

FIRST EDITION (E)

ISBN NO: 978-981-07-4297-3
© 2012 by Vance Group Ltd

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This book is published for educational and non-commercial purposes. It is written based on the author's and publisher's selection, study and review of available published research, scientific articles, reviews, news reports and documents of similar nature. While we have attempted to limit the sources of our information to those generally considered to be legitimate, and have attempted to represent the facts, opinions and conclusions of those sources as accurately as possible, we make no representations, nor do we express warranties in respect to the information stated herein. We do not guarantee the accuracy, completeness, efficacy, or timeliness of the information contained in this book and each user must evaluate and bear all risks associated with the use of such information. Suggestions for the use and application of any associated product and guide formulations are given in good faith, for information purposes only, and strictly without commitment or warranty. Reference to any product, process, or service does not constitute or imply endorsement, recommendation, or favoring by us.

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Preface

Officially discovered by Evans and Scott Bishop in 1922, Vitamin E is today a globally-sought dietary supplement. Although the generic form of Vitamin E that is commonly used is a form called α-tocopherol, very few are aware that another form of Vitamin E called the tocotrienols exists. This is not surprising given that the latter is rarely studied and thus much less heard of, until very recently.

As scientific research begins to give prominence to the tocotrienol form of Vitamin E, more and more discoveries about their vast range of health benefits are being made. As a result, tocotrienols are now being hailed as "the superior form of Vitamin E". With all these research findings invariably supporting the consumption of tocotrienols, consumer interest has also been rising in tandem.

To better cater to this growing interest and the increasing sophistication of end-users (who are constantly striving to make educated and informed choices), we hit upon the idea of publishing this book in 2011, which was founded upon two fundamental principles.

Firstly, to consolidate the known research results and data, as thoroughly as possible, so that the different pieces may fit together cohesively to reinforce each other in a bigger overall picture. To our knowledge, although reviews and articles of similar (albeit far more abbreviated) nature to this document exist, an endeavor on such a scale (in the form of a book) has hitherto been absent until now and herein.

Secondly, to explain the complexity of this information in a manner that is easily understood, but without diluting its essence. While it is not difficult to compile all the findings and facts into a single tome, and for the scientifically or biologically inclined to make sense of the voluminous information involved, the same cannot be said for the rest of us. In order for the greater population to appreciate the value of tocotrienols, we therefore felt it was important to study, evaluate and simplify these findings as best as we can, in a language that we can all understand. Having said that, we do nonetheless encourage readers to study the cited articles in their original form, to read them as the original authors have intended.

As a result, we have here a book that brings you through the A-to-Z of tocotrienols over several chapters.

Chapter 1 would first introduce the tocotrienol molecule itself, and discuss how it has grown to be recognized as the "superior form of Vitamin E" compared to its conventional "cousin", the α-tocopherol isomer.
The following six chapters will then discuss the six main health benefits of tocotrienols and tocotrienol rich fractions (TRF). A common theme which readers will inevitably encounter is how tocotrienols, in almost all mentioned accounts, will emerge to be significantly superior to the generic α-tocopherol in conferring these benefits.

Chapter 2 talks about tocotrienols as excellent antioxidants, a function that Vitamin E is well-known for, and which action is responsible directly and indirectly for many (but not all) of the other health benefits ascribed to tocotrienols.

Chapter 3 discusses cardiovascular health (the health of the heart and blood delivery system) - a subject that is immense in both depth and breadth. The chapter also reveals how the well-known cholesterol-lowering property of tocotrienols is merely a starting point for many other cardiovascular benefits.

Chapter 4 moves on to explain neuroprotection, a benefit that has ignited significant interest in the research community subsequent to pioneering ground work laid more than a decade ago. More studies are now in progress, and the protection conferred by tocotrienols to the brain and nerve cells is seen to show significant promise in various brain diseases, including stroke.

Chapter 5 addresses the anti-cancer characteristics of tocotrienols. It will discuss how tocotrienols display attributes that antagonize the fundamentals of cancer growth, showing efficacy against the many different forms of, arguably, the world's most dreaded disease.

Chapter 6 focuses on the cosmeceutical and skin care benefits of tocotrienols. It will elaborate on how tocotrienols simultaneously display the 5 main properties highly sought for in topical ingredients: skin lightening, ultraviolet protection, moisturizing, anti-ageing and safe topical use.

Chapter 7 explains the anti-inflammatory effects of tocotrienols. This is important as inflammation is regarded as the mechanism for tissue damage in almost all diseases.

Last but not least, Chapter 8 fittingly concludes the book by addressing the bio-availability of tocotrienols, i.e., how well the nutrient is absorbed by the body, and subsequently, how well it is delivered throughout the human body - an issue that had no real determination until recently.

It is our hope that this book will spark further interest towards tocotrienols within its readers, who may then embark on their own journey to discover more about this wonderful molecule.

The Authors
Table of Contents

Chapter 1 Introduction to Tocotrienols and Tocotrienol Rich Fractions (TRF).

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(i) What are tocotrienols and tocotrienol rich fractions?

(ii) Tocotrienols have multiple health benefits.

(iii) Tocotrienols are the superior form of Vitamin E.

Chapter 2 Antioxidant Properties.

(i) What are antioxidants and why are they needed?

(ii) Tocotrienols are extremely efficient antioxidants, far superior compared to the alpha-tocopherol isomer.

Chapter 3 Cardiovascular Health Benefits.

(i) What are cardiovascular diseases and why are they so detrimental?.

(ii) Tocotrienols help protect the structural integrity of blood vessels.

(iii) Tocotrienols help lower blood pressure.

(iv) Tocotrienols help lower blood cholesterol, and also maintain a healthy lipid profile within the body.

(v) Tocotrienols help protect against atherosclerosis.

(vi) Tocotrienols help protect against inappropriate blood clots (thrombosis) and platelet aggregation.

(vi) Tocotrienols help protect against reperfusion injury.

(vii) Special Article 1: Tocotrienols help in diabetes.

(viii) Special Article 2: Tocotrienols help in non-alcoholic fatty liver disease (NAFLD).

Chapter 4 Neuroprotective Properties.

(i) What is a stroke and how is it linked to neuronal death and glutamate-induced toxicity?

(ii) Tocotrienols help protect against glutamate-induced toxicity.

(iii) The potency and efficacy of tocotrienols' neuroprotective effects.

(iv) Inside the mechanisms of tocotrienols' neuroprotective effects.

Chapter 5 Anti-Cancer Properties.

(i) What is cancer and why is it such a serious problem?

(ii) Tocotrienols possess multiple anti-cancer characteristics.

(iii) Tocotrienols and pancreatic cancer.

(iv) Tocotrienols and breast cancer.

(v) Tocotrienols and prostate cancer.

(vi) Tocotrienols and liver cancer / hepatocellular carcinomas.

(vii) Tocotrienols and colon cancer.

(viii) Tocotrienols and skin cancer / skin neoplasm.

Chapter 6 Cosmeceutical Benefits.

(i) What is the cosmeceutical industry and what is the industry looking for?

(ii) Tocotrienols have skin lightening & whitening properties.

(iii) Tocotrienols have photo-protection properties (protection from ultraviolet damages).

(iv) Tocotrienols have skin moisturizing properties.

(v) Tocotrienols have anti-ageing properties.

(vi) Tocotrienols are effectively absorbed and safe for topical application.

(vii) Special Article 3: Tocotrienols can offer protection from radioactivity.

Chapter 7 Anti-Inflammatory Properties.

(i) What is inflammation and why are anti-inflammatory properties desired?

(ii) Tocotrienols have anti-inflammatory properties.

Chapter 8 Bio-availability of Tocotrienols

(i) What is bio-availability and why is it important?

(ii) Resolving the controversies on the bio-availability of tocotrienols.

(iii) Contrasting the bio-availability of chemically-modified and natural Vitamin E.

(iv) The safe consumption of tocotrienols.

(v) Co-consumption of tocotrienols and tocopherols: a balanced approach in supplementation.

What is next in Tocotrienols?

References & Citations.

Chapter 1 Introduction to Tocotrienols and Tocotrienol Rich Fractions (TRF)

## What are tocotrienols and tocotrienol rich fractions?

Vitamin E has long been renowned for its antioxidant property, and as such, has wide applications in the healthcare industry, be it in food, health or cosmetic products. Even though the form of Vitamin E that is most commonly found in these applications is the isomer called α-tocopherol, the Vitamin E family actually consists of eight different isomeric forms, which include:

(i) four isomers of tocopherols (differentiated as alpha-, beta-, gamma- and delta-tocopherol); and

(ii) four isomers of tocotrienols (differentiated as alpha-, beta-, gamma- and delta-tocotrienol).

This means that tocotrienols are one-half of the Vitamin E family tree. And in terms of natural and commercial availability, tocotrienols are often present as high-concentration mixtures. These mixtures, called tocotrienol rich fractions (TRF), are natural liquid fractions highly enriched for more than one isomer of tocotrienol. The exact content and ratio of individual tocotrienol isomers may vary depending on the source from which these fractions are derived from (e.g., from palm, rice bran or annatto); and the mixture may occasionally also contain tocopherols.

## Tocotrienols have multiple health benefits.

Although the term "tocotrienol" was first coined in 1964, the molecule received little scientific attention, and as a result, remained obscure till the 1990s. Up till 1998, there were only 56 research articles on tocotrienols. Comparatively, research on tocopherols had already produced roughly 25,000 published articles by that time.

It was not until the end of the 20th century that the momentum for tocotrienols started to pick up. In fact, more than two-thirds of all the literature on tocotrienols within PubMed (a comprehensive database containing references and abstracts on life sciences and biomedical topics) were published after the
year 20002, 3 (Figure 1 below illustrates this surge in momentum very clearly). This drastic change was largely underpinned by discovery of the cholesterol-lowering and anti-cancer potential of tocotrienols1.

Figure 1: Statistics from the PUBMED database showing the increase of research publications after the year 2000.

As more studies are being conducted, it is becoming apparent that the cholesterol-lowering and anti-cancer potential of tocotrienols are only a fraction of the many health benefits tocotrienols can offer. Figure 2 below summarizes more of these health benefits that have been discovered to date, and will be separately discussed in later sections of this book.

Figure 2: The multiple benefits of tocotrienols & tocotrienol rich fractions.

However, the list above is by no means exhaustive. There are other benefits currently being explored (such as the maintenance of eye health, the prevention of hair loss, the repair of muscle, etc.), and we look forward to writing about them in future editions of this book, when the respective concepts and mechanisms underlying them are eventually better established.

In short, the value of tocotrienols lies in its multiple yet potent benefits, some of them even deemed to be on par with conventional medications or certain drugs. However, it is important to appreciate that tocotrienols, or tocotrienol rich fractions (TRF), are supplements, not drugs. The pharmaceutical industry does not fall short of drugs capable of providing exceptional benefits, but these are often designed to tackle only one or two specific problems. Furthermore,
these drugs almost invariably carry along toxic side effects. Tocotrienols represent the potential to simultaneously confer multiple health benefits. And as vitamins, they represent a class of natural nutrients that humans have been consuming safely for hundreds of years.

## Tocotrienols are the superior form of Vitamin E.

If there is a single most important take-away from this book, it is this: even though tocopherols are currently the conventional and more commonly-known form of Vitamin E used, tocotrienols, while yet to reach the same level of mainstream mindshare, are in fact by far superior in function and potency.

The molecular structure of Vitamin E (both tocopherols and tocotrienols) consists of a "polar chromanol head group" and a "long isoprenoid side chain" (see figure 3 below). The structure of the isoprenoid side chain is what distinguishes a tocotrienol molecule from a tocopherol molecule: while the side chains of tocopherols have saturated single bonds (a phytyl chain), the side chain of tocotrienols have unsaturated double bonds (a geranylgeranyl chain).

Figure 3: The molecular structure of tocotrienols & tocopherols.

It is this unsaturated molecular structure that allows tocotrienols to outperform tocopherols in the antioxidant-linked benefits they have in common. Furthermore, it also allows tocotrienols to deliver unique benefits independent of antioxidant pathways, many of which the more ordinary α-tocopherol cannot. Figure 4 below illustrates these superior abilities.

Tocotrienols constantly outperform α-tocopherol in the benefits they have in common | Tocotrienols also possess benefits not displayed by α-tocopherol

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(i) Tocotrienols possess far superior antioxidant activities, protecting biological systems from free radical damage up to 40-60 times more efficiently than α-tocopherol4.

(ii) They also suppress pigment producing enzymes in skin cells (which contributes to skin darkening) >3.5 times better than α-tocopherol. The SPF (sun protection factor) of δ-tocotrienol is also more than double of the SPF of α-tocopherol6.

(iii) They suppress a number of additional enzymes that contribute to all inflammatory processes within the body. | (i) Tocotrienols can lower the LDL "bad" cholesterol levels in the body.

(ii) They also confer neuroprotection against brain damage at nanomolar concentrations (10-9 M)5.

(iii) They suppress the proliferation & growth of cancer cells, and also trigger the death of cancer cells.

Figure 4: The superior abilities of tocotrienols.

# Chapter 2 Antioxidant Properties

## What are antioxidants and why are they needed?

While oxygen is required by the human body to stay alive, it is ironically also capable of exerting oxidative stress on the body, through the formation of reactive oxygen species (ROS). Generally, any oxygen-containing free radical is known as a reactive oxygen species (ROS). Free radicals are atoms, molecules, or ions with unpaired electrons, and these unpaired electrons cause radicals to be highly chemically-reactive.

As a result, ROS are chemically-reactive molecules generated in the human body as a result of both the body's normal metabolism (the collective chemical processes that occurs within a cell), as well as its exposure to external agents (food, pollutants, sunrays, etc.). Examples of ROS include hydrogen peroxide (H2O2), hypochlorous acid (HClO), the hydroxyl free radical (•OH), and the superoxide anion (O2−).

Figure 5 below illustrates a typical cell and its main components, all of which are highly susceptible to the oxidative damage inflicted by ROS.

(i) Oxidation of lipids (lipid peroxidation) by ROS causes their degradation.

(ii) Oxidation of DNAs by ROS causes mutations to the genes.

(iii) Oxidation of proteins and enzymes by ROS leads to their malfunction.

Figure 5: The human cell and its main components.

As a result, ROS and the associated oxidative stress are highly implicated in various diseases and conditions, such as cancer, cardiovascular diseases, inflammation, etc.

Figure 6: Conditions and diseases associated with ROS.
Antioxidants are powerful molecules that prevent ROS from being formed, or remove them before they are able to cause any damage at the cellular level. Although the body itself has natural antioxidant systems and enzymes, these are very often exhausted due to environmental factors, stress, lifestyle habits and ageing. Dietary supplementation of antioxidants therefore plays a very important role in maintaining and sustaining healthy body functions. Below is an example of how antioxidants help prevent lipid peroxidation (i.e., oxidation of lipids).

