Vitamin E is a fat-soluble antioxidant that can only be produced by plants through photosynthesis, hence it needs to be obtained from foods or supplements, as it plays a crucial role in human nutrition. The distribution of vitamin E in palm oil was reported to be approximately 30% tocopherols and 70% tocotrienols [1]. Tocotrienols have similar structures to tocopherols, with a chromanol head denoted by α, β, γ, or δ, which depends on the position number and methyl groups on the chromanol ring [2]. Tocotrienols differ structurally from tocopherols by the degree of saturation at the side chains, with three double bonds at carbons 3, 7, and 11, whereas tocopherols have saturated phytyl side chains. It was discovered to naturally occur at higher levels in certain cereals and vegetable oils, including palm oil, rice bran oil, barley germ, wheat germ, and annatto ^{[3][4][5][6][7]}[3,4,5,6,7].

Tocotrienols are valuable nutraceuticals due to their numerous pharmacological properties, particularly in preventing or treating non-communicable diseases, including cardiovascular, musculoskeletal, metabolic, and skin disorders, as well as cancers [8]. Tocotrienols have biochemical functions that are directly or indirectly impacted by their antioxidant properties, which prevent the non-enzymatic oxidation of various cell components by molecular oxygen and free radicals. In several studies, tocotrienols have been shown to inhibit free radical production via the induction of enzymes such as superoxide dismutase ^[9][10][9,10] and glutathione peroxidase, which neutralise superoxide radical-produced free radicals [11]. In addition, tocotrienols have been extensively studied for their anti-inflammatory properties, and promising scientific evidence has been presented. In light of the fact that inflammation is a full array of physiological responses to a foreign organism, it has been established that inflammation is a significant element in the progression of numerous chronic diseases and disorders. Tocotrienols have been demonstrated to inhibit the expression of inflammatory mediators such as tumour necrosis factor-alpha [10], interleukin [IL]-1 [12], IL-6 [13], and nitric oxide synthase [14]. Antioxidants protect tissues from damage against reactive oxygen species and other free radicals, indirectly preventing unwanted inflammatory responses from occurring in the first place.

Despite their medicinal value, translating tocotrienols into a viable medicinal product remains challenging due to their low oral bioavailability. Oral administration remains the preferred choice for drug delivery due to its safety, patient compliance, and self-administration capacity. Although oral delivery is the most convenient, it is also limited by a number of barriers in the gastrointestinal tract (GIT) [15]. Upon oral administration, drug solubilisation in the GIT is essential for absorption, as insufficient dissolution can cause incomplete absorption, low bioavailability, and high variability [16]. Tocotrienol has unfavourable physicochemical properties as a highly viscous oil that is nearly insoluble in water and readily oxidised by atmospheric oxygen. Other limitations exist, such as saturable intestinal absorption and the selectivity of the transfer protein ^[17][18][17,18]. These physicochemical, biological, physiological, and anatomical factors act independently and in concern to limit tocotrienol bioavailability and prevent effective oral delivery [19]. Using a rat model, an in vivo study found that intraperitoneal and intramuscular administration of tocotrienols resulted in minimal absorption, whereas oral administration resulted in incomplete absorption of tocotrienols [20]. Researchers have invented various methods to improve the bioavailability of these compounds. One of the innovations is a self-emulsifying drug delivery system (SEDDS), an isotropic mixture of oils, surfactants, co-surfactants, and co-solvents. SEDDSs can improve the oral absorption of highly lipophilic drug compounds by maintaining them in a solubilised state, thereby preventing an improperly water-soluble compound from solubilising and subsequently dissolving [21]. The oral bioavailability of each tocotrienol form was reported low in previous studies, with α-tocotrienol at 27.7%, γ-tocotrienol at 9.1%, and δ-tocotrienol at 8.5% [22]. Therefore, without α-tocopherol, tocotrienol absorption is virtually nonexistent without suitable conditions and optimal fat levels. The findings also showed that the elimination half-lives of several tocotrienol forms ranged between 2.3 and 4.4 h, much shorter than α-tocopherol elimination half-lives, which lasted 48 to 72 h ^[23][24][25][23,24,25]. The poor and inconsistent oral bioavailability of fat-soluble compounds in the GI led researchers to explore solutions to overcome these issues and ensure positive therapeutic effects in humans.

