective human diseases. Red bubbles: tocopherol treatment; green bubbles: tocotrienol treatment; yellow bubbles: tocopherol and tocotrienol treatment.

64

ACCEPTED MANUSCRIPT

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Table 1 Natural sources of tocopherols and tocotrienols

Bixa orellana L. a Hevea brasiliensis Müll.Arg. a Delphinium ajacis L. b Rosmarinus officinalis L. a Vitis vinifera L. a Aesculus hippocastanum Hort. b Litchi chinensis Sonn. a

Lipstick tree Rubber tree Larkspur Rosemary Grape Horse chestnuts Litchi (lychee)

seed latex seed seed seed seed seed

Tocopherol (TP, µg/g tissue) α β γ δ 1758 5.0 120 2883 18.0 195 124.1

Oryza sativa L. e Secale cereal f Amaranthus ssp. e Triticum sp. a

Total tocols

Coconut oil Palm oil Peanut oil Rice bran oil Safflower oil Soybean oil Barley Corn Oat Black rice Brown rice White rice Rye Sorghum Wheat

36 890 367 860 774 958 76.1 66.9 15.8 93.7 26.4 7.4 27.87 17.9 45

α

5 152 130 324 387 101 8.6 3.7 5.4 12.4 4.2 0.7 11.46 1.4 15.4

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Cocos nucifera L. c Elaeis guineensis L. c Arachis hypogaea L. c Oryza sativa L. c Carthamus tinctorius L. c Glycine max L. c Hordeum vulgare L. d Zea mays L. d Avena sativa L. a

Common Name

AC

Family

TE D

Plant Oils and Cereals

528.1 1.10 78 435.2 9.2

CR

Tissue

606.5 241.6 83 30.8 1.9 88 59.2

US

Common Name

MA N

Species

IP

Plants and Fruits

Tocopherol (TP, µg/g) β γ 18 0.9 0.2 2.28 5.0

216 53 387 593 5.6 45.0 3.2 2.5 1.2 0.1 13.3 -

174.7 506.3 17.4

Tocotrienol (T3, µg/g tissue) α β γ δ 18.7 522.4 566 560.5 5.0 97 -

δ

α

6 21 264 0.7 1.0 38.6 0.2 -

5 205 116 40.3 5.3 4.2 13.8 5.6 0.8 5.94 2.1 5.0

1.84 153 300.3 -

534.7 196.7 109.4 25.0 626 3.37

Tocotrienol (T3, µg/g) β γ 1 8.7 8.19 19.6

19 439 349 10.4 11.3 2.1 26.4 15.4 5.8 0.8 -

% of T3

977.9 1870 336 488.0

53.9 77.6 71.9 22.5 60.1 78.9 70.1

δ

% of T3

94 0.9 0.4 0.9 0.1 -

69.4 82.9 54.1 79.2 25.4 45.6 42.9 79.5 89.2 50.7 16.8 54.7

Tocols: tocopherols and tocotrienols. Reference: a (Horvath et al., 2006), b (Matthaus et al., 2003), c (http://tocotrienol.org/), d (Panfili et al., 2003), e (Choi et al., 2007), f (Zielinski et al., 2001). 65

ACCEPTED MANUSCRIPT Table 2 Molecular targets modulated by tocopherols and tocotrienols beyond cancer

Bcl-2 Caspase-3 IL-1β IL-4, IL-5 IL-6 IL-13 G-CSF TNF-α

↑ ↓ ↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓

↓

TGF-β

↓↓

↔ ↑ ↑ ↓ ↓ ↓ ↔

G-CSF (radiation) Alkaline phosphatase

↑ ↑ ↑↑ ↓ ↓ ↓ ↓

↓↑

HO-1

↑↑

↓ ↓ ↓ ↑ ↔ ↔ ↓ ↔ ↔ ↔ ↔ ↔ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓

12-LOX, PLA2 c-Src, ERK MAPKα, Src MAPKβ PI3K PK6 PKC C/EBP NF-κB Nrf2 PPARα STAT6 Akt Apolipoprotein A1 Apolipoprotein B C-reactive protein