Lipid Peroxidation and Cell Damage

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The cell membrane generally consists of lipids (also called polyunsaturated fatty acids), which unfortunately, are highly susceptible to damage from lipid peroxidation. The figure below illustrates this process.

1. During lipid peroxidation, lipids first react with hydroxyl radicals (a form of ROS generated within the body) to form lipid radicals. These then go on to react with oxygen to form lipid peroxyl radicals.

2. A chain reaction is subsequently initiated where different lipids, lipid radicals, and lipid peroxyl radicals react with each other to eventually form lipid peroxides.

3. As more and more lipids are consumed by this reaction, the cell membrane eventually gets destroyed. Additionally, lipid radicals may also react with and destroy nearby proteins and DNA.

The lipid peroxidation process

Figure 7: Lipid peroxidation and how antioxidants help prevent them.

Antioxidants help stop the formation of lipid free radicals (and other ROS), by being the "scapegoat" for lipids. Rather than lipids being converted into free radicals, the antioxidants get converted instead. However, unlike lipid free
radicals, antioxidant free radicals are unreactive, and do no or little damage to cell components.

## Tocotrienols are extremely efficient antioxidants, far superior compared to the alpha-tocopherol isomer.

Figure 8 below depicts the molecular structure of Vitamin E. As seen, both tocotrienol and tocopherol molecules have in common a phenolic hydroxyl group, located at the C6 position of their aromatic ring. It is this hydroxyl group that exerts the antioxidant properties of Vitamin E, allowing it to be the "scapegoat" against free radical formation.

Figure 8: The molecular structure of tocopherols and tocotrienols.

But if tocopherols and tocotrienols have the same chromanol head that is responsible for conferring antioxidant functions, then why do they perform so differently?

The structural difference between tocopherols and tocotrienols boils down to the saturation of their "tails" (also termed as side-chain), and it is this unsaturated structure that gives tocotrienols their enhanced performance over α-tocopherol. The unsaturated double bonds in tocotrienols essentially cause the tail of the molecule to "pucker", and this contracted structure in turn facilitates a few advantages over tocopherols12.
(i) A higher recycling efficiency from the exhausted form.

Vitamin E stops free radical formation by being itself turned into relatively harmless free radicals. This "exhausted free radical form" of Vitamin E can subsequently be recycled back into its "active form" within cells. Research shows that this regeneration rate is approximately one-and-a-half times faster with tocotrienols.

(ii) A higher mobility and a more uniform distribution within the cell membrane layer.

The unsaturated tail of tocotrienols allows them to move with greater ease and freedom within cell membranes (these are the external coverings of cells), facilitating better uptake, transfer and incorporation. When absorbed into cell membranes, tocotrienols are also found to be better distributed and less clustered compared to tocopherols. This improves interaction with free radicals and regeneration components.

(iii) A greater ability to disorder the cell membrane layer.

Cell membranes tend to be highly organized and tightly-packed, thus hindering the access and action of anti-oxidants in general. The unsaturated tails of tocotrienols are able to disorder such an orderly structure. Again, this facilitates interaction with free radicals, especially those which are situated deep within the core of the membrane.

As a result, research has established that tocotrienols (especially δ-tocotrienol) are far better than tocopherols in protecting the body from free radical and oxidative damage. Below is a summary of the various protection avenues tested to date that describes the relative efficacy of tocotrienols compared to α-tocopherol.

(i) Tocotrienols are better at protecting liver membranes from lipid peroxidation.

When microsome (vesicular fragments) made up of animal liver membranes were exposed to lipid peroxidation, α-tocotrienol was seen to be forty times better than α-tocopherol in protecting against "Fe2+ \+ NADPH-induced lipid peroxidation", and sixty times better in protecting against "ascorbate + NADPH-induced lipid peroxidation"4.

(ii) Tocotrienols are better at protecting LDL (Low Density Lipoprotein) from oxidation.

Oxidation of LDL contributes to its deposit onto blood vessel walls. This subsequently disrupts blood flow via the formation of atherosclerotic
plaques. When oxidation of LDL was induced by copper, treatment with tocotrienol rich fraction (TRF) significantly delayed the formation of TBARS (these are oxidation markers), and the magnitude of this delay was two times longer compared to the effect of α-tocopherol 7, 8.

(iii) Tocotrienols are better at protecting liver enzymes from lipid peroxidation.

The liver enzyme cytochrome P450 is extremely sensitive to deactivation and degradation by lipid peroxidation. When these enzymes were exposed to "Fe2+ \+ NADPH-induced lipid peroxidation", the protection provided by α-tocotrienol was six times better than α-tocopherol4.

(iv) Tocotrienols are better at protecting red blood cells from free radical damage, lipid peroxidation and oxidative stress.

Oxidative damage is known to impair the flexibility and elasticity of red blood cells, potentially causing cell rupture and death. In experiments comparing α-tocopherol and α-tocotrienol, the prevention of rupture and death by α-tocotrienol was seen to be 50% better. Flow within vessels was also 20-40% faster, indicating better cell elasticity and flexibility. Additionally, lipid peroxidation (measured by TBARS formation) was also three times lower9.

(v) Tocotrienols are better at protecting blood vessel walls from oxidative stress.

When vessel-lining cells were exposed to oxidative stress, treatment with tocotrienol rich fraction (TRF) significantly reduced lipid peroxidation. This effect was 46% greater when compared to treatment with α-tocopherol, and three-and-a-half times better when compared to treatment with Vitamin C (which is also a well-known antioxidant)7.

(vi) Tocotrienols are better scavengers of peroxyl radicals.

In the presence of peroxyl radical generators, the free radical scavenging ability of tocotrienol rich fraction (TRF), δ-, γ- and α-tocotrienol was found to be eight times, four times, three times and three times better then α-tocopherol respectively10, 11.

(vii) Tocotrienols provide better protection against auto-oxidation.

Compared to α-tocopherol, α, γ and δ-tocotrienols were found to be able to prevent the auto-oxidation of β-carotene and linoleic acid 22% - 31% more effectively11.

Figure 9: Comparing the antioxidant efficacy of tocopherols and tocotrienols.

# Chapter 3 Cardiovascular Health Benefits

## What are cardiovascular diseases and why are they so detrimental?

The cardiovascular ("cardio"= heart, and "vascular"= blood vessels) system is arguably the most important system in our body, as it functions to supply the entire body with the nutrients and hormones it needs. Because of this extensive bearing on human well-being, cardiovascular diseases are unsurprisingly the most common cause of morbidity and mortality in humans, especially in men and women above 60 years old.

(i) Every year, the condition results in 17 million deaths worldwide 13.

(ii) According to the Center for Disease Control and Prevention in USA, it is the number one killer in the United States with more than
2,600 deaths every day, which translates into one death every 33 seconds. Heart disease kills more Americans than cancer.

(iii) In Australia, cardiovascular diseases are the largest cause of premature deaths, accounting for just over one-third of all deaths in 200714.

With cardiovascular diseases, it is important to realize that the two most common forms of the disease are stroke and heart failure. And the main underlying cause for both of these is atherosclerosis.

Atherosclerosis is a process marked by an abnormal build-up of fat and cholesterol in the inner lining of the arteries. This build-up consequently narrows the blood's passageway and obstructs a healthy blood supply. The outcome is devastating when this happens in the heart (causing angina, heart attack, heart failure) or in the brain (causing stroke). Unfortunately, by the time these problems manifest, the disease is usually already at its advanced stages, and has been progressing for decades.

There is therefore an increased emphasis on prevention of atherosclerosis over treatment, and tocotrienols have been shown to achieve this by diverse pathways. The molecule tackles almost every stage of the disease's progression, and also suppresses many of the known associated complications. These include kidney failure (which is closely associated to high blood pressure), diabetes and non-alcoholic fatty liver disease (which are closely associated to high blood cholesterol).

Figure 10: Tocotrienols help prevent stroke and heart attack by targeting multiple stages of the diseases' progression.

## Tocotrienols help protect the structural integrity of blood vessels.

The average adult has about five liters of blood flowing through an intricate network of blood vessels comprising of arteries, veins and capillaries. The elasticity (and stiffness) of an individual's arteries is important in ensuring a
smooth, non-disrupted blood flow, and can be measured via his or her arterial compliance.

Arterial compliance is generally poor in smokers or diabetics, and is known to decline with age and menopause, even in healthy individuals. This means that regardless of an individual's health condition, blood vessels tend to gradually lose their ability to distend with pressure. This condition may initiate a vicious cycle that causes hypertension (high blood pressure), aggravates atherosclerosis, and increases cardiovascular risk15.

In animal studies, rabbits on diets designed to cause hardening of the arteries show improved arterial structure when supplemented with TRF for ten weeks. Results show that the continuity of elastic components within these arteries was better preserved in the presence of TRF, and these arteries better retained their elastic properties. On the contrary, thickening of the arterial wall, as well as disruptions in the internal layer of these walls can be observed without TRF supplementation 16.

Apart from animal studies, a human study also showed that supplementation of TRF for 8 weeks can significantly improve arterial compliance. This was assessed not only by the pulse wave velocity of these subjects, but also by the value of their augmentation index17, both of which are recognized classical indices of arterial stiffness (AS). Pulse wave velocity measures the speed at which the blood pressure pulse travels from the heart to the peripheral artery after blood rushes out during contraction, and augmentation index calculates the ratio of this ejection pressure from the heart to the resulting reflection pressure from the arterial system.

## Tocotrienols help lower blood pressure.

High blood pressure (also known as hypertension) increases the heart's workload, causing the heart muscles to enlarge and thicken, but nevertheless weaken over time. Because hypertension has few early symptoms, it often goes undetected and fails to be properly controlled. As a result, the condition can be a precursor to many different diseases and complications, including heart attack, heart failure, kidney failure, stroke and even premature death. Maintaining an optimum blood pressure is therefore essential, and even a moderate elevation of arterial pressure is known to shorten life expectancy significantly.
Under normal circumstances, an intricate network of factors within the body (which involves a complex interplay between various chemicals, hormones and the nervous system) work to regulate blood pressure. Among them is an important mechanism called endothelium-dependent vascular relaxation. To briefly summarize, endothelial cells lining the blood vessel produce Nitric Oxide (NO), and NO functions to keep the blood vessels in a relaxed state, thus preventing unnecessary elevations in blood pressure.

Under certain circumstances, endothelium-dependent vascular relaxation may be impaired, and this leads to hypertension. A few factors are known to cause this. The first is an increased removal of NO due to excess free radicals, and the antioxidant property of tocotrienols protects against this.

The second is a debilitated Nitric Oxide Synthase (NOS) activity. This is the enzyme responsible for producing Nitric Oxide (NO), which in turn keeps the blood vessels relaxed. Supplementation of γ-tocotrienol has been shown to boost NOS activity within spontaneous hypertensive rats. These were rats which were genetically modified for lower NOS activity, and therefore had extremely high systolic blood pressures (this refers to blood pressure when the heart beats while pumping blood; as opposed to diastolic blood pressure which measures pressure when the heart is at rest between beats). Twelve weeks of tocotrienol supplementation significantly alleviated the condition, reducing systolic blood pressures from ~210 mmHg to ~123 mmHg18. Note that the optimal systolic blood pressure in an adult human is somewhere between 90 mmHg - 120 mmHg, and anything >180 mmHg is considered a hypertensive emergency, which can result in irreversible organ damage.

## Tocotrienols help lower blood cholesterol, and also maintain a healthy lipid profile within the body.

The cholesterol-lowering property of tocotrienols is one that is very heavily studied. To appreciate this benefit in its entirety, an accurate understanding of blood cholesterol, triglycerides and the other players of the blood lipid profile is first needed.

Contrary to popular belief, cholesterol is not a lipid/fat (also known as triglyceride). Cholesterol contains no calories, so the body cannot derive any energy from it. Nevertheless, cholesterol is a component of the cell membrane, and is therefore essential for animal life. Triglyceride, on the other hand, is the form in which most fat exists in the body, and the body uses it for energy.
Despite their importance for human function, high levels of both cholesterol and triglyceride are associated with artery damage, atherosclerosis and, by extension, the risk of heart disease, stroke and various cardiovascular diseases. This means that when it comes to these components, it is about maintaining a careful balance, one that is neither too high nor too low.

Although high levels of triglyceride in the blood is commonly caused by a high fat or high carbohydrate diet (other causes include diseases such as diabetes, kidney failure or alcoholism), this is not the same for cholesterol. In fact, a common misperception is that dietary cholesterol predominantly determines blood cholesterol levels. What most people fail to realize is that the majority (~80%) of the blood's cholesterol is actually produced by our bodies (mainly in the liver) and not from the food we consume. Reports from more than ten clinical trials have also shown that dietary cholesterol has little effect on an individual's plasma cholesterol level, or even the risk of cardiovascular diseases19.

This means that controlling one's cholesterol intake would be of nominal help in keeping blood cholesterol levels low. This advocates the consumption of anti-cholesterol agents to help maintain healthy cholesterol levels.

The cholesterol-lowering properties of tocotrienols were first identified in 1986, when chickens fed with tocotrienols were found to have livers that produced less cholesterol20. Since then, research and clinical studies have affirmed the potency of tocotrienols, not only in cholesterol lowering, but also in conferring a healthy lipid profile. Lipid profile is the collective term given to the estimation of, typically, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides, etc.

In over 50 published scientific articles, tocotrienols and TRF have been tested effective in enhancing the blood's lipid profile in a number of ways.

(i) By lowering of triglycerides.

(ii) By lowering of total cholesterol.

(iii) By lowering of Low Density Lipoproteins (LDL) cholesterol. This is the form of cholesterol generally transported from the liver (the body's main distribution hub) to other parts of the body. When in excess, LDL gets deposited in the walls of arteries, causing atherosclerotic plaques. This plaque gradually narrows the arteries until blood supply to the vital organs is eventually occluded. This is why LDL is commonly known as the "bad cholesterol".

(iv) By increasing High Density Lipoproteins (HDL) cholesterol. This form of cholesterol is known as "good cholesterol" because it travels like a cleaner through the bloodstream, picking up excess cholesterol to be eliminated.

(v) By lowering of Apolipoprotein B (ApoB). This protein is involved in the formation of LDL and its transport to tissues. There is considerable evidence that APOB levels may be a better indicator of heart disease risk compared to total cholesterol or LDL.

The effects of tocotrienols & TRFs on the blood's lipid profile

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The effects of tocotrienols & TRFs on blood cholesterol and blood lipid have been tested in various animal models (pigs, rabbits, mice, and hamsters) 21, 22, 23, 24, 25, 26 as well as in humans (both in healthy subjects 27, 28 and subjects with high-cholesterol 29, 30, 31, 32). The table below summarizes these results clearly.

In many of these studies, the cholesterol-lowering effect of γ-tocotrienol was shown to be the most potent, in the order of γ-tocotrienol > α-tocotrienol > δ-tocotrienol22, 23, 25, 30.

It is important to realize that these properties are exclusive to tocotrienols, and are not observed with the generic α-tocopherol.