2. Pharmacokinetics of Tocotrienol

Tocotrienol exhibits many beneficial effects, but its poor oral bioavailability aids in its application as a future therapeutic agent. Many factors influence tocotrienol pharmacokinetics, such as their solubility, absorption, distribution, and elimination. Absorption is the process by which drugs or substances enter the body. Given any method other than intravenously, drugs or substances molecules must pass tissue barriers such as the skin epithelium, subcutaneous tissue, gut endothelium, and capillary wall to enter the blood. Meanwhile, distribution refers to how drugs or other substances are transported throughout the body. It is then followed by the metabolism or biotransformation of toxicants that function to detoxify, which is divided into two stages: Phase I and Phase II. Phase I includes oxidation, hydrolysis, and reduction mechanisms, where these reactions, catalysed by hepatic enzymes, generally convert foreign compounds into phase II derivatives. Phase I products can be excreted as such if polar solubility permits translocation. Meanwhile, Phase II involves the conjugation or synthesis reactions. Standard conjugates include glucuronides, acetylation, and glycine combinations [26]. Lastly, the excretion removes metabolic waste from the body, mainly through various processes performed by various body parts and internal organs. The effectiveness of vitamin E in those tissues is significantly influenced by the delivery of orally administered vitamin E to vital organs. Thus, mechanisms underlying the delivery of absorbed vitamin E to tissues have been the subject of active investigation. As the palm vitamin E mixture contains both α-tocopherol and tocotrienols, interactions between these compounds would affect their pharmacokinetics. Of note, α-tocopherol is suggested to affect the bioavailability of tocotrienol significantly. In a study by Qureshi et al., 33 healthy male subjects were supplemented with δ-tocotrienol at 125, 250, or 500 mg/d, dose dependently increasing the plasma area under the curve (AUC). The findings indicated that tocotrienols augmented in the absence of tocopherols, as δ-tocotrienol had better bioavailability, thus enhancing therapeutic properties by reducing the levels of cytokines related to inflammation [23].

2.1. Absorption of Tocotrienol

The bioavailability of an orally administered compound comprises three parts products, including the dissolved and the part that escapes degradation in the lumen. Next is the fraction that absorbs and penetrates the entire intestinal membrane and escapes the intestinal first-pass metabolism. The last part is the fraction that escapes the liver’s first-pass metabolism [27]. Tocotrienols are natural lipophilic compounds that exist as oily liquids. Their oral bioavailability was poor and erratic due to low aqueous solubility and miscibility [20]. It is widely believed that all forms of vitamin E are absorbed by passive diffusion ^[28][29][28,29]. This type of diffusion is defined as a substance moving toward the concentration gradient without any energy input from a higher concentration area to a lower concentration area. Thus, the therapeutic level of tocotrienol might be difficult to reach in the blood and target tissue using oral administration [30]. Previous studies have discovered that taking tocotrienols with food increases the amount of tocotrienols absorbed through the intestinal wall. Food like a fatty meal enhances tocotrienol solubility due to the formation of mixed micelles that increase the area of absorption in the intestines caused by stimulating bile salts and pancreatic enzyme secretion ^[25][31][25,31]. Indeed, mixed micelles can solubilise hydrophobic components and diffuse into the unstirred water layer (glycocalix) to approach the brush border membrane of the enterocytes. In addition, food also increases the lymph lipid precursor pool inside the enterocytes, eventually enhancing lymphatic transport. Vitamin E does not stabilise for long in the digestive tract after being ingested, leading to a rapid attack by the digestive enzyme pancreatic lipase before breaking down fat molecules into tiny pieces called fatty acids and mono-glycerides. According to Fairus et al., tocotrienols disappear rapidly from plasma within 24 h, raising questions about their biological activities [32]. This might be partly due to the low affinity of the α-tocopherol transport protein (α-TTP) for tocotrienols. The findings showed that the repackaging of α-tocopherol in the liver into very low-density lipoprotein cholesterol results in a longer shelf life and higher plasma concentrations of α-tocopherol. A different pathway for tocotrienol absorption has been proposed, which is independent to TTP [33]. In addition, some researchers identified that absorption of γ-tocotrienol was not complete, although an increase in solubility after being administrated with food significantly enhanced bioavailability [20]. Increased levels of γ-tocotrienol in the intestinal lumen cause saturation of tocotrienol absorption, which explains why increasing the dose does not increase tocotrienol bioavailability, indicating barriers and specific mechanisms of oral absorption transport by primary enterocytes. The intestinal uptake of γ-tocotrienol is inversely proportional to its presence in the intestinal lumen, which strongly suggests that the transportation across the intestinal membrane involves a carrier-mediated process [34]. Studies showed that the intestinal permeability of γ-tocotrienol decreased at a concentration of more than 25 µM, indicating that the intestinal uptake of the same was saturable, and a carrier-mediated process was involved. The in situ findings revealed the role of Niemann-Pick C1-like 1 (NPC1L1) as an intestinal transporter in both γ-tocotrienol and α-tocopherol intestinal uptake ^[34][35][34,35]. NPC1L1 is a critical mediator of cholesterol absorption and is mainly found in the GIT epithelial cells and hepatocytes.