Osteopontin

↓↓ ↓↓ ↓↓ ↑ ↓ ↓ ↓ ↓ ↓ ↑ ↑ ↓ ↑ ↑↑ ↓ ↓ ↓↓ ↓ ↓ ↓

PDE2, PGE2

↓↓

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Transcription factor

Others

GTPase

↓

(Kuhad & Chopra, 2009b; Miyoshi et al., 2005) (Devaraj et al., 2008; Kuhad & Chopra, 2009b) (Peh et al., 2015; Wagner et al., 2008) (Chin & Ima-Nirwana, 2014b; Wang & Jiang, 2013) (Peh et al., 2015; Wang et al., 2012) (Peh et al., 2015; Wang & Jiang, 2013) (Chin & Ima-Nirwana, 2014b; Devaraj et al., 2008; Qureshi et al., 2010) (Jiang et al., 2011; Kuemmerle et al., 1997; Kuhad & Chopra, 2009b) (Li et al., 2013; Satyamitra et al., 2011; Singh et al., 2014a) (Norazlina et al., 2000) (Matough et al., 2014; Musalmah et al., 2002; Peh et al., 2015; Venditti et al., 2011) (Jiang, 2014; Jiang et al., 2008) (Das et al., 2008; Freedman et al., 2000; Mukherjee et al., 2008) (de Diego-Otero et al., 2008; Mo et al., 2012) (Berbée et al., 2009; Deng et al., 2014; Parker et al., 1993; Qureshi et al., 2002) (Das et al., 2008; Jenkins et al., 2001; Mukherjee et al., 2008; Niess et al., 2000) (Khanna et al., 2010)

MA

eNOS

D

HMG-CoA reductase

TE

Kinases

COX-2, 5-LOX

CE P

Enzymes

Catalase, GPx, SOD

(Lekli et al., 2010; Mukherjee et al., 2008)

T

↑ ↑ ↔ ↓ ↓ ↓ ↓ ↓

References

IP

Apoptotic regulator

Cytokines

T3

NU

TP

SC R

Molecular Targets Modulated

Classification

Collagen type I α1 Histamine LTB4, LTC4, LTD4

(Sen et al., 2000) (Das et al., 2008; Mukherjee et al., 2008) (Das et al., 2008; Mukherjee et al., 2008) (Nakaso et al., 2014) (Li et al., 2013) (Freedman et al., 2000; Tsuduki et al., 2013) (Wang & Jiang, 2013) (Peh et al., 2015; Wang & Jiang, 2013) (Li et al., 2012; Peh et al., 2015) (Fang et al., 2010) (Wang et al., 2012) (Lekli et al., 2010; Mukherjee et al., 2008) (Cloarec et al., 1987; Daud et al., 2013; Nicod & Parker, 2013; Qureshi et al., 1991) (Devaraj & Jialal, 2000; Haghighat et al., 2014) (Chin & Ima-Nirwana, 2014b; Chojkier et al., 1998; Jiang et al., 2011; Norazlina et al., 2000) (Gueck et al., 2002; Tsuduki et al., 2013) (Jiang, 2014; Jiang et al., 2008) (Chin & Ima-Nirwana, 2014b; Jenkins et al., 2001; Norazlina et al., 2000) (Gueck et al., 2002; Jiang, 2014; Jiang et al., 2008)

TP: tocopherol; T3: tocotrienol; ↓: moderate decrease; ↓↓: substantial decrease ↑: moderate increase; ↑↑: substantial increase; ↔: no effect; ↓↑: contradicting results where both ↓ and ↑ were reported.

66

ACCEPTED MANUSCRIPT Table 3 Comparison of tocopherols versus tocotrienols in multiple diseases beyond cancer Disease

Tocopherol

Tocotrienol

References

Conflicting results regarding bone resorption, bone formation, bone density and bone mass.

γ-tocotrienol: ↓osteoblast cell apoptosis, ↓MDA, ↑SOD, CAT and GPx, ↓mevalonate pathway, ↑GTPase, ↓HMG-CoA reductase, ↓pro-inflammatory cytokines, ↑genes related to bone formation.

(Abd Manan et al., 2012; Chai et al., 2008; Chin & Ima-Nirwana, 2014b; Deng et al., 2014; Fujita et al., 2012; Iwaniec et al., 2013; Nizar et al., 2011)

IP

SC R

Osteoporosis

T

Bone Diseases

γ-tocotrienol > TRF >> α-tocopherol: ↑trabecular thickness; ↓eroded surface.

Postmenopausal osteoporotic bone loss

α-tocopherol ≈ tocotrienols: Improved parameters for bone morphology and preserved bone microarchitecture.

Bone metabolism and formation

Palm tocotrienol >> α-tocopherol: in suppressing lipid peroxidation and activating antioxidant enzymes. Tocotrienols >> tocopherols: for bone formation; among tocotrienols, γ-tocotrienol > δ-tocotrienol.

MA

NU

Nicotine-induced osteoporosis

Cardiovascular Diseases

Myocardial ischemia reperfusion injury

α-tocopherol: ↓myocardial infarct size; ↓cardiomyocyte death; ↑p42/44 ERK; ↑p38 MAPKβ; ↑Akt-Bcl-2 pathway; ↓p38 MAPKα; ↓JNK, ↓c-Src; ↑antioxidant enzymes.