One of the human studies conducted even associates "the magnitude of HDL increase" seen with a "22.5% risk reduction in cardiovascular complications"27.

The cholesterol lowering effect in another animal study persisted as long as 10 weeks, after reversion to a non-TRF diet22.

Some of these studies also demonstrate tocotrienols' ability to work synergistically and safely with other cholesterol-lowering agents, such as citrus flavonoids and Lovastatin29, 32 (a commonly used cholesterol-lowering drug).

Figure 11: The effects of tocotrienols & tocotrienol rich fractions on the blood's lipid profile.
So how exactly do tocotrienols lower cholesterol levels?

As mentioned earlier, majority of the body's cholesterol is actually produced by the body itself, and tocotrienols function by targeting this production.

The starting point for cholesterol production within the liver (the main site of cholesterol production) involves an enzyme called 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase). The enzyme helps make mevalonate \-- a compound that is subsequently converted by the body to sterols, including cholesterol.

Conventional anti-cholesterol drugs, like statins, lower cholesterol by rendering HMG-CoA reductase non-functional. However, completely ablating the function of this enzyme also brings about adverse side effects, including muscle breakdown, nerve damage and sexual dysfunction. The severity of this has even resulted in certain drugs being withdrawn from the market (e.g. Cerivastatin).

Tocotrienols, on the other hand, have a more benign mechanism: they modulate the level at which HMG-CoA reductase is present within the liver. As a result, they carry none of these side effects. It has been experimentally shown that tocotrienols (but not tocopherols) can33:

(i) reduce the production of HMG-CoA reductase by up to 50%; and

(ii) increase the degradation rate of the enzyme by more than two-fold.

The two mechanisms cooperate synergistically to produce very significant reduction in enzyme levels (up to 75%). The inhibition of cholesterol synthesis achieved by γ-tocotrienol (known to exhibit the strongest effect among all isomers) is exceptionally effective, to the extent that it has been observed to be 30-times better than the second most potent isomer (α-tocotrienol)34.

## Tocotrienols help protect against atherosclerosis.

As discussed, atherosclerosis occurs when excess LDL-cholesterol in the blood gets deposited onto the walls of arteries, occluding blood flow.

So how exactly does this occur?
During atherosclerosis, oxidized or damaged LDL-cholesterol within the blood is first engulfed by immune cells of the body. These cells (also called monocytes) in turn lodge themselves to endothelial cells Single layer of cells that line the internal lining of blood vessels. They are involved with control of vascular tone and blood pressures along with exchange fo gas and metabolic products and extravasation of immune compounds. that line the blood vessel walls, and subsequently migrate within the sub-layer of these walls. The aggregation of these LDL-cholesterol-engorged cells forms the core of the atherosclerotic plaque, which is filled with lipids, cholesterol crystals and dead cell debris.

Figure 12: The stages of atherosclerosis.

As most vital organs within the body are inevitably heavily dependent on a healthy blood supply, the compromise in blood supply eventually damages them. As a result, the two most common outcomes of atherosclerosis are stroke (brain cell death when flow to the brain is compromised) and myocardial infarction (heart cell death when flow to the heart is compromised).

In that respect, animal trials have provided clear evidence of tocotrienols' efficacy in protection against atherosclerosis. In fact, not only could
tocotrienols help in preventing the condition, they have been observed to even bring about regression of pre-existing conditions.

(i) When supplemented with palm TRF for 90 days, mice genetically programmed to develop atherosclerosis were found to have their atherosclerotic lesions almost completely eradicated24. Similar results were also observed in studies using different animals16.

(ii) In another instance, rabbits that were fed with a high cholesterol diet showed regression of atherosclerotic conditions after 30 days of treatment with individual tocotrienol isomers23. Among the isomers, γ-tocotrienol was found to be the most potent, followed by α-tocotrienol and δ-tocotrienol23 . This is illustrated in the diagram below.

Figure 13: The regression of atherosclerosis after tocotrienol treatment.

In some of these studies, atherosclerosis regression was observed to be independent of plasma cholesterol levels24. This means that tocotrienols protect against atherosclerosis directly, and not just by lowering cholesterol levels. Indeed, tocotrienols can directly intervene in two of the processes leading to atherosclerotic plaque formation.
Firstly, as potent antioxidants, tocotrienols decrease the oxidation of LDL-cholesterols. This is important because only oxidized (damaged) LDL-cholesterols contribute to plaque formation.

Secondly, tocotrienols also reduce the lodging of LDL-laden monocytes to blood vessel walls. This process is highly dependent on "adhesive molecules" on the surface of endothelial cells that line the blood vessel walls. By down-regulating a gene called NF-κB (NF-κB helps control the production of these adhesive molecules), tocotrienols hamper the production as well as the surface expression of the three main "adhesive molecules" - VCAM-1, ICAM-1 and E-selectin35:

(i) Inhibition of production was found to be between 40% and 77%.

(ii) Inhibition of surface expression was found to be up to 63%.

A particular study found the inhibition of VCAM-1 by α-tocotrienol to be 10 times that of the generic α-tocopherol36. Having said that, α-tocotrienol is not the most potent isomer, as studies suggest that δ-tocotrienol is in fact one-and-a-half to two times better than α-tocotrienol in this respect37.

## Tocotrienols help protect against inappropriate blood clots (thrombosis) and platelet aggregation.

More often than not, atherosclerosis (and the concomitant narrowing of blood vessels) causes blood clots to form within the blood vessels, a condition also known as thrombosis.

While the narrowing of blood vessels in atherosclerosis is alone sufficient to cause stroke and myocardial infarction (i.e. heart attacks), thrombosis compounds both the risk and the severity of these outcomes. This is the reason why patients prone to blood clotting are placed on aspirin-containing medications, because aspirin acts as a "blood-thinner" to reduce the likelihood of these highly detrimental blot clots forming.

To understand how tocotrienols can help protect against thrombosis, one must first understand the process itself.

The initiation of thrombosis is facilitated by small cell fragments within the blood called platelets. Platelets function to initiate the blood clotting cascade upon "agitation" (for example in atherosclerosis, agitation occurs upon contact
with collagen molecules from damaged vessel walls). Apart from secreting clot-stimulating factors, activated platelets will also start aggregating together, and the outcome of this is invariably the formation of a platelet plug, which then occludes blood flow.

Figure 14: Thrombosis and atherosclerosis.

As inhibitors of platelet aggregation, tocotrienols and TRF help protect against thrombosis. In studies conducted using different aggregation-inducing agents (using different inducing agents, such as collagen or adenosine diphosphate to mimic different biological processes within the body that initiates platelets aggregation and blood clotting), TRF was found to be two to four times more effective than α-tocopherol in inhibiting platelet aggregation, showing up to a 92% inhibition in the most effective instance38.

Treatment

(250 μg) | Reduction in Platelet Aggregation

---|---

Collagen-Induced | Adenosine diphosphate-Induced

TRF | 92% | 42%

α-tocotrienol | 59% | 34%

α-tocopherol | 22% | 16%

Figure 15: Reduction of platelet aggregation by Vitamin E.

How tocotrienols manage this remarkable feat is very complex, and the underlying mechanisms have thus yet been fully delineated. Nonetheless, studies do suggest that tocotrienols can21, 30:

(i) inhibit the key components and processes of platelet activation (such as the calcium mobilization process, the protein kinase enzyme, etc.);

(ii) inhibit the clot-stimulating factors made and secreted by activated platelets (such as thromboxane A2, thromboxane-B2, platelet factor 4, etc.); and

(iii) increase the production of blood factors that function to prevent inappropriate clotting (such as prostacyclin PGI2).

## Tocotrienols help protect against reperfusion injury.

While the lack of blood supply (also termed ischemia) is commonly known to be detrimental to vital organs, it is less known that the restoration of blood flow to these previously ischemic tissues also causes injury. This is known as "reperfusion injury", which is especially detrimental at the heart, causing severe conditions such as irregular heartbeats (arrhythmia), impaired heart contraction, death of heart cells and heart failure.

The individual isomers of tocotrienols have been tested to demonstrate protection from reperfusion injuries using rat models. When treated with the individual tocotrienol isomers, the protection from cell death (apoptosis) was observed to be in the potency order of γ-tocotrienol > δ-tocotrienol > α-tocotrienol39.

In separate experiments using rabbits, consumption of tocotrienols for four weeks rendered the hearts of hypercholesterolemic rabbits relatively resistant to ischemic reperfusion injury. Not only were the heart's damaged regions visibly reduced, recovery of heart function (including blood flow and pressure generation) was also improved23.

Restoration of blood circulation to ischemic regions typically results in inflammation and oxidative damage, which in turn incapacitate the restoration of normal tissue functions. Tocotrienols help circumvent this via a few different pathways23.
(i) As inherently strong anti-oxidants and anti-inflammatory agents, tocotrienols alleviate these stressful conditions, reducing inflammation and oxidative damage.

(ii) Tocotrienols also upkeep the activity of proteasomes. These are components within the cell that help degrade unneeded or damaged proteins which accumulate during ischemia.

(iii) Tocotrienols modify the function of proteins called Caveolin. As a result, these proteins start to sequester "death signalling components" that are released during stressful conditions. By hindering the mobility and function of these components, the likelihood of cell survival during reperfusion increases.

(iv) Tocotrienols bolster key components essential to the heart's function. For example, γ-tocotrienol is shown to increase the levels of heat-shock proteins, and this helps other proteins attain proper shape and function during stress conditions. Other beneficial components which levels can be increased by tocotrienols include fatty-acid-binding proteins (important for high exercise tolerance) and heart muscle proteins.

## Special Article 1: Tocotrienols help in diabetes.

Diabetes or diabetes mellitus is a metabolic disease that affects a large number of people worldwide. According to projections by World Health Organization (WHO), more than 340 million people worldwide will likely be affected by the condition within the next 20 years (this is roughly 4% of the projected population).

Under normal conditions, when glucose (sugar) level rises in the blood (e.g. after a meal), a hormone called insulin "instructs" the liver and muscles to take up the excess blood glucose for subsequent storage. Diabetes occurs when the body does not produce enough insulin (Type 1 Diabetes), or when the body loses sensitivity in responding to the "instructions" of insulin (Type 2 Diabetes, also called insulin resistance).

The result is a high glucose level in the blood, which consequently causes oxidative and inflammatory damage to the blood vessels.

(i) Damage in the larger blood vessels (such as the arteries) may lead to atherosclerosis, stroke and myocardial infarction (i.e. heart attack). In fact, diabetes doubles the risk of cardiovascular disease, and 80% of type 2 diabetes patients die of an atherosclerotic event40.

(ii) Damage in the smaller blood vessels may lead to diabetic retinopathy (reduced vision and potentially blindness), diabetic nephropathy (kidney failure), diabetic neuropathies (nerve damage), etc. Neuropathy contributes to the risk of diabetic foot ulcers, which occasionally requires amputation.

Tocotrienols help the condition in three main ways. They help circumvent: the underlying causes of the disease, the propagation of damage during disease progression and the resulting symptoms of the disease.

(I) Circumventing the underlying causes of the disease. As mentioned, Type 1 Diabetes results from the lack of insulin. In diabetic studies22, dietary TRF has been shown to increased insulin levels by as much as two times. Additionally, it can also reduce the level of the glucagon hormone (while insulin "instructs" for sugars to be stored, glucagon does the exact opposite: it "instructs" for sugars to be released back into the blood).
In animal models, administration of tocotrienols has been observed to achieve up to a 32% reduction of blood sugar levels. Additionally, it also prevented the development of glucose intolerance in animals induced for diabetes 41, 42 (glucose intolerance is a pre-diabetic state in which the body's blood glucose is higher than normal but not high enough to warrant the diagnosis of diabetes).

On the other hand, Type 2 Diabetes occurs when the body becomes insensitive to insulin (a condition known as insulin resistance), a condition known to be closely associated with elevated levels of reactive oxygen species (ROS). Tocotrienols, as antioxidants, can suppress this effect. γ-tocotrienol has been shown to improve insulin sensitivity in animals studies43, 44.

Furthermore, tocotrienols can also modulate proteins called peroxisome proliferator-activated receptors (PPARs), and this also helps improve insulin sensitivity. Well known diabetic drugs like rosiglitazone and pioglitazone also target these components. In culture assays, α, γ, and δ-tocotrienols can activate PPARs, and they do this by enhancing the interaction between PPARs and their co-activators43.

(II) Circumventing the propagation of damage during disease progression. In diabetics, the prolonged elevation of blood glucose causes auto-oxidation and glycation.

(a) Glucose auto-oxidation is the spontaneous reaction of glucose with oxygen. The reaction produces ROS.

(b) Glycation of proteins or lipids occurs when glucose randomly attaches itself to proteins, forming advanced glycosylation end-products (AGE). The process also generates free radicals, and glycated components are typically impaired in function. For example, glycation of haemoglobin impairs the supply of oxygen in the body (haemoglobin is the protein responsible for carrying oxygen in the blood).

The accumulation of free radicals and AGE eventually results in oxidative and inflammatory damage, to blood vessels, tissues and organs.

In diabetic models, dietary TRF have been shown to reduce oxidative damage.
(a) They decrease the levels of lipid peroxidation and oxidative stress in the blood vessel by more than half, to a level even lower than that of non-diabetic, healthy rats45;

(b) They also up-regulate the manufacture of glutathione peroxidase in the kidney (this is an enzyme whose main biological role is to protect against oxidative damage); and

(c) They prevent the increase of AGE in normal rats (30% lower), and decrease the levels of glycated haemoglobin in diabetic models (20 % lower) 41, 42.

Apart from oxidative damage, there is also inflammatory damage. This causes diabetic nephropathy (i.e. kidney damage), which affects 30–40% of Type 1 Diabetes patients and 15% of Type 2 Diabetes patients. It also impairs cognitive performance in the brain (characterized by diminishing mental speed, mental flexibility, learning and memory). Patients with diabetes mellitus are two times more likely to develop dementia46.

In diabetic models, TRF has been proven to suppress the NF-κB signalling pathway. This is the main inflammatory pathway in biological systems (this will be further explained in Chapter 7 of this book). As a result, in diabetic models, dietary TRF have been shown to:

(a) reduce kidney cell damage and death (by up to 45%)47; and

(b) protect the brain from cell death (reduction by up to 62%) and improve cognitive performance. Interestingly, diabetic rats treated with tocotrienols showed improved memory and learning, when finding the escape platform within Morris Water Maze studies48.

(III) Circumventing the resulting symptoms of the disease. Most complications in diabetes involve damage to the blood vessels. Very often, an elevated blood pressure can also be observed.

(a) In diabetic models, dietary TRF has been shown to protect the blood vessels, resulting in vessel walls with a smoother surface and fewer defects42.
(b) They also prevented the increase in blood pressure associated with diabetes47, and reduced the high blood pressure in rats with glucose intolerance49.

Diabetes is also seen to be highly associated with dyslipidaemia. The condition involves abnormally high levels of lipids and cholesterols in the blood.