2.2. Distribution of Tocotrienol

After oral administration, tocotrienol is absorbed from the intestine and transported to the systemic circulation through the lymphatic pathway. It is then incorporated into triglyceride-rich chylomicrons, and some of it is transported to other organs. The distribution of tocotrienol was examined by considering the tolerable upper intake level of vitamin E. The experiment using rats set the limit for tocotrienol daily intake to not exceed an amount in rats equivalent to the acceptable daily intake for humans, approximately 600–800 mg/day [36]. A study by Ikeda et al. reported that two isomers of tocotrienol (α and γ) were distributed in the adipose tissues and skin of the rats fed with palm tocotrienol diet [37]. However, these palm tocotrienols were not detected in the brain, liver, kidney, or plasma. A different source of tocotrienol from rice bran also showed that γ-tocotrienol was predominantly present in the adipose tissue and highly in the skin and heart [38]. Other findings demonstrated that γ-tocotrienol was predominant in adipose tissue on day 3 and was still higher on day 14 after administering a single dose of emulsified γ-tocotrienol subcutaneous injection. This finding aligned with previous studies in which γ -tocotrienol was administered in the diet and showed a preference for accumulation in adipose tissue regardless of the route of administration. Interestingly, γ-tocotrienol levels in the heart and spleen increased significantly on day 14 compared to day 3 [39]. Of note, tocotrienols predominantly accumulated in various sites of white adipose tissues, including epididymal fat, perirenal fat, and visceral fat, as well as in the skin after supplementation with a few concentrations of tocotrienol [40]. Increasing the tocotrienols intake increased its concentration in most tissues, although no dose-dependent effects were observed in the brain site. Despite unclear mechanisms on how tocotrienols selectively accumulate in these tissues, the researchers hypothesise that it depends on the affinity of tocotrienol towards the vitamin E-binding proteins and also due to cytochrome P450 expression level in each organ. A long-term dietary intake of tocotrienol leads to the accumulation in adipose tissue for 8 weeks [41]. Interestingly, the dietary α-tocopherol reduced the α-tocotrienol but not γ-tocotrienol concentration in various tissues. The findings demonstrated a significant amount of α- and γ-tocotrienol accumulated in the perirenal adipose tissues and epididymal fat of rats fed with a tocotrienol mixture. In contrast, other findings showed that γ-tocopherol concentrations were lower in the adipose tissues than in some tissues, including the adrenal gland, liver, and spleen of rats fed with γ-tocopherol [42]. Although the affinity of α-tocotrienol for α-TTP is nearly identical to γ-tocopherol, it does not explain the tissue-specific accumulation of tocotrienol for α-TTP. Furthermore, it was observed that the concentrations of α- and γ-tocotrienol in perirenal adipose tissue were 9.4- and 36.4-fold greater, respectively, than those in the serum. In contrast, the concentrations of α- and γ-tocopherol were only 0.9- and 1.7-fold higher in adipose tissue compared to serum. The findings suggest that adipose tissue preferentially absorbed or stored tocotrienol than tocopherol.