CE P

TE

D

Atherosclerosis

α-tocopherol: ↓aortic and coronary wall thickening; ↓IL6, CRP, VEGF;

AC

Platelet thrombosis and aggregation

(Hermizi et al., 2009; Norazlina et al., 2007) (Muhammad et al., 2012)

(Maniam et al., 2008; Mehat et al., 2010)

TRF: ↓aortic MDA; ↓Intimal thickening; ↓internal elastic lamina; ↑PPARs/apolipoproteins; ↑cholesterol transporters.

(Li et al., 2010; Nafeeza et al., 2001; Shirpoor et al., 2013; Wade et al., 2013)

γ-tocotrienol > α-tocotrienol ≈ δtocotrienol: differential interaction of MAPK with caveolin 1/3 in conjecture with proteasome stabilization; ↑autophagy; ↑Akt-Bcl-2 pathway; ↑p42/44 ERK; ↑p38 MAPKβ; ↓p38 MAPKα; ↓JNK, ↓c-Src.

(Das et al., 2008; Lekli et al., 2010; Mukherjee et al., 2008; Venditti et al., 2011)

TRF > α-tocotrienol >> α-tocopherol: ↓platelet aggregation by 92%, 59% and 22% respectively; ↓cyclic flow reduction.

(Qureshi et al., 2011)

Diabetes mellitus

Type II diabetes mellitus

Type II diabetes mellitus Diabetes-associated wound healing Diabetes-associated cognitive decline Diabetes-induced nephropathy Eye diseases Cataractogenesis

α-tocopherol: normalized glucose tolerance; ↓blood pressure, cardiac collagen deposition and ventricular stiffness; ↑GPx activity; ↓plasma MDA.

TRF: normalized glucose tolerance; ↓blood pressure, cardiac collagen deposition and ventricular stiffness; ↓total cholesterol, LDL and total lipids; ↓CRP and urine microalbumin.

(Baliarsingh et al., 2005), (Haghighat et al., 2014), (Patel et al., 2011), (Musalmah et al., 2002)

α-, γ-, and δ-tocotrienols but not α-tocopherol: ↑PPARα; ↑interaction with PGC-1α LXXLL peptide.

(Fang et al., 2010)

α-tocopherol: ↑wound healing

(Musalmah et al., 2002)

-

Combined treatment of TRF and α-tocopherol: ameliorated glutamate cytotoxicity in astrocytes. TRF, not α-tocopherol: ↓inflammation, ↓NF-κB activation, ↓plasma TNF-α, TGF-β, IL-1β and caspase-3.

(Kuhad et al., 2009; Kuhad & Chopra, 2009b; Selvaraju et al., 2014)

Tocotrienol >> α-tocopherol: at restoring renal function.

(Kuhad & Chopra, 2009a)

α-tocotrienol >> α-tocopherol: at intraocular penetration to ↓oxidative and nitrosative stress.

67

(Tanito et al., 2004)

ACCEPTED MANUSCRIPT α-tocotrienol >>> α-tocopherol: ↓growth of Tenon’s capsule fibroblasts.

Glaucoma Inflammatory diseases Prostaglandininduced inflammation

γ-tocotrienol ≈ δ-tocopherol > γ-tocopherol >> α-/βtocopherol: ↓PGD2 and ↓PGE2.

α-tocopherol: ↓oxidative stress; ↓structural damage on colon.

Rheumatoid arthritis

α-,γ-tocopherol: ↓IL-1β, ↓inflammation, but disease onset remained unchanged.

Lipid disorders

Neurological diseases

-

D

Hypercholesterolemia and hyperlipidemia

TRF: ↑apoptosis and autophagy; ↓extracellular matrix production; ↓TGF-β1. γ-tocotrienol: ↓paw edema; ↓CRP; ↓TNF-α; ↑GSH and SOD. δ-tocotrienol >> glucosamine: ↓paw edema, ↓CRP; ↓collagenspecific T-cells.

Tocotrienol >> Tocopherol: ↓total cholesterol, lipid peroxidation and LDL.

TE

Hypercholesterolemia

SC R

Inflammatory bowel disease

TRF: ↓PKC; ↓dermal thickening; ↓mast cell degranulation; ↓serum histamine levels.

NU

α-tocopherol: ↓swelling of skin rashes in patients.

MA

Dermatitis

IP

T

γ-tocotrienol >> α-tocopherol: ↓inflammatory cytokines; ↑antioxidants activity; ↓NF-κB and STAT6 activation; ↑Nrf2 activation; and ↓airway hyperresponsiveness.