(a) In Type 2 Diabetic humans, dietary TRF have been shown to reduce serum total lipids by 23%, total cholesterol by 30%, and LDL-cholesterol by 43%137.

(b) The typical goal in Type 2 Diabetes is to reduce LDL-cholesterol levels to below 100 mg/dl. Tocotrienols have been shown to achieve reductions from an average of 179 mg/dl to 104 mg/dl.

## Special Article 2: Tocotrienols help in non-alcoholic fatty liver disease (NAFLD).

Non-alcoholic fatty liver disease (NAFLD) is another very common condition that occurs when excessive fat (triglycerides) is deposited in liver cells (also called hepatocytes). While this may not be harmful in general, damage subsequently occurs when fat accumulation exceeds 5-10% of the liver weight. When this happens, the liver cells eventually get destroyed via oxidative and inflammatory damage, and they are then gradually replaced by scar fibers (causing conditions such as liver fibrosis or liver cirrhosis).

Although 30% of the global population is estimated to have NAFLD, there is at the moment no drug that effectively cures NAFLD.

However, studies are now beginning to demonstrate tocotrienols' prophylactic effect against NAFLD.

(a) In animals with fatty liver and liver fibrosis, TRF suppressed both the accumulation of fatty triglycerides in the liver, as well as the resulting liver damage, both by about 50%50.

(b) A double-blind placebo controlled human study reported that consumption of palm-based TRF for one year successfully cured the condition in 50% of the NAFLD subjects they studied51.

(c) In another study of humans with end stage liver disease (which is the progressive destruction of the liver due to various conditions, including NAFLD and viral hepatic cirrhosis), TRF supplementation improved the MELD score in 50% of all subjects. This MELD scoring system (Model for End-Stage Liver Disease) is clinically used to assess the severity of chronic/end-stage liver diseases, and is seen as a reliable marker for mortality52.

# Chapter 4 Neuroprotective Properties

## What is a stroke and how is it linked to neuronal death and glutamate-induced toxicity?

Stroke is a condition that occurs when the brain neurons die as a result of disrupted blood supply, either due to a blockage in the blood vessels, or when bleeding occurs. The condition is said to be the number one cause of human disability, and the third leading cause of death in the world.

In the event of a stroke (or any brain trauma for that matter), a substance called glutamate is released from the brain, and glutamate-induced toxicity is what causes the subsequent neuronal death and degeneration (neurodegeneration).
Tocotrienols can protect the brain from stroke damage and glutamate-induced-toxicity. They have been shown to rescue the cells from death, and improve recovery after the event.

Furthermore, since glutamate-induced-neurotoxicity is also implicated in brain traumas, seizures and other chronic diseases such as AIDS-dementia complex, amyotrophic lateral sclerosis, Huntington's disease, and possibly Alzheimer's disease54, tocotrienols are suggested to be beneficial in these circumstances too.

## Tocotrienols help protect against glutamate-induced toxicity.

Blood capillaries within the brain, unlike those that exist within the general circulation, consist of tighter junctions -- endothelial cells that form the brain's capillary walls are stitched together more tightly. This forms a selective barrier (also known as the blood-brain-barrier) that hinders the access of certain harmful substances to the brain.

Unfortunately, this barrier also presents a challenge to a large number of nutrients and drugs that target the brain. Very often, beneficial or therapeutic molecules that might otherwise have been effective fail to cross the blood-brain-barrier in adequate amounts.

Studies have firmly established that orally consumed tocotrienols and TRF do reach the brain57. As a result, they are capable of protecting the brain from stroke damages and glutamate-induced-toxicity.

(i) In cell culture studies, the presence of nanomolar α-tocotrienol protected neurons from glutamate-induced toxicity, and these neurons maintained a healthy growth and motility even in the presence of excess glutamate56.

(ii) 250 nm of α or γ-tocotrienol reduced neuronal death from about 90% (without treatment) to about 10% (with treatment) in one study55, and from 70% (without treatment) to almost 0% (with treatment) in another54.
(iii) In rats with high blood pressure, consumption of α-tocotrienol reduced stroke-induced infarction (death and damage) of the brain by up to 25%57.

(iv) In dogs inflicted with brain ischemia (restriction of blood supply), consumption of α-tocotrienol significantly reduced stroke-induced lesion and infarction (by more than 80%)58. Not only that, the loss of the brain's white matter fibre tract was also reduced (the white matter fibre tract forms the brain's communication network; its well-being indicates how much of the brain's "sensing and responding" function is affected, and how well recovery is taking place).

(v) In a clinical trial of 200 human volunteers with white matter lesions (oxygen-starved brain regions), tocotrienol consumption, after a year prevented further deterioration of the condition. The increase in oxygen-starved areas was observed to be seven times less in supplemented volunteers. Moreover, by the second year, regression of lesions was observed59.

These neuroprotective properties hold, not only in response to glutamate challenge but also in response to other chemical insults. Cell culture studies show that α-tocotrienol can confer neuroprotection against chemical insults like homocysteic acid and linoleic acid. These compounds are known to reduce the level of glutathione (the brain's natural anti-oxidant enzyme) and induce oxidative stress within the body5.

Among all tocotrienol isoforms, α-tocotrienol appears to be the strongest isomer in neuroprotection. This is despite the fact that absorption efficiencies into neurons were found to be in the order of γ-tocotrienol > α-tocotrienol > α-tocopherol.

## The potency and efficacy of tocotrienols' neuroprotective effects.

In most instances of neuronal damage and death, glutamate-induced neurotoxicity is invariably involved (such conditions include stroke, brain traumas, seizures, chronic diseases like AIDS-dementia complex, amyotrophic lateral sclerosis, Huntington's disease, and possibly Alzheimer's disease).

From a biochemical point of view, glutamate triggers neurodegeneration via 2 mechanisms: (i) by death signaling mechanisms (known as excitotoxicity);
and (ii) by oxidative stress mechanisms (known as oxytosis). The brain is known to be an organ that is highly susceptible to oxidative stress and lipid peroxidation damages53. This is because it receives a large percentage of oxygen (required to sustain its high metabolic demand), but is relatively deficient in certain antioxidant enzymes.

By protecting neurons from glutamate-induced-toxicity, tocotrienols confer protection against stroke damage, and improve recovery in the aftermath of such threatening situations. They achieve this in a number of different ways, including both antioxidant and antioxidant-independent mechanisms.

(i) Antioxidant mechanism: at micromolar (10-6 M) concentrations, tocotrienols function as chain-breaking antioxidants, thus protecting the brain from the oxidative stress mechanisms triggered by glutamate. Even during normal, non-stroke circumstances (in the absence of glutamate), tocotrienols are capable of protecting the brain from general lipid peroxidation caused by exposure to free radicals and ROS.

(ii) Antioxidant-independent mechanism: at nanomolar (10-9 M) concentrations, they can also block the death signalling mechanisms triggered by glutamate.

(iii) Apart from that, tocotrienols can also help trigger additional blood supply to areas of the brain affected by stroke.

(iv) Tocotrienols may even help prevent blood vessels in the brain from leaking, thus preventing edema (water accumulation, i.e., swelling) in the brain.

Figure 16: The ways by which tocotrienols achieve their protective benefits within the brain.

The above shows that the protective actions of tocotrienols go beyond antioxidant pathways. It also means that some of these benefits are specific to the tocotrienol molecule, and no other antioxidants (not even tocopherols) are able to achieve them. Furthermore, not only do tocotrienols act on the brain cells, they also act on the blood vessels supplying nutrients to these cells.

Tocotrienols are thus superior compared to α-tocopherol, firstly because α-tocopherol requires at least micromolar (10-6 M) concentrations to confer neuroprotective effects. Secondly, even at micromolar (10-6 M) concentrations, α-tocopherol is still a weaker anti-oxidant compared to tocotrienols. Furthermore, tocopherols have been found to be less effectively absorbed by neurons55.

Figure 17: Comparing the neuroprotective properties of tocotrienols & tocopherols.

From previous studies, the concentration needed for tocotrienols to confer its neuroprotective effect is approximately one hundred nanomols (10-9), and blood plasma levels after supplementation are known to be about ten to twenty times this value5, 52. Peak plasma concentration of α-tocotrienol, after oral intake, is known to reach about three micromols (10-6). This is more than sufficient to achieve the neuroprotective properties established, via both antioxidant and antioxidant-independent mechanisms5.

It should be appreciated that the neuroprotective effects of tocotrienols at nanomolar concentrations represent the most potent biological function of all Vitamin E forms characterized so far. Generally, nutrients are required at high micromolar (10-6) or millimolar (10-3) concentration levels to achieve biological responses.

## Inside the mechanisms of tocotrienols' neuroprotective effects.

Glutamate-induced neurotoxicity and stroke typically involves a few different components.

(i) An early death-signalling component (known as excitotoxicity)5, which in turn involves cellular pathways like (i) the Eicosanoid pathway, and (ii) the ERK pathway.
(ii) A late oxidative stress component (known as oxytosis).

(iii) Leakage within the brain's blood vessels, causing edema (water accumulation) and swelling.

(iv)Disruption in the brain's blood supply.

Tocotrienols are able to address these different components effectively.

Figure 18: Glutamate induced neurotoxicity.
(I) Tocotrienols help block the early death-signaling component during glutamate-induced neurotoxicity: the Eicosanoid pathway and the ERK (extracellular-signal-regulated kinase) pathway. The underlying mechanism of glutamate-induced neurotoxicity is said to follow certain steps.

(i) During brain trauma, ischemia (lack of oxygen) or stroke, a sudden surge of glutamate molecules is released from the brain.

(ii) This excess of glutamate inhibits the uptake of cysteine into neurons, and because cysteine is used to make glutathione (GSH), this reduces the cell's level of glutathione. Low GSH levels go on to trigger the activation of an enzyme called 12-lipoxygenase (also known as 12-LOX). Meanwhile, glutamate also causes the activation of an enzyme called cPLA2, which leads to an increase of free Arachidonic Acids (AA) in the cell. AA is then used by activated 12-LOX to produce molecules called eicosanoids54. This represents the eicosanoid pathway.

(iii) Eicosanoids cause the oxidative degradation of lipids (lipid peroxidation), which is one of the main sources of cell damage and death. This represents the late oxidative-damage component during glutamate-induced neurotoxicity.

(iv) On the other hand, a glutamate surge can also activate the Src kinase enzyme, which then activates a series of ERK proteins (extracellular-signal-regulated kinases) implicated in cell death. This represents the ERK pathway.

(v) In certain situations, the Src kinase enzyme can also activate the 12-LOX enzyme of the eicosanoid pathway.

At nanomolar (10-9) concentrations, tocotrienols, specifically α-tocotrienol (but not tocopherols), can block the action of these three enzymes within neurons5, 54, 55, 56.

(i) The cPLA2 enzyme. Suppressing this enzyme reduces the production of eicosanoids, which in turn reduces the oxidative damage it causes.
(ii) The 12-LOX enzyme. Docking studies have suggested that α-tocotrienol hinder the access of AA to the 12-LOX enzyme, and thus AA cannot be processed by the 12-LOX enzyme to make eicosanoids. The roles of 12-LOX enzymes are so crucial that neurons without the enzyme essentially became resistant to glutamate-induced death; and

(iii) The Src kinase enzyme. Suppressing this enzyme also suppresses the production of eicosanoids by the 12-LOX enzyme. Additionally, edema is also suppressed.

(II) Tocotrienols help block the late oxidative damage component during glutamate-induced neurotoxicity. As mentioned, glutamate-induced neurotoxicity also involves a late oxidative-stress/lipid peroxidation component downstream from the eicosanoid pathway.

Apart from glutamate-induced neurotoxicity, oxidative damage is also implicated in various neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, and Parkinson's disease, etc. Furthermore, lipid peroxidation is also known to be a major contributor in disorders such as epilepsy and brain trauma.

Under such circumstances, micromolar concentrations (10-6M) of tocotrienols can offer protection via its strong antioxidant properties56, 60. As mentioned, α-tocotrienol has been proven to be very effective against lipid peroxidation and oxidative stress in the brain. While these antioxidant mechanisms may not be specific to tocotrienols, other antioxidants like tocopherols tend to be absorbed less well by neurons, and are thus weaker antioxidants55.

(III) Tocotrienols help block possible vascular leaks and edema in the brain during stroke or ischemic injury. During stroke or ischemic injury (injury due to lack of oxygen supply), the blood-brain-barrier is often disrupted, and this increases vascular permeability. Cells forming the blood vessel walls become less tightly sealed together, and certain blood contents may begin to leak, subsequently causing excess water accumulation (edema) and damage in the brain.

Vascular permeability and vascular leak in the brain is known to be regulated by the Src kinase enzyme, and as mentioned, tocotrienols can block the activation of this enzyme in neurons55, 61. In certain studies, mice treated with Src kinase inhibitors show less vascular leak, and as a result, have reduced levels of edema and cell death in the event of a stroke.

Figure 19: Disruption of the blood brain barrier and edema.

(IV) Tocotrienols help provide additional blood supply to the stroke affected areas. Ultimately, stroke occurs when a disrupted blood flow in the brain causes neuronal death.

Typically, when blood arteries fail to function (e.g., in the presence of a blockage or bleeding), collateral blood vessels exist to act as the brain's back-up vascular system. The competency of this back-up mechanism is known to be a good predictor of stroke outcomes (i.e., brain damage is usually more severe in patients with poor back-up mechanisms) 58.
Tocotrienols are able to bolster this mechanism. They help "activate" the nearby collateral blood vessels in the event of a disrupted blood flow. This is achieved by inducing the expression of genes responsible for arteriogenesis (arteriogenesis refers to an increase in the diameter of existing arterial vessels). This subsequently increases the production of proteins that help to remodel pre-existing collateral blood vessels into larger, new brain arteries (e.g., the TIMP1 protein, tissue inhibitor of metalloprotease 1 protein).

# Chapter 5 Anti-Cancer Properties

## What is cancer and why is it such a serious problem?

The human body is made up of trillions of cells. These cells are programmed to multiply as the body grows or renews itself, and to stop multiplying when sufficient cells are present. Apart from these, cell death is also an integral part of the body's growth and renewal process, whereby old cells that die are constantly being replaced by new cells.

Cancer occurs when cell growth is unregulated, that is, when cells) start to divide and grow uncontrollably, or when they refuse to die when their time comes. These cells accumulate to form an abnormal mass of tissue (also called a tumor), which may then invade and spread to distant parts of the body through the bloodstream or the lymphatic system (like the bloodstream, the lymphatic system is another interconnected system of vessels in the body by which the white blood cells circulate).
Cancer affects people of all ages, and the risk of developing it generally increases with age.

(i) In the year 2007, about 13% (7.9 million) of all deaths worldwide was caused by cancer.

(ii) The number of new cancer cases is set to nearly double by the year 2050 (National Cancer Institute, US).

(iii) It has been statistically predicted that more than 1 in 3 people will develop some form of cancer during their lifetime (Cancer Research UK).

