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Vitamin E exists in eight isoforms. α, β, γ and δ tocopherols and α, β, γ and δ tocotrienols. Tocopherols and tocotrienols have similar structures in possessing a chromanol ring. However, the unsaturated farnesyl side chain of tocotrienols makes them more active than tocopherols which have a saturated phytyl side chain. Tocopherols and tocotrienols also differ in the number of methyl groups present on the chromanol ring (Sen et al., 2006). Tocopherols do not have double bonds in the phytyl side chain, whereas tocotrienols have three double bonds in the farnesyl side chain. Tocotrienols are chemically more active than tocopherols because of the unsaturated tail. Hence, the current research focuses on α, β, γ and δ tocotrienols.

Figure 2.1 Structure of α-tocotrienol

α, β, γ and δ tocotrienols are similar in structure. However, the difference lies in the number of methyl groups on the chromanol ring. Tocotrienols are basically composed of a chromanol ring with one chiral centre, which is attached to the farnesyl side chain with three double

44 bonds. α-tocotrienol has three methyl groups at 5, 7, 8 positions of the aromatic ring as shown in Figure 2.1. β-tocotrienol has two methyl groups at 5, 8 of the aromatic ring as shown in Figure 2.2.

Figure 2.2 Structure of β-tocotrienol

Figure 2.3 Structure of γ-tocotrienol

γ-tocotrienol has two methyl groups at 7, 8 positions as depicted in Figure 2.3. δ-tocotrienol has only one methyl group at position 8 which is represented in Figure 2.4. The difference in the number and position of methyl groups is the reason for the existence of four different isoforms of tocotrienols. This difference leaves a significant change in their binding affinities with the same receptors.

45 Figure 2.4 Structure of δ-tocotrienol

2.3.1.1. Metabolism of Tocotrienols

Evidence of tocotrienols metabolism in the body is relatively limited compared to tocopherols (Gee, 2011). According to the experiments conducted by Fairus et al., after the administration of 1011mg of Tocotrienol Rich Fraction (TRF) in healthy subjects, tocotrienols were transported in triacyl glycerol rich fractions (Fairus et al., 2006). In this study it was observed that considerable amounts of α, γ and δ-tocotrienols were detected in both plasma and lipoproteins. In another clinical study it was found that higher concentrations of tocotrienols were observed in adipose tissue surrounding benign than malignant breast cancer tumour (Nesaretnam et al., 2007). Recently Fu et al. revealed through their experiments that tocotrienols disappeared from plasma after 24 hours of administration due to the low affinity of α-Tocopherol Transport Protein (α-TTP) for tocotrienols (Fu et al., 2014). Another study has suggested that tocotrienols might be metabolized by an alternate independent α-TTP pathway (Gee, 2011).

2.3.1.2. Biological Activity of Tocotrienol

Tocotrienols are capable of preventing cancer development and reduce the risk of atherosclerosis (Hendrich et al., 1994). It was indicated from the past in vitro research that tocotrienols can be used in the treatment of breast cancer (Guthrie et al., 1997). In another

46 study it was noticed that TRF obtained from rice bran oil has remarkably reduced the risk of cardiovascular diseases by inhibiting cholesterol synthesis (Qureshi et al., 1997). A previous study performed by Aggarwal, et al., suggested that tocotrienols have positive health effects on bone health, brain health, blood sugar metabolism and cancer (Aggarwal et al., 2010). The findings of a study performed in patients with hyperlipidemia and carotid stenosis concluded that the anti-oxidation properties of tocotrienols may influence the course of carotid atherosclerosis (Tomeo et al., 1995). Tomeo et al., have investigated the anti- oxidation role of α and γ tocotrienols in patients with carotid atherosclerosis. The effect of tocotrienols on hepatocarcinogenesis is tested in rats as a part of research performed previously by Ngah et al. The results from this experiment suggested that tocotrienol administered in rats reduced the severity of hepatocarcinogenesis (Ngah et al., 1991). It was identified in another study that γ tocotrienol is the most potent inhibitor of cholesterol synthesis and helps in lowering serum cholesterol in hypercholesterolemic patients (Qureshi et al., 1991). This study has concluded that tocotrienols, being the natural food products can be easily administered and accepted by humans, and may well prove to have fewer side effects than do many other medications (Qureshi et al., 1991). The medicinal values of tocotrienols imply the need for further research on them.

2.3.1.3. Interactions of Tocotrienols with Different Proteins

Naturally occurring tocotrienols exhibit anti-carcinogenic properties due to the presence of isoprenoid (2-methyl-1-3-butadiene) units in the lypophilic side chain (Packer et al., 2001). The unique structure of tocotrienols helps them to enter easily into cells and confer their better anti-oxidant properties (Richard, 2014).

The recent research on diabetic mice has shown that after binding to peroxisome proliferator activated receptor (PPAR), tocotrienols can regulate the PPAR target genes and improve

47 the consumption of whole body glucose and insulin sensitivity (Fang et al., 2010). Steroid Xenobiotic Receptor (SXR) binds to tocotrienols resulting in the activation of gene expression by forming a tocotrienol-SXR-RXR complex. It was noticed in an in silico

docking study that α-tocotrienol binds to the opening cavity close to the active site of 12-LOX and hinders the access of arachidonic acid to the catalytic site (Khanna et al., 2003). These studies lend further support to α-tocotrienol as a neuroprotective form of vitamin E. The docking of all four tocotrienols along with all four tocopherols was performed and the study selected the adenosine triphosphate (ATP) binding site of P-glycoprotein (Upadhyay, 2009). The data from this study has revealed that α and δ tocotrienols have highest affinity at the ATP site of P-glycoprotein. The interaction of α-tocopherol transfer protein, tocopherol associated protein, human serum albumin, and P-glycoprotein with tocotrienols and tocopherols present valuable information about their binding mode during metabolism, absorption, transport and efflux. The unsaturated side chain of tocotrienols makes them more potent than tocopherol (Upadhyay, 2009). The binding of tocotrienols with Farnesoid Xenobiotic Receptor (FXR) and RAR still needs to be tested.

It was observed in a recent study that tocotrienols are more potential cancer agents than tocopherols (Ling et al., 2012). Tocotrienols could be used as potential anti-cancer therapeutic agents as they were shown to have chemosensitization and anti-cancer stem cell effects. The research is now focusing on tocotrienols for future chemoprevention and cancer treatment, because of the disappointing results of tocopherols in previous clinical studies. Although the medicinal importance of tocotrienols was proved by previous research, the action of tocotrienol as a potential drug target is studied less (Ling et al., 2012).

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After performing a thorough literature review, PPARs, RAR, RXR, LOX were predicted to have a strong binding affinity with tocotrienols. Interestingly, all the receptors selected for the current study have medicinal value and so the ability of tocotrienols to act as their ligands is further tested in the current research. Figure 2.5 depicted the known targets of tocotrienols with different targets and enzymes.