Asthma

(Meyenberg et al., 2005)

(Jiang et al., 2008)

(Peh et al., 2015; Wang et al., 2012) (Javanbakht et al., 2010; Kosari et al., 2010; Tsuduki et al., 2013) (Aghdassi et al., 2003; Bitiren et al., 2010; Luna et al., 2011a; Luna et al., 2011b) (Bandt et al., 2002; Haleagrahara et al., 2014; Jiang et al., 2009; Radhakrishnan et al., 2014)

(Daud et al., 2013; Magosso et al., 2013; Qureshi et al., 2002; Teoh et al., 1994)

TRF: ↓MDA; ↓degree of intimal thickening; ↓cholesterol accumulation in macrophages; ↓lipids level.

(Nafeeza et al., 2001; Rashid Khan et al., 2015)

α-tocopherol (nM): not effective.

α-tocotrienol (nM): ↓neuronal cell death; ↓c-Src/ERK; ↓12-LOX; ↓cytosolic PLA2.

(Khanna et al., 2010; Khanna et al., 2003; Khanna et al., 2005b; Sen et al., 2000)

External agentsinduced neurotoxicity

α-tocopherol: cytoprotective against Fe2+-Ca2+-induced oxidative damage; ↓neuronal death; ↑TRPV1 channel; ↑adenine nucleotide hydrolysis.

γ-,δ-tocotrienol: cytoprotective against MPP+ and other toxicities; bind to ERβ/caveolin-1; ↑PI3K/Akt.

(Crouzin et al., 2010; Gutierres et al., 2012; Nakaso et al., 2014)

Ischemic stroke

γ-tocopherol > αtocopherol: ↓cerebral infarct size. δ-tocopherol: no effect.

α-tocotrienol: ↓cerebral infarct size; ↑MRP1; ↓miR-199a-5p; γ-,δ-tocotrienol: no effect.

AC

CE P

Glutamate-induced neurotoxicity

Radiation damage Hematopoietic syndrome

γ-tocotrienol >>> α-tocopherol: as radioprotectant.

Gastrointestinal syndrome

-

γ-,δ-tocotrienol: ↑G-CSF for survival of white blood cells; ↓HMG-CoA reductase; ↓IL-1β, IL-6 and PK6; ↓apoptosis.

TRF: tocotrienol-rich fraction.

68

(Mishima et al., 2003; Park et al., 2011)

(Ghosh et al., 2009) (Kulkarni et al., 2013b; Li et al., 2013; Satyamitra et al., 2011; Singh et al., 2014b; Suman et al., 2013)

ACCEPTED MANUSCRIPT Table 4 Ongoing and completed clinical trials of tocotrienols* Status (last updated)

Clinical trial

Completed (2014)

NCT01571921

I

δ-Tocotrienol

Pancreatic cancer

I

Tocotrienols

Pharmacokinetics

I

Tocotrienols

Attention Deficit Disorder

Tocotrienols

IP

Metastatic breast cancer

Ongoing (2015)

NCT01450046 NCT01446952 NCT00985777 NCT01185769

III

Completed (2013)

NCT01855984

Cognitive impairment in Type 1 and 2 diabetes mellitus

IV

Ongoing (2015)

NCT01973400

Tocotrienols

Ischemic stroke

III

Ongoing (2014)

NCT02263924

Tocotrienols

Ovarian cancer

II

Ongoing (2015)

NCT02399592

Tocotrienols

Osteoporosis

II

Ongoing (2015)

NCT02058420

Tocotrienols

Pharmacokinetics

I

Completed (2014)

NCT00678834

Tocotrienols Topical and Oral Tocotrienols γ-/δ-TRF

Stroke

-

Recruiting (2015)

I

Ongoing (2015)

Metabolic syndrome

-

Recruiting (2012)

NCT01858311 NCT01579227, NCT00700791 NCT01626430

TRF

Breast cancer

Pilot trial

Completed (2010)

NCT01157026

TRF

Cerebrovascular disorders

II

Completed (2014)

NCT00753532

TRF

Cholesterol lowering

III

Ongoing (2014)

NCT02142569

TRF

Chronic haemodialysis Platelet aggregation in metabolic syndrome

-

Recruiting (2015)

NCT02358967

I

Completed (2013)

NCT01631838

Friedreich's Ataxia

II

Ongoing (2015)

NCT01962363

Leber's hereditary optic neuropathy

Single patient

Completed (2014)

NCT02300753

EPI-743

Leigh syndrome

II

Ongoing (2015)

NCT02352896

EPI-743

Mitochondrial disorders

II

Ongoing (2015)

NCT01642056

EPI-743

Pearson syndrome

II

Ongoing (2015)

NCT02104336

TRF

CE P

TE

D

Scar

MA

Completed (2010)

NU

δ-Tocotrienol and TRF

T

Phase

SC R

Drug Disease conditions Naturally occurring Tocotrienols

Modified Tocotrienols EPI-743

AC

EPI-743

TRF: tocotrienol-rich fraction; *: Data obtained from http://clinicaltrials.gov/ in September 2015.

69

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

70

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

71

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

72

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

73