The prevalence of cancer is attributable to its numerous, complex and highly ubiquitous risks factors. Besides hereditary factors, these include environmental pollutants and toxins, smoking, lack of physical activity, excessive sunlight exposure, poor diet, obesity and so on. It is thus unsurprising that the number of cancer victims continue to rise, in tandem with mass lifestyle changes within fast developing societies.

While treatments for cancer are available, treatment outcomes are unguaranteed or tied to shortcomings of one kind or another.

(i) Surgery is commonly used to physically remove tumor masses, but its effectiveness drastically declines once cancer cells have begun to spread.

(ii) Chemotherapy is the treatment of cancer by a drug or a combination of drugs, but its use and effectiveness is often limited by its toxicity to the healthy non-cancer tissues in the body.

(iii) Radiation therapy (also termed radiotherapy) involves the destruction of cancer cells using ionizing radiation, but side effects usually include skin damage, hair loss, infertility, cognitive decline, and in certain circumstances, heart disease.

Figure 20: The dilemma cancer poses and how tocotrienols may help provide a solution to these problems.

Tocotrienols, particularly γ- and δ-tocotrienol, possess properties that antagonize tumors and cancers within the body, both suppressing their growth and triggering their deaths. More importantly, they do so without harming healthy cells within the body.

## Tocotrienols possess multiple anti-cancer characteristics.

Cancer can develop in almost any organ or tissue within the human body (such as the lung, stomach, pancreas, colon, breast, skin, bones, or nerves), and depending on the part of the body that is afflicted, the prospects of mortality differ.

This means that a certain treatment may be effective, say for instance, in breast cancer, but not prostate cancer. Tocotrienols, on the other hand, have been shown to potentially antagonize cancer growth in a wide range of cancer types.

Through extensive research work, scientists are now beginning to understand the mechanisms by which tocotrienols exhibit their anti-cancer properties
(discussed in detail below). While some of these are general mechanisms common to a wide range of cancers, others have been found to be more relevant for a specific cancer type.

(I) Mechanism 1: Antioxidant properties. Cancer is a disease of genes (DNA) gone awry. Oxidative damage inflicted by free radicals to the genes (especially to those genes that regulate cell multiplication and death) may thus contribute to the disease62. Consequently, many known carcinogens (cancer causing molecules) are generators of free radicals, and cancer cells are almost always observed to have a disrupted antioxidant balance63.

According to the National Cancer Institute of USA, there is considerable evidence from chemical,  cell culture, and animal studies indicating that antioxidants may slow or possibly prevent the development of cancer.

Therefore, regular supplementation of powerful antioxidants, such as tocotrienols, is desirable. Nevertheless, the antioxidant attributes of tocotrienols are not the only aspects involved, and even modified tocotrienols, devoid of any antioxidant properties, have been found to remain effective in causing the death of cancer cells 64.

(II) Mechanism 2: Inhibition of the mevalonate pathway. Earlier on, we discussed about how tocotrienols can inhibit the HMG-CoA reductase enzyme (the rate limiting enzyme responsible for cholesterol production in the body) to help lower the body's cholesterol levels. In fact, the same pathways involved are fostered by cancer cells to produce components for growth and survival65, 66.

These pathways are constantly in overdrive within cancer cells. While healthy cells are capable of automatically suppressing these pathways when cholesterol production exceeds a certain threshold (via feedback regulation mechanisms), cancer cells, on the other hand, have defective feedback mechanisms. The resulting overactive state works to support the cancer's aggressive growth.

Suppression of the HMG-CoA reductase enzyme by tocotrienols (by reducing its production and increasing its degradation)33, 67 may thus potentially (i) prevent cancer cell formation; (ii) stop its growth; (iii) cripple its aggressive attributes; or (iv) kill it.
Healthy cells, on the other hand, are less dependent on the enzyme for its survival and are thus insensitive to this suppression. This gives tocotrienols the unique ability to differentiate between healthy cells and cancer cells.

(III) Mechanism 3: Inhibition of the NFκB pathway (the pathway for cancer survival and spread) and induction of the p21 protein. The NF-κB pathway (nuclear factor-kappaB) is a pathway that supports cell survival and proliferation, and as a result, is often elevated in cancer cells. The pathway is undoubtedly crucial to cancer progression, and numerous lines of evidence even suggest that it contributes to chemotherapy-resistant tumors68.

Tocotrienols have been found to suppress the NFκB pathway within cancer cells, and they do this by suppressing the IKK enzyme (ΚB kinase enzyme) which activates it 69.

Furthermore, tocotrienol rich fractions have also been found to induce the activity of the p21 protein, another protein which, under normal circumstances, functions to keep cell proliferation in check and under control. Cancer cells subsequently enter a dormant non-replicating state, effectively stopping cancer growth, invasion and spread.

Figure 21: Tocotrienols can help by inhibiting cancer growth, proliferation, spread and invasion.

(IV) Mechanism 4: Activation of the Caspase pathway (the pathway for organized cell death). Apoptosis (organized cell death) is the process by which stressed or injured cells "commit suicide". The process forms part of a "quality assurance" mechanism within the body, ensuring that unfit cells do not remain within the body.

Tocotrienols can trigger apoptosis in cancer cells, which then commence self-destruction in an organized manner. When this happens, the DNA within the cell gets degraded, and the cell eventually "blebs" into smaller fragments for uptake by surrounding cells. Cell death in an organized manner is important, because an unorganized death process (also termed necrosis) causes spillage of cell contents and harmful chemicals, which is potentially damaging to surrounding cells.
Initiation of apoptosis is known to be triggered by the Caspase pathway. Like most pathways, this is a complicated process that involves a series of steps and components, many of which are targets that tocotrienols have effect upon. Cancer cells treated with tocotrienols have been seen to experience70:

(i) an activation of the JNK enzyme. This activation process acts as an internal stress signal that alarms the cell of its own burdened state;

(ii) an activation of both Caspase-8 and Caspase-9, which are initiator caspases that activate effector caspases (caspases-3, -6, and -7). Caspases are enzymes capable of breaking down proteins;

(iii) an increased synthesis of Bax and BID proteins (these are pro-apoptotic proteins);

(iv) an increased Bax: Bcl2 ratio, of which a high ratio favours apoptosis; and

(v) a suppression of the PI3K/PDK-1/Akt pathway (a pathway which prevents apoptosis from occurring in healthy cells).

Figure 22: Tocotrienols help by inducing cancer cell death.

(V) Mechanism 5: Inhibition of the telomerase enzyme (the enzyme that immortalizes cancer cells). Within the body, cells that die are constantly being replaced by new cells formed from the division of other cells. However, a typical human cell can only divide so many times before it finally hits a limit and loses this ability (as observed in the normal ageing process).

In the nucleus of each cell, DNA molecules are packaged into structures called chromosomes. Each time a cell divides, DNA situated at the edge of the chromosomes (called telomeres, see illustration below) gets eroded away. As a result, a part of the genetic material is lost forever with each subsequent cell cycle. Although an enzyme called telomerase functions to protect this erosion
from occurring, telomerase production diminishes with age, and the cell eventually loses so much genetic material that it can no longer divide.

Cancer cells often possess abnormally over-active telomerase enzymes. Consequently, tumor cells generally show no net loss of genetic material despite successive cell divisions71, and this supports their ability to divide endlessly and uncontrollably.

Tocotrienols can act as telomerase inhibitors in cancer cells, and they do this by inhibiting a supporting protein that helps it function 66. On the other hand, telomerase in healthy cells are not inhibited. In fact, studies show that tocotrienols are able to restore the diminished activity of telomerase in aged cells, thereby conferring anti-ageing effects (see Chapter 6 for more details).

Figure 23: Tocotrienols can help by suppressing the unregulated and potentially endless division of cancer cells.

(VI) Mechanism 6: Inhibition of angiogenesis – the formation of new blood vessels. Angiogenesis, the formation of new blood vessels, is a critical step in cancer development, because blood vessels are crucial for supplying oxygen and nutrients to cancer cells. Furthermore, these vessels also act as conduits for tumor spread.
The angiogenesis process typically progresses in a number of particular stages, and tocotrienols have been found to suppress these different stages68, 74, with potency in the order of: δ > β> γ> α-tocotrienol66, 72, 73.

Figure 24: The stages of angiogenesis and its suppression by tocotrienols.

Figure 25: Angiogenesis and its role in cancer progression and spread.

Furthermore, oxidative stress stimulates and induces angiogenesis, and tocotrienols can potentially circumvent this by being strong antioxidants.

Recently, studies have also reported that HMG-CoA reductase inhibitors interfere with angiogenesis74. As discussed, tocotrienols are capable of suppressing the HMG-CoA reductase enzyme by reducing its production and increasing its degradation within cells33, 67.
(VII) Mechanism 7: Disruption of Metastatic Spread and Invasion. The spread of cancer cells to distant sites (also termed metastasis) is a highly damaging aspect of cancer. Responsible for 90% of all cancer-related deaths, it is also one of the greatest impediments in cancer treatment75.

Very often, cancer cells produce a substance known as matrix metalloproteinase (MMPs), which breaks down and remodels the extracellular matrix, facilitating their invasion and spread into surrounding tissues. γ-tocotrienol has been shown to reduce the production of MMPs, and increase the production of "anti-MMPs substances". Anti-MMPs substances (such as tissue inhibitors of metalloproteinases, TIMPs), form complexes with MMPs, rendering MMPs non-functional75.

Besides that, tocotrienols have also been shown to restore the production of E-cadherin and gamma-catenin in cancer cells76. Both of these are proteins located at the cell surface that function to hold and bind cells to their correct location. Failure (or reduction) in its production is typically observed in cancer cells, and this enables cancer cells to invade and spread to other locations.

Figure 26: The invasion and spread of cancer cells (also called metastasis).

The following section will discuss the different types of cancer, and how research show that tocotrienols may be able to help.

Figure 27: Tocotrienols and the various types of cancer

## Tocotrienols and pancreatic cancer.

The pancreas is a large organ located behind the stomach. It makes and releases enzymes into the intestines, helping the digestion of food (especially fat).

Pancreatic cancer is the most lethal cancer of all, with an estimated five-year survival rate of about 4%.

(i) When it is diagnosed, it is often already at very advanced stages. More than 80% of patients, when diagnosed with pancreatic cancer, have already experienced tumor spread.

(ii) Very few pancreatic tumors can be completely removed by surgery.

It responds poorly to chemotherapy. The only treatment agents approved by the U.S. Food and Drug Administration (FDA) are gemcitabine and erlotinib, and both produce responses in less than 10% of patients, have multiple adverse effects and are known to be associated with drug resistance.

What do scientific studies show about the effects of tocotrienols?

Tocotrienols inhibit pancreatic cancer growth and proliferation | In cell culture studies and mice models, δ- and γ-tocotrienol suppressed the proliferation of various pancreatic cancer cell lines, irrespective of their different underlying genetic faults68, 77.

---|---

Tocotrienols induce pancreatic cancer death | In cell culture studies, δ-tocotrienol caused apoptosis (organized cell death) in various human pancreatic cancer cells77.

Preliminary results of human trials also show that δ-tocotrienol is able to increase cell death in pancreatic tumors without causing toxicity78.

Tocotrienols work synergistically with pre-existing treatment strategies | In mice models implanted with human pancreatic tumors, γ-tocotrienol enhanced the antitumor activity of gemcitabine (the standard treatment for patients with pancreatic cancer)68.

In terms of the mechanisms involved, γ-tocotrienol has been shown to inhibit the NF-κB pathway in pancreatic cancer cells. This subsequently down-regulates downstream proteins that support survival, proliferation, invasion and spread. Since the pathway is also postulated to give certain pancreatic cancer cell lines their resistance to chemotherapy, tocotrienols may also be effective in potentiating chemotherapeutic drugs.

## Tocotrienols and breast cancer.

Breast cancer originates from the breast tissue, most commonly from the inner lining of milk ducts) or the lobules that supply the ducts with milk. Worldwide, breast cancer comprises 22.9% of all cancers in women (excluding non-melanoma skin cancers, which are rarely fatal). In 2008, breast cancer caused 458,503 deaths worldwide79.

What do scientific studies show about the effects of tocotrienols?

Tocotrienols can prevent the occurrence of breast tumor | Tocotrienols, but not tocopherols, delayed and reduced the occurrence of tumor in rats exposed to potent inducers of breast cancer (7, 12-Dimethylbenz[a]anthracene, DMBA)80, 26. Similar results were observed even when induction of the cancer was attempted by physically transplanting breast cancer cells onto mice81.

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Tocotrienols inhibit breast cancer growth and proliferation | Breast cancers are usually categorized depending on whether the cells have estrogen receptors (ER) present on their surface, which then determines the specific treatment regime (i.e., ER+ cancers rely on estrogen for their growth, and are thus treated with drugs that block the effects of estrogen, e.g. Tamoxifen). Tocotrienols were found to inhibit the growth of human breast cancer cells, irrespective of their estrogen receptor status82, 83.

The potency of this inhibition follows the order of: γ- ≈ δ- > α-tocotrienol84, with almost no inhibition observed with tocopherols. Studies also show that cancer cells preferentially take up and accumulate tocotrienols compared to tocopherols 85, 69.

Tocotrienols induce breast cancer death | Tocotrienols, especially γ-tocotrienol, was shown to induce apoptosis (organized cell death) in breast cancer cells within twenty-four hours of incubation86, 87.

Tocotrienols work synergistically with pre-existing treatment strategies | Drugs like statins inhibit tumor growth by inhibiting the HMG-CoA reductase enzyme, but clinical applications has thus far been limited by heart toxicity and muscle breakdown at high dosages. Tocotrienols target the same enzyme, but via a different mechanism, and do not cause any toxic side effects. Moreover, γ-tocotrienol has been shown to work synergistically with statins (thus allowing usage of a much lower dose of statins)88.

1:1 combinations of γ- or δ-tocotrienol with tamoxifen showed synergistic growth inhibition on both ER+ and ER- breast cancer cells84.

Tocotrienols have been also shown to enhance the effect of breast cancer drugs like Erlotinib (by four times), Gefitinib (by greater than six times) 89, and celecoxib (which excessive doses are linked to gastrointestinal and cardiovascular toxicity)90.

In terms of the mechanisms involved, tocotrienol rich fractions (TRF) and γ-tocotrienols were shown to activate the caspase pathway in breast cancer cells, causing apoptosis. They do so by suppressing the PI3K/PDK-1/Akt pathway in breast cancer cells, which help keep caspases inactive 70, 91, 92, 93. γ-tocotrienol has also been shown to suppress the NFκB pathway in breast cancer cells 69.

## Tocotrienols and prostate cancer.

The prostate is a gland in the male reproductive system. Although highly prevalent, most cases of prostate cancers are slow-growing (with long latency periods) and symptom-free. They therefore usually go undetected and untreated.