2.3. Metabolism of Tocotrienol

Vitamin E has several forms, containing the same chromanol ring and a hydrophobic side chain 13 carbons long. Tocotrienols differ from tocopherols in that they have an unsaturated side chain that contains three double bonds at the 3′, 7′, and 11 positions, as opposed to tocopherols. Upon supplementation, tocotrienols accumulate only in the skin and adipose tissues, whereas tocopherols can be found in most tissues ^[43][44][43,44]. These findings show that tocotrienols are metabolised and eliminated more extensively and quickly than tocopherols. Once tocotrienols and tocopherols are absorbed and delivered to the liver, their fates will likely undergo metabolism and excretion. Findings demonstrated that the tocotrienols are metabolised essentially, like tocopherols, as few metabolites degraded from γ-tocotrienol, like carboxyethyl hydroxychroman, carboxymethylbutyl hydroxychroman, carboxymethylhexenyl hydroxychroman, and carboxydimethyloctenyl hydroxychroman, were identified, similar to α-tocopherol [45]. Tocopherols and tocotrienols are metabolised without modifying the chromanol ring through oxidative degradation of the hydrophobic side chain. The mechanism involved was cytochrome P450 catalysed, hydroxylation, and oxidation of the 13′-carbon to form 13′-carboxy chromanol (13′-COOH), followed by a series of stepwise β-oxidations to remove a 2- or 3-carbon moiety from the side chain each cycle ^[46][47][46,47]. Recently, researchers discovered that γ-tocotrienol undergoes metabolism to produce novel metabolites, including sulphated 9′, 11′, and 13′-carboxy chromanol, both in human lung epithelial A549 cells and in rats [48]. In this study, sulfation likely occurs parallel to β-oxidation during tocopherol metabolism, indicating tocotrienol is metabolized much faster and more extensively. Similarly, the plasma concentrations of most metabolites were higher in rats supplemented with γ-tocotrienol than in rats fed with tocopherol [49].

2.4. Excretion of Tocotrienol

There are two main routes through which vitamin E is excreted. The most common route of excretion is through bile, which is then excreted in faeces after passing through the liver. Alternatively, vitamin E can be excreted in the urine after chain shortening, similar to β-oxidation, making it more water soluble. Due to a lack of TTP, α-tocopherol is not secreted back into the bloodstream. Therefore, an excess of vitamin E is not accumulated in the liver because it does not accumulate toxic amounts of vitamin E, which leads it to be metabolised and excreted in the bile [50]. A recent investigation revealed that α-tocopherol is found in urine as the metabolite α-carboxyethyl-hydroxychroman (CEHC), where the urinary α-CEHC excretion increased by 0.086 μmol/g creatinine with every 1 mg (2.3-μmol) increase in dietary α-tocopherol [51]. CEHC in urine increased in response to tocotrienol supplements, suggesting that tocotrienols are metabolised in the same way as tocopherols [52]. A study by Lodge et al. found that the γ-CEHC excretion time course increased urinary γ-CEHC at 6 h and a peak at 9 h following ingestion of 125 mg γ-tocotrienyl acetate [52]. However, the percentage of the dose recovered as metabolites in the urine is low (only up to 8%). In addition the long-chain carboxychromanols, especially 13′-COOHs, were found in tissues and faeces in animals supplemented with γ-tocopherol, δ-tocopherol, and γ-tocotrienol [53]. Both tocotrienol and tocopherol are bioavailable in the plasma according to research on vitamin E forms in rats, and they are primarily excreted as unmetabolized forms and long-chain metabolites, including 13′-COOHs in faeces, with more metabolites from tocotrienols than from tocopherols [53].