Nevertheless, aggressive prostate cancers do exist, and they account for more cancer-related mortality than any other cancer except lung cancer. Aggressive prostate cancer is also identified as the most common cause of death in men over age 75.

What do scientific studies show about the effects of tocotrienols?

Tocotrienols inhibit prostate cancer growth and proliferation | Tocotrienols and TRF have been established to cause growth inhibition in at least 3 different prostate cancer cell lines (LNCaP, DU145, and PC-3, derived from different metastatic sites), and in mice models as well94, 95.

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Tocotrienols induce prostate cancer death | Studies have shown that tocotrienols selectively accumulate in prostate cancer cells, causing the apoptosis (organised cell death) of these cancer cells without harming healthy cells94, 95.

Tocotrienols work synergistically with pre-existing treatment strategies | Tocotrienols have been shown to inhibit the expansion of prostate tumors in mice models; in combination with the cancer drug Docetaxel (by up to 50%), and in combination with radiotherapy (by up to 40%)94, 95.

## Tocotrienols and liver cancer / hepatocellular carcinomas.

The liver is known to be crucial in a wide range of body processes, including detoxification, protein synthesis, and digestion. Unsurprisingly, liver cancer is extremely deadly, and will kill almost all of its patients within a year. 80 – 90% of hepatocellular carcinomas fail to be completely removed using surgery, and the disease is usually fatal within three to six months if the cancer is not completely removed from the body.
The number of inflicted patients continues to rise (due to rising incidences of obesity, diabetes and chronic hepatitis) and the disease is already the third most common cancer in the world96.

What do scientific studies show about the effects of tocotrienols?

Tocotrienols can prevent the occurrence of liver tumor | In mice genetically-modified to promote tumor development, the emergence of liver tumors is seen to be five times lower with TRF consumption97.

In rats exposed to potent liver carcinogens (Acetylaminofluorene, AAF or diethylnitrosamine, DEN), those that were fed with tocotrienols showed less liver damage and cancerous irregularities26, 98, 99.

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Tocotrienols inhibit cancer growth and proliferation | Compared to tocopherols, liver cancer cells take up tocotrienols faster100, and the inhibition exerted on these cells followed the potency order of: δ-> β- > γ- ≥ α-tocotrienol97. Healthy cells were not affected.

Tocotrienols induce liver cancer death | Within 6 hours of treatment, TRF (but not α-tocopherol) caused the apoptosis (organized cell death) of hepatocellular carcinomas in mice cell cultures101.

Similar to the case in breast cancers, tocotrienols have been shown to induce apoptosis in liver cancer cells via induction of both Caspase-8 and Caspase-9100, 102. An increased synthesis of the Bax (Bcl-2–associated X protein) and Bid proteins (BH3 interacting-domain death agonist) can also be observed, both of which are important triggers for Caspase-9 activity102.

## Tocotrienols and colon cancer.

Colon Cancer

Mostly triggered by ageing and lifestyle habits (such as a high fat & alcohol diet, obesity, smoking and a lack of physical exercise), colorectal cancer occurs at either the large intestine (also called the colon) and/or the rectum (at the end of the colon). It is one of the leading causes of cancer-related deaths in the United States (American Cancer Society). In the year 2008, 1.23 million new cases of colorectal cancer were clinically diagnosed worldwide, and the number of deaths in the same year was 608,00096.

What do scientific studies show about the effects of tocotrienols?

Tocotrienols inhibit colon cancer growth and proliferation | Tocotrienols, but not tocopherols, has been shown to inhibit growth and colony formation of colon cancer cell cultures103.

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Tocotrienols induce colon cancer death | Tocotrienols, but not tocopherols, has also been shown to trigger the apoptosis (organized cell death) of colon cancer cell cultures, as early as 18 hours after initiation of treatment103.

In terms of the mechanisms involved, TRFs have been shown to increase the Bax:Bcl2 ratio in colon cancer cells, consequently triggering apoptosis (while the Bax protein induces Cytochrome C release and causes apoptosis, the Bcl2 protein blocks cytochrome C release and supports cancer survival).

Research also showed that tocotrienol rich fractions (TRF) can induce the p21 protein within cancer cells, causing these cells to enter a dormant non-replicating state, effectively stopping cancer growth. Under normal circumstances, the p21 protein functions to keep cell proliferation in check and under control.

In a separate study, tocotrienols (but not tocopherols) have also been shown to inhibit the telomerase activity within human colorectal carcinomas. This suppresses the cancer's ability to divide endlessly and uncontrollably, and the potency order of this was established to be: δ- > β- > γ- ≥ α-tocotrienol104.

## Tocotrienols and skin cancer / skin neoplasm.

Skin cancer is the most common of all human cancers (in the United States alone, one million new cases are diagnosed each year). The three major types of skin cancer are:  basal cell carcinoma (BCC),  squamous cell carcinoma (SCC), and melanoma. While basal cell carcinoma and squamous cell carcinoma are more common, they are less likely to spread. Melanoma, on the other hand, spreads aggressively, and is often fatal when not treated early.

In terms of the risk factors, sunlight or ultraviolet (UV) ray exposure is arguably the most frequent cause of skin cancer, especially in humans with fair skin (these people tend to  freckle or get  sunburnt easily). Those with numerous unusual moles may also be at risk, as moles, although not cancerous per se, can become cancerous105.

What do scientific studies show about the effects of tocotrienols?

Tocotrienols inhibit skin cancer growth and proliferation | In mice implanted with melanoma cells, consumption of tocotrienols inhibited the subsequent cancer growth. The order of potency for this was found to be: δ- > γ- > α-tocotrienol67, 97.

In another study, treatment with γ-tocotrienol was also seen to render melanoma cells less invasive (causing the level of invasion to decrease by more than 50%)76.

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Tocotrienols induce skin cancer death | Tocotrienols have been shown to induce apoptosis in melanoma cells as early as 24 hours after initiation of treatment, and the order of potency for this was shown to be: γ- > δ- > β- > α-tocotrienol76.

Tocotrienols work synergistically with pre-existing treatment strategies | When combined with chemotherapeutic drugs such as Docetaxel or Dacarbazine, γ-tocotienol boosted the efficacy of these drugs by up to two-fold76.

In terms of the mechanisms involved, γ-tocotrienol has been shown to induce the caspase pathway within skin cancer cells by activating upstream components within the pathway, such as the JNK enzyme76 (see figure 22 illustrating the caspase pathway). The same study also established γ-tocotrienol's ability to suppress the NFκB pathway in these cells (this interferes with cell survival), as well its ability to restore the production of E-cadherin and gamma-catenin in them (this suppresses cancer invasion and spread)76.

Apart from that, suppression of the HMG-CoA reductase enzyme by tocotrienols has also been shown to occur in skin cancer cells, as early as forty-eight hours after initiation of treatment88. As mentioned, the HMG-CoA reductase enzyme (responsible for cholesterol production in the body) is fostered by cancer cells to produce components for growth and survival.

# Chapter 6 Cosmeceutical Benefits

## What is the cosmeceutical industry and what is the industry looking for?

Cosmeceuticals (a combination of cosmetics and pharmaceuticals) are cosmetic products that contain biologically active ingredients with medical benefits. The industry is estimated to be worth more than USD 6.5 billion in the United States alone today.

These products work on the skin, which is not only the largest organ in the human body, but also the foremost factor in dictating an individual's appearance. Consequently, there is a strong, constant and growing demand for ingredients that are capable of lightening, protecting, or rejuvenating the skin.

Vitamin E has had a long history of use in the cosmeceutical industry. And although α-tocopherol is historically the conventional isomer used, there is now increasing evidence indicating tocotrienols to be comparatively superior in cosmeceutical applications.

The cosmeceutical benefits of tocotrienols are founded on four of its main effects, namely:

(i) skin lightening;

(ii) photo-protection (protection from ultraviolet radiation);

(iii) moisturization; and

(iv) anti-ageing.

## Tocotrienols have skin lightening & whitening properties.

The skin layer is made up of 2 types of cells:

(i) Keratinocytes, the typical skin cells; and

(ii) Melanocytes, special cells responsible for the production of melanin, the pigment that gives human skin its color. The process is also called melanogenesis ("melano" for melanin, and "genesis" meaning "the making of"). Skin darkening is invariably triggered by an overproduction of melanin.
Normally, sunrays or ultraviolet (UV) rays reaching the keratinocytes (and the subsequent free radical production) stimulates these cells to secrete hormones called α-melanocyte stimulating hormone (α-Msh), which triggers nearby melanocytes to increase melanin production. The melanin produced subsequently gets transported back into the keratinocytes, causing them to darken.

Two types of melanin can be produced: eumelanin (a brown black pigment) and pheomelanin (a yellow red pigment). The balance and the ratio of both melanin pigments determine skin color and intensity.

Figure 28: The darkening of the skin by production of melanin pigments (melanogenesis).

Because free radicals can encourage the overproduction of melanin and cause skin darkening, powerful free radical scavengers such as tocotrienols can prevent this from occurring.

Furthermore, tocotrienols can also directly inhibit melanin production (melanogenesis) within skin cells. Eventually, as the old-darkened skin cells gradually slough off, newer cells with less melanin reach the surface of the skin, giving the skin a lighter and whiter complexion.

Skin cells have been tested to show greater than 50% reduction of melanin content106 when treated with δ-tocotrienol (for 48 hours), and greater than 40% reduction of melanin content when treated with TRF (for 24 hours)6.
In addition, the melanin-inhibition potency of tocotrienols is seen to be far higher than some of the currently used skin whitening ingredients agents, such as:

(i) Kojic Acid, a by-product of rice fermentation, used in cosmeceutical products for its melanin inhibition properties107, 108; and

(ii) Sodium Lactate, a multi-functional material that is used in many skin lightening products, suggested to have skin lightening properties in combination with ascorbic acid (vitamin C)6.

Below are some of the results obtained from experimental studies. They show that roughly 150 times the amount of kojic acid, or sodium lactate, is required to achieve melanin suppressions that are merely comparable to that of tocotrienols. The potency of α-tocopherol (if any at all), on the other hand, lags behind all three of these6.

Whitening Agents | Decrease in the skin's melanin content (after 9 days)

---|---

γ-Tocotrienol (20 μM) | 60% decrease

δ-Tocotrienol (20 μM) | 25% decrease

Kojic Acid (3.5 mM)* | 30% decrease

Sodium Lactate (4.5 mM)* | 30% decrease

α-Tocopherol (20 μM) | No decrease

*these levels are > 150 x the amounts of 20 μM

In terms of the mechanism involved, tocotrienols work by targeting not only one, but at least three important enzymes needed in the pathway of melanin production.

(i) Tocotrienols decrease the activity of the tyrosinase enzyme activity, the first and most critical "rate limiting" enzyme within the pathway (experimental result below).

Agents Used | Period of Treatment | Decrease in Tyrosinase Activity

---|---|---

δ-Tocotrienol (50 μM) | Within 48 hours106 | > 75% decrease

TRF (20 μM) | Within 72 hours6 | > 25 % decrease

γ-Tocotrienol (20 μM) | After 9 Days

(establishing efficacy even after long term exposure)6 | > 90% decrease

δ-Tocotrienol (20 μM) | > 65% decrease

Kojic Acid (3.5 mM)* | > 90% decrease

Sodium Lactate (4.5 mM)* | > 60% decrease

*these levels are > 150 x the amounts of 20 μM

(ii) Tocotrienols also decrease the content of the TRP-1 enzyme (tyrosinase-related protein-1) and the TRP-2 enzyme (tyrosinase-related protein-2)106 within melanocytes (experimental result below).

Treatment | Decrease of enzymes in melanocytes

---|---

δ-Tocotrienol (50 μM) | Up to 75% decrease in TRP-1 present

Up to 52 % decrease in TRP-2 present

Below is an illustration of the melanin production pathway in skin cells, and the role of these enzymes within the pathway.

Figure 29: Tocotrienols inhibit the enzymes within the melanin production pathway.

As a result, tocotrienols can be beneficial, as both therapeutic and preventive agents, against various skin darkening conditions.

(i) Hyperpigmentation. This is the darkening of the skin caused by sun damage, inflammation, or other skin injuries, including those related to acne scars.
(ii) Darkening of large skin areas associated with hormonal changes. This is often seen with pregnancy, consumption of birth control pills, steroids, etc.

(iii) Solar lentigenes (age or liver spots). These are dark spots found on the hands or the face, mostly due to frequent exposure to the sun.

## Tocotrienols have photo-protection properties (protection from ultraviolet damages).

Because the skin forms the outermost barrier of the body, it is the most exposed organ, and therefore subjected to a variety of environmental insults, in particular, to ultraviolet (UV) radiations from the sun.

When exposed to UV rays, the skin produces a range of reactive oxygen species (ROS). These ROS, upon depleting the cell's natural antioxidant defenses, go on to inflict oxidative damage on the lipids, proteins and DNA within these cells. This may lead to a number of consequences6.

(i) Wrinkle formation. Free radicals are said to be the primary cause of wrinkles.

(ii) Sagging skin. Oxidation of lipids (lipid peroxidation) weakens connective tissues within the skin, thus destroying its elasticity.

(iii) Sunburn (also known as skin inflammation).

(iv) Photo-ageing (ageing due to exposure to the sunlight). This is said to be the main cause (90%) of a damaged skin appearance (source: U.S. Environmental Protection Agency).

(v) Tumor and cancer formation. Statistics show that 1 in every 5 Americans will develop some form of skin cancer within their lifetime (source: American Cancer Society). Globally, the incidence of skin cancer is about 3 million each year (the condition accounts for 1 in every 3 cancers), and doctors believe that exposure to UV radiation (specifically UV-B) is the most important cause of this.

Tocotrienols offer protection against these consequences, via a variety of different ways.
(i) Vitamin E is known to absorb radiation strongly in the UV-B frequency, and can therefore act as effective sunscreens.

(ii) As powerful free radical scavengers, tocotrienols can protect the skin from oxidative damage.

(iii) Tocotrienols also possess anti-ageing, anti-inflammatory (sunburn) and anti-tumor properties.

In animal models, the depletion of tocotrienols within the skin during the course of UV-irradiation has been observed. This effectively demonstrates the real-time consumption of these "scapegoat" molecules as they are spent while conferring the protective benefits6.

In a separate study where animals were exposed to UV-B rays daily, consumption of TRF greatly reduced the extent of damage to their skins. TRF was observed to confer a greater protection compared to α-tocopherol, and this was in spite of a higher α-tocopherol content found within the skin cells (although present at about 10 times the concentration compared to tocotrienols, α-tocopherol was seen to be merely half as effective)111. Below is a summary of the results.

Reduction in damage caused by UV-B exposure | With consumption of tocotrienol rich fraction (TRF) | With consumption of α-tocopherol

---|---|---

Reduction of TBARS   
(under normal conditions) | 50% reduction | 30% reduction

Reduction of TBARS  
(when exposed to UV-B) | 80% reduction | 30% reduction

Protection against sunburn (when exposed to UV-B) | Strong | Weak

Reduction of tumor incidences (when exposed to UV-B) | 40% reduction | 20% reduction

** TBARS (thiobarbituric acid reactive substances) are oxidative stress markers.

Among all tocotrienol isomers, the protection conferred by δ-tocotrienol and γ-tocotrienol is found to be the highest. In fact, the SPF value of δ-tocotrienol (this is a measure of protection against UV-B) was determined to be twice that of α-tocopherol. In simplified terms, this implies that δ-tocotrienol will allow subjects to stay twice as long under the sun, before getting sunburnt6.

## Tocotrienols have skin moisturizing properties.

Moisturization refers to the ability of a substance to retain and increase the water content within the skin's outermost layer (this layer is also called the stratum corneum). As an oil-based nutrient, Vitamin E has always been recognized as a cosmeceutical ingredient with outstanding skin hydration properties.

Studies conducted on the human skin have shown that direct application of TRF (in the form of a 5% emulsion) can increase the skin's hydration value by more than 40%, as early as 90 minutes after application112. It should also be appreciated that these effects hold, not only when TRF is being topically applied, but also when it is orally consumed. Reduction of the skin's water loss, as a result of tocotrienol consumption, has been detected to be as high as 50%111.

Apart from the above, tocotrienols are also known to work well with other cosmeceutical ingredients. A double-blind, placebo controlled human study showed that palm-based TRF works in synergy with Astaxanthin (this is a naturally occurring carotenoid with antioxidant properties) 112, 113. Supplementation of both ingredients has been shown to significantly increase the skin's moisture levels, by up to 10 times (at the eye region). This result, observed within 4 weeks of supplementation, was also accompanied by an improvement of skin elasticity, a reduction of fine wrinkles, and a reduction of swelling under the eyes.

## Tocotrienols have anti-ageing properties.

As opposed to photo-ageing that is caused by sunlight, the chronological ageing of the human body (this includes the skin) is a highly complex process. It can be explained by numerous theories, each of which is supported by a multitude of experimental evidence.

In simplified terms, all living cells invariably possess a limited capacity for division and proliferation. This means that after a set (and finite) number of cell divisions, all cells inevitably experience senescence (meaning "old"), where they enter a dormant state and lose the ability to divide any further. Cell senescence is said to underlie the body's chronological ageing processes.
Tocotrienols have been shown to confer protection against senescence and ageing, for instance, in the human diploid fibroblast (HDF) cell. These cells are responsible for synthesizing collagen as well as the extracellular matrix, both of which are supporting structures in firm, taut and youthful skin114.

(i) Typically, after undergoing roughly 50 cycles of cell division, the HDF cell enters a senesced state, which causes them to enlarge, flatten, and attain irregular shapes.

(ii) However, when directly incubated with TRF, these senesced cells reversed their morphology, back to that which resembled young cells (those that have only undergone less than twelve cell cycles).

The anti-ageing mechanisms of tocotrienols can generally be divided into 2 categories: (i) protection against oxidative stress, and (ii) protection against telomere aging.

(I) Protection against oxidative stress. According to the free radical theory of aging, oxidative stress and damage, caused by reactive oxygen species (ROS), contribute not only to aging, but also to age-related diseases.

ROS primarily originate from the mitochondria (the cell's own "energy reactor"), where up to 4% of the oxygen consumed will inevitably be converted into ROS. There is therefore a constant generation of ROS within the body, and as time passes, the accumulated damage they cause (on proteins, lipids and DNA) leads to a decline in the cell's renewal and division capability, causing senescence.

Ageing can thus be seen as a balance between "the accumulations of ROS" versus "the cell's antioxidant defenses".

Tocotrienols, by virtue of their powerful free radical scavenging and antioxidant characteristics, are therefore desirable. Indeed, genetic modifications that boost antioxidant defenses, as well as supplementation of external antioxidants, have been demonstrated to increase the lifespan of certain organisms115, 116.

(i) In cell culture studies, treatment with TRF reduced the DNA damage within "old" cells (those that have undergone greater than fifty-five cell cycles), to a level comparable to that of "young" cells (those that have only undergone less than twelve cell cycles)114.
(ii) In models where primitive animals were subjected to Ultraviolet B irradiation to induce ageing, treatment with tocotrienols reduced the cell's protein carboxylation (an oxidation process indicative of ageing) and extended lifespan by up to 19%117.

(II) Protection against telomere shortening. Apart from oxidative stress and damage, telomere shortening is also highly implicated in the process of ageing and senescence.

As discussed in the previous chapter, within the nucleus of each cell, DNA molecules are packaged into structures called chromosomes. Each time a cell divides, DNA situated at the edge of the chromosomes (also called telomeres) gets eroded away. As a result, a part of the genetic material is lost forever with each subsequent cell cycle. While the enzyme telomerase protects and minimizes this corrosion, telomerase production diminishes with age, and (at a critical limit), the cell eventually loses so much genetic material that it can no longer divide.

Figure 30: Cell ageing and senescence.
Diminished telomerase activities, as well as telomere shortening, are therefore common attributes of old-senesced cells.

Tocotrienols have been shown to reduce the extent of telomere shortening within senesced human diploid fibroblast (HDF) cells, by restoring (essentially almost doubling) their telomerase activity114. (Note that this effect on the telomerase enzyme is exactly the opposite when it comes to cancer cells, as mentioned in the previous chapter).

Moreover, because oxidative damage may also contribute to telomere shortening118, the potent antioxidant properties of tocotrienols are again beneficial. Indeed, pre-treatment of fibroblast cells with γ-tocotrienol prevented telomere shortening upon subsequent exposure to oxidative stress114.

## Tocotrienols are effectively absorbed and safe for topical application.

The cosmeceutical benefits of tocotrienols would be in vain without evidence of its effective absorption, and more importantly, its safe use. Without these, a cosmeceutical product essentially loses its value, regardless of the number of benefits it may possess.

In terms of absorption, the skin has been shown to have a natural affinity for tocotrienols, and these molecules tend to accumulate selectively within the skin, whether applied topically or consumed orally109, 110.

(i) Animal models have shown that topically applied tocotrienols can penetrate the skin effectively. Within 2 hours of application, 20 μL of 5% TRF successfully increased the skin's level of α-tocotrienol (by up to eighty-fold) and γ-tocotrienol (by up to fifty-fold) 109.

(ii) These isomers accumulate at the stratum corneum. This is the outermost layer of the skin, and incidentally, where benefits such as photo-protection (protection against UV rays) are most required.

Another point to note is that, most skin care products currently in the market contain the acetate version of Vitamin E (such as α-tocopherol acetate). However, the acetate form of Vitamin E may not be as effective as thought: esterification of α-tocopherol into its acetate form essentially locks it in an "inactive" state. This "inactive" acetate form is thought to possess an increased
shelf life. Antioxidant efficacy, however, would then depend on its reversion back into its active form by hydrolysis.

(i) Recently, studies are showing that the extent of this hydrolysis is in fact, minimal. In one particular study, no hydrolysis was observed even after 5 hours of application of the acetate form, and as a result, none of the corresponding protective effects were present.

(ii) The same study subsequently concluded that even after 5 consecutive days of application, the percentage of hydrolysis was found to be below 1%1.

(iii) Another study separately showed that even after four months of topical application, α-tocopherol acetate failed to elicit changes in the levels of the molecule present, at both the skin and in the blood119.

In term of safe use, tocotrienols have been tested in many ways.

(i) Animal studies have shown that direct exposure of cells to as high as 50 μM of tocotrienols produces no toxic effects, even after 72 hours of exposure106.

(ii) In human studies, topical application of 5% tocotrienol rich fractions (TRF) elicited no undesirable skin reaction, even after 96 hours112.

(iii) Cumulative patch studies in humans (these test for allergic responses) found that their subjects display minimal reaction towards topical application of tocotrienol rich fractions (TRF)112.

## Special Article 3: Tocotrienols can offer protection from radioactivity.

**(I) Aftermath of a nuclear disaster: how dangerous is radioactivity exposure?** Triggered by the 2011  Tohoku earthquake-tsunami, the events of the Fukushima Daiichi nuclear disaster resulted in the release of large amounts of radioactive isotopes from the crippled  Fukushima Daiichi Nuclear Power Plant, into the Pacific Ocean. Shortly after, the scientific community found itself in a heated debate on the danger levels of radioactivity exposure. Experts were brought in, and numerous studies were conducted to evaluate the extent of the threat. Some of these conclusions are shown below.
(i) The incident is rated at level 7 on the International Nuclear Event Scale. The total amount of iodine-131 and caesium-137 released into the atmosphere has been estimated to exceed 10% of the emissions from the 1986 Chernobyl disaster.

(ii) Following the event, trace amounts of radiation (including iodine-131 and caesium-134/137) have been detected around the world, including in New York State, Alaska, Hawaii, California, Montreal and Austria.

(iii) According to reports from various sources, including the US Environmental Protection Agency, there is food with radionuclides being found in the food supply in America (note that radioactive particles that are consumed are many times more dangerous compared to external exposure).

(iv)Experts generally agree that there is no such thing as a safe level of radiation exposure. An associate professor from the University of Melbourne's Nossal Institute for Global Health remarked that even though there is a threshold for the other damaging effects of radiation, there is none for cancer. Any radiation exposure, however little, can contribute to increased cancer risks.

Because it is practically impossible to accurately analyze and definitively determine the actual threat levels of radioactivity exposure, fear and anxiety on the adverse effects of radiation prevails not only in Japan but also around the world. A global rush for iodine pills right after the incident testifies to this: the premise is that the greatest risk of radiation comes from iodine-131, which is concentrated by the body in one spot -- the thyroid. Consumption of iodine tablets was thus hoped to saturate the thyroid with so much non-radioactive iodine that it will no longer take up radioactive iodine-131. However, the World Health Organization (WHO) warned against consumption of iodine pills without consultation, following reports of people being admitted to poison centers after taking iodine tablets.

**(II) Tocotrienols as radioprophylactic agents. **At present, development of good radioprophylactic agents (agents that can prevent the diseases cause by radiation exposure) remains a challenge, mostly due to limited efficacies and the side effects involved. At present, the only substance approved for this purpose is called amifostine.
(i) However, amifostine has a very narrow window of effect: it must be administered within a certain time after radiation exposure to be effective.

(ii) Furthermore, common side effects include diarrhoea, nausea, vomiting, low blood pressure, severe allergy and loss of consciousness.

Interestingly, analogs of tocotrienols are currently under development as radioprophylactic agents. They are said to be ideal candidates because of their high efficacy, as well as their lack of toxicity.

γ-tocotrienol, particularly, is of interest, because it generally accumulates to a greater extent within endothelial cells compared to the other isomers. Studies have shown γ-tocotrienol to be effective in decreasing radiation-induced stress and improving post-irradiation survival120. Administered as a single dose (24 hours before exposure to ionizing radiations), it increased the survival rates of mice (from 0% to 88%, thirty days after exposure).

(i) Blood vessel damage is an important cause of collateral damage under these circumstances. For example, damage of the vessels in the gut can promote injury to the gut lining and subsequently cause radiation enteropathy (disease of the intestine). Tocotrienol treatment is seen to reduce the extent of this oxidative damage (by reducing the levels of oxidants like peroxynitrite) by up to 60%.

(ii) While blood cell count typically starts declining as early as four hours after irradiation, treated animals exhibited a recovery from this by the fourteenth day.

(iii) The treatment also reduced the damage within the gastrointestinal tract, and increased the extent of its recovery by up to 20%.

In terms of the mechanisms involved, tocotrienols can provide protection not only by virtue of their antioxidant properties, but also due to their ability to inhibit the HMG-CoA reductase enzyme. Statins, which are drugs that inhibit the same enzyme, have been shown to not only attenuate lung and intestinal injury after irradiation, but also reduce rectal toxicity after pelvic radiation therapies. Tocotrienols have an edge over statins, as they do not possess the toxic side effects commonly caused by statins.

# Chapter 7 Anti-Inflammatory Properties

## What is inflammation and why are anti-inflammatory properties desired?

Inflammation is a physical condition in which the body (or part of the body) experiences redness, warmth, swelling, and pain. The process forms part of the body's biological immune response to injury, infection or invasion by harmful substances (e.g., toxins, bacteria and foreign objects).

During inflammation, there is a cascade of release of signalling chemicals (also called inflammatory factors). These molecules (such as cytokines and prostaglandins) go on to subsequently recruit and stimulate the cells of the immune system, which then kill, neutralize, or digest the threat present (the cells of the immune system that carry out these roles are mostly phagocytes or lymphocytes).

As mentioned, although inflammation forms part of the body's biological immune response, it is unfortunately usually concomitant with redness, warmth, swelling, and pain of the tissues involved.

(i) Redness, heat and swelling are caused by an increased blood flow.

(ii) Pain may be caused by either an expansion of tissue (which mechanically pressures nerve cells) or the presence of pain-mediating cytokines.

(iii) Sometimes, inflammatory factors (such as prostaglandin PGE2 and cytokine IL-1) can also affect the brain's regulation of the body's temperature, causing fever. The part of the brain responsible for regulating this set-body-temperature is called the hypothalamus.

As a result, persistent, excessive or inappropriate inflammations are pathologically damaging. These may occur if the pertinent threat that triggered the inflammatory event fails to be eliminated, or if the body continues to respond even after the threat has been resolved.
Anti-inflammatory agents are thus commonly used for a number of purposes.

(i) To remedy pain. In fact, half of all medical pain-killers are anti-inflammatory agents.

(ii) To remedy symptoms such as redness, swelling, stiffness or loss of function.

(iii)To help circumvent fever.

Existing anti-inflammatory agents mostly work by blocking the COX-2 enzyme (cyclo-oxygenase 2) within the body. This is the enzyme which helps produce prostaglandins. However, there are certain drawbacks to this.

(i) Some anti-inflammatory agents end up causing stomach ulcers or bleeding, because they are not specific enough. Apart from blocking the COX-2 enzyme, these agents end up blocking the COX-1 enzyme as well. COX-1 enzymes help protect the lining of the stomach from stomach acid.

(ii) Other common side effects of existing anti-inflammatory agents include nausea, diarrhoea, rashes, headache, dizziness, nervousness, depression, drowsiness and insomnia. Occasionally, pre-existing conditions such as high blood pressure, heart failure, or kidney failure may be worsened.

Recently, scientists are beginning to discover that an alternate form of inflammation, called silent inflammation, plays a key role in most age-related pathological conditions, such as stroke, diabetes, atherosclerosis, rheumatoid arthritis, and even cancer.

(i) When a person ages, the body's weakened ability to remove reactive oxygen species (ROS) triggers an inflammation that occurs at low but continuous levels.

(ii) This chronic and persistent inflammation, also called silent inflammation, causes cell death and tissue destruction

(iii) This is said to be the link between ageing and age-related diseases, even cancer. The diagrams below further illustrate this.

Inflammation & Cancer (e.g., Gallbladder Carcinoma)

---

(i) During inflammation, cells of the immune system produce signalling chemicals that trigger and activate each other, stimulating growth and multiplication. This multiplication effect aids in the neutralization of the threat present.

(ii) Unfortunately, the same signals occasionally also trigger, no doubt inappropriately, the active division of other cells. This may contribute to cancer progression, as, ultimately, cancer cells are cells that divide and replicate uncontrollably.

Figure 31: Silent inflammation and its implication in various age-related conditions.
Numerous scientific studies have demonstrated the anti-inflammatory potential of tocotrienols. . These can be achieved without any of the side effects present within existing anti-inflammatory agents. Consumption of δ-tocotrienol for four weeks has been shown to visibly reduce inflammation within the liver of animal models placed on a high fat diet (inflammation can be triggered by the excessive accumulation and infiltration of lipids within the liver)122. Furthermore, since inflammation is also implicated in most age-related diseases, tocotrienols may thus help prevent or alleviate some of the symptoms involved121.

## Tocotrienols have anti-inflammatory properties.

Even though the forms and outcomes of inflammation tend to vary, a single pathway ultimately forms the core of all its complex processes. This is the NF-κB pathway, a pathway that is well established as the key regulator of all inflammatory processes. Tocotrienols have been found to suppress this pathway via diverse mechanisms. These are summarized in the diagram below.

(i) TRF consumption has been shown to lower the level of the NF-κB protein within the kidney (by up to 75%)47. The NF-κB protein is the main protein component within the pathway that facilitates inflammation.

(ii) Additionally, γ-tocotrienol (but not tocopherols) has been found to suppress the IKK (IκBα Kinase) enzyme, the enzyme needed for activation of NF-κB167. Reactive oxygen species are known to trigger the enzyme, and as powerful antioxidants, tocotrienols suppresses this effect122.

(iii) Tocotrienols (but not α-tocopherol) can also reduce the activity of the proteasome enzyme123. These enzymes degrade proteins that hold back the function of NF-κB (i.e., the I-κB protein). Below are results from studies that were conducted.

Isomers | Reduction of proteasomes activity

---|---

δ-tocotrienol | Up to 55% reduction

α-tocotrienol | Up to 36% reduction

γ-tocotrienol | Up to 32% reduction

α-tocopherol | No reduction

(iv) The HMG-CoA Reductase enzyme (or rather, its product, the compound mevalonate) is known to play a supportive role in the NF-κB pathway. By suppressing the enzyme, tocotrienols obstruct this support122.

(v) Tocotrienols also boost levels of the adrenocorticotropic hormone and the corticosterone hormone in animals during inflammation (the former stimulates the production of the latter, which in turn obstructs the function of NF-κB)123. Below are results from studies that were conducted.

 | Increase in the Adrenocorticotropic hormone | Increase in the

Corticosterone hormone

---|---|---

δ-tocotrienol | Up to 145% | Up to 41%

γ-tocotrienol | Up to 118% | Up to 31%

α-tocotrienol | Up to 81% | Up to 19%

Figure 32: Tocotrienols can help by inhibiting the inflammatory pathway at multiple levels.

Consequently, tocotrienols have been found to suppress the resulting inflammatory processes and outcomes of the pathway.
(i) They reduce the levels of both the COX-2 enzyme and the iNOS enzyme within body (their respective functions are shown in the diagram above)123, 124, 125. More importantly, the COX-1 enzyme is not affected (this enzyme helps protect the lining of the stomach from stomach acid).

(ii) Animals fed with tocotrienols also produce less inflammatory molecules, both under normal circumstances, and when exposed to inflammation-triggering factors. δ-tocotrienol has been found to achieve this exceptionally well123. Below are study results showing the reductions involved.

*Genetic expression is the process by which information from a gene is used in the synthesis of a functional product, in this case, inflammatory molecules.

Figure 33: Tocotrienols reduce the production of inflammatory molecules.

# Chapter 8 Bio-availability of Tocotrienols

Figure 34: The absorption and delivery of tocotrienols.

## What is bio-availability and why is it important?

The bio-availability of a substance or nutrient describes the degree to which it becomes available to the target tissues, after administration or consumption. This is predominantly determined by how well the nutrient is absorbed by the body, and subsequently, how well it is delivered throughout the body. Oral consumption of 100 mg of a particular nutrient does not necessarily mean that all 100 mg of it will reach the relevant organs to confer the intended health benefits. In fact, depending on the bio-availability of the nutrient, a significant portion of it may actually end up being excreted by the body.

In the past chapters, we have discussed the 6 main health benefits of tocotrienols at great length. Nonetheless, none of these benefits would ever be realized should the nutrient fail to reach its target tissues within the body. Affirming the bio-availability of tocotrienols is thus important, without which, this book would not be complete.
When orally consumed, all 8 isomers of Vitamin E are shown to be well absorbed by the human intestine. The majority of these end up in the liver. The liver then selectively releases the isomers into the bloodstream by selectively incorporating them into structures called very low density lipoproteins (VLDL). Only upon entering the bloodstream do these isomers become bio-available for delivery to various tissues and organs within the body2, 127. The rest are eventually metabolized and excreted from the body.

The selective enrichment of VLDLs with Vitamin E is therefore conventionally thought to be the bottleneck that determines the bio-availability of a particular isomer. Within this process, specific liver proteins bind to Vitamin E molecules, facilitating their incorporation into VLDLs. It is believed that the main protein that does this is the α-tocopherol transport protein (α-TTP).

## Resolving the controversies on the bio-availability of tocotrienols.

It was previously wrongly held that the only Vitamin E isomer that is capable of entering the blood circulation in abundant amounts is α-tocopherol128. The α-TTP route was assumed to be the sole mechanism by which all Vitamin E isomers gained access to the bloodstream; and the α-tocopherol transport protein was found to bind to α-tocopherol eight-and-a-half times much better than α-tocotrienol. This led to the notion that orally consumed tocotrienols are poorly enriched in the VLDLs by the liver, and are thus unable to reach the vital organs.

However, further studies later confirmed that while α-tocopherol transport protein do bind to tocotrienols to facilitate their secretion into the bloodstream, the bio-availability of tocotrienols does not depend solely on them. Even in studies using mice without α-TTP proteins, orally consumed tocotrienols do eventually reach their target tissues, and the benefits do subsequently manifest (these include the restoration and improvement of fertility)129. Another absorption mechanism therefore exists but is hitherto unstudied and undiscovered.

Notwithstanding this, it has always been known, through various studies and clinical trials, that consumption of tocotrienols does provide their intended benefits (as described numerous times throughout this book). There could be no better evidence. These positive results illustrate the body's ability to absorb
and deliver tocotrienols; and also prove that orally consumed tocotrienols can and will reach their different body destinations in beneficial quantities.

On that note, it is worth mentioning that the achievable blood plasma levels of tocotrienols are shown to be many times higher than that required for attainment of these health benefits.

(i) The plasma concentration of tocotrienols after oral consumption has been shown to go as high as 1-3 μmol/L (10-6M) in humans, and this, for instance, is twelve to thirty fold higher than that required to completely prevent stroke-associated neurodegeneration 130, 52, 131.

(ii) Furthermore, these concentrations are typically achievable within three to five hours after consumption; after which they drop with a half-life of two to five hours. This advocates the twice-a-day dosing regimen that is commonly recommended132.

Furthermore, organ samples that were removed from supplemented human subjects have been shown to contain significant amounts of tocotrienols. These include the skin, the liver, the heart (by up to sixty times the content during non-supplementation), the brain (up to thirty-seven times the content during non-supplementation) and the adipose tissues (up to eight times the content during non-supplementation) 52, 131. The same was also observed in animal studies.

(i) 1 - 12 nmol/g of tocotrienols could be found at various organs (this includes the lungs, the skeletal muscles, etc.).

(ii) In the skin, long term supplementation has been known to result in a marked increase in α-tocotrienol levels. This indicates a build-up over time, and in turn, the presence of an effective transport mechanism that is capable of absorbing and retaining the molecule within the skin129.

Among the isomers, there is an emerging consensus that α-tocotrienol has the highest oral bioavailability, followed by γ-tocotrienol and δ-tocotrienol. This is observed in both humans and animals. Incidentally, the clearance rate (the rate by which a substance is removed from the body, mostly by excretion) of tocotrienols is found to be in the reversed order: δ- > γ- > α-tocotrienol132, 133, 130, 17. These differences amongst the isomers are attributable to differences in
lipid solubility (α-tocotrienol has the highest lipid solubility, followed by γ and δ-tocotrienol)25, 134.

Studies also suggest that consumption of tocotrienols along with food may more than double (2.4 to 3.6-folds) the extent of absorption. Not only that, the onset of absorption also starts earlier. Intake of food is said to trigger bile secretion within the liver, and this is known to aid the process of digestion and absorption132.

## Contrasting the bio-availability of chemically-modified and natural Vitamin E.

Due to various reasons, certain manufacturers may choose to chemically modify the form of their Vitamin E, and one commonly seen modification is the conversion of Vitamin E into Vitamin E acetates. Nonetheless, it should be pointed out that chemically modifying the molecule may not only alter its activity, but also its bio-availability.

Figure 35: The structure of tocotrienols and tocopherols.

"Acetate-modification" replaces the free phenolic OH group in the vitamin E molecule with an acetate group, locking the molecule in an "inactive" state (with no/less antioxidant activity). This is said to increase the molecule's shelf life. Efficacy would nonetheless depend on subsequent conversion (by the hydrolysis process) back into the active form.
(i) As mentioned, topical skin studies have found the extent of this hydrolysis to be minimal in α-tocopherol acetate. No hydrolysis was observed after 5 hours of application, and the extent of hydrolysis remained < 1% even after 5 consecutive days of application1.

(ii) Moreover, conversion into acetates may also affect the oral bio-availability of tocotrienols. This can be inferred indirectly by comparing results from different studies. While these separate studies may not be entirely comparable, they indicate that subjects who consumed three to four times less the "natural-non-acetate form" of tocotrienols have two to three times higher blood tocotrienol levels 128, 130.

## The safe consumption of tocotrienols.

Another issue that is often closely associated with bio-availability is safe consumption. The pharmaceutical industry does not fall short of drugs claimed to be able to provide exceptional benefits, but these drugs quite frequently carry along toxic side effects.

In that respect, it is important to appreciate that tocotrienols, or tocotrienol rich fractions (TRF), are supplements, not drugs. As vitamins, they represent a class of nutrients that humans have been consuming safely for hundreds of years.

(i) To date, human clinical studies have yet to report any undesirable effects resulting from tocotrienol consumption, be it after up to 6 months of elevated intake29, or up to 400mg/day of intake52.

(ii) On top of that, exposure to up to 5 μg/ml of tocotrienols showed no toxicity in cell culture studies124, 130, and blood levels found in humans after consumption of tocotrienols are typically around 0.5 - 2.1 μg/ml132, 133.

(iii) A particular study even estimates the conservative dose of tocotrienols for human consumption to be up to 1,000mg/day135.

## Co-consumption of tocotrienols and tocopherols: a balanced approach in supplementation.

It is commonly claimed that α-tocopherol, because of its higher affinity to the α-tocopherol transport protein, has a higher bioavailability compared to tocotrienols132. Not only that, it is also often advocated that co-consumption of too high a tocopherol content compromises the bio-availability of tocotrienols (as both forms may end up competing for the same delivery mechanism) 129.

There are a number of points that need to be highlighted to set the record straight.

First of all, it has been proven that "co-consumption of both tocotrienols and tocopherols" does not ablate the absorption of tocotrienols. Human clinical studies have shown tocotrienols to be absorbed and accumulated by the vital organs, even when consumed together with α-tocopherol. This therefore unequivocally dispels the claim that tocopherols prevent the absorption of tocotrienols52.

Secondly, although it may appear that the generic α-tocopherol is the preferably absorbed isomer, it is important to bear in mind that certain organs (like the skin and the brain) are known to have preferable "affinity" or "liking" for tocotrienols, and these molecules are accumulated and retained over time129, 136.

(i) After long-term supplementation, the levels of α-tocotrienol in the skin of tocotrienol-fed animals were folds higher compared to the levels of α-tocopherol in the skin of tocopherol-fed animals.

(ii) In other animal models, preferential affinity for tocotrienols was also observed within the brain. TRF supplementation for nine days increased α-tocopherol content by 0.1-fold, but increased α-tocotrienol content by five-fold (TRF contains both tocotrienols and tocopherols). When fed to pregnant rats, this value was found to be twenty-fold for α-tocotrienol.

Thirdly, compared to tocopherols, tocotrienols are required in far lower amounts to confer their health benefits.
(i) As antioxidants, α-tocotrienol is shown to be up to forty to sixty times better than α-tocopherol in protecting liver cell membranes from lipid peroxidation4.

(ii) Tocotrienols are capable of protecting the brain at concentrations much less than one tenth that of α-tocopherol (nanomolar concentration levels compared to micromolar concentration levels).

Therefore, rather than totally eliminating the presence of α-tocopherol within one's supplementation regime, a balanced approach should be adopted instead: by consumption of the full spectrum of tocotrienols plus a moderate amount of α-tocopherol. As a rule of thumb, the consumption of a TRF supplement that has a ratio of tocotrienols-to-tocopherols in excess of a 2-to-1 ratio is consistent with such a balanced approach.

# What is next in Tocotrienols?

This book has attempted to consolidate the facts and findings to date on tocotrienols, based on the multitude of research data available. However, we cannot stress enough that the body of research on tocotrienols continues to grow daily, so much so that the greatest challenge faced in putting together the contents of this book is undoubtedly in keeping abreast of new and evolving developments.

Today, with the known health benefits of tocotrienols firmly established and affirmed based upon cell culture and animal studies, a lot of attention around the world is now progressively focused on human trials and clinical studies. The table below provides a snapshot on these current progressions to give a glimpse of what one can look forward to in the near future.

The clinical trials | The areas of interest

---|---

Neuroprotection

Trials on patients with mini-stroke (i.e. transient ischaemic attacks - subjects are statistically projected to be stricken with stroke within the next year). | To determine if stroke incidence, and the extent of damage, can be reduced by the treatment of tocotrienols3.

Diabetes

Trials on nerve degeneration in diabetes (diabetes often cause degeneration in the peripheral nervous system, leading to a loss of sensation in the limbs). | To determine if treatment with tocotrienols can slow or prevent the condition in diabetics (300 volunteers, 3 years)59.

Brain associated disorder

Trials on attention deficit hyperactivity disorder (ADHD). | To determine if the symptoms in ADHD children who are hyperactive, and often have trouble focusing, can be alleviated by the treatment of tocotrienols.

Cancer

Trials on advance metastatic breast cancer. | To determine if γ-tocotrienol can prevent tumor progression and death.

Trials on metastatic castration refractory prostate cancer. | To determine if γ- and δ tocotrienols can prevent or delay tumor progression.

Trials on colorectal cancer (pre-clinical studies). | To determine if γ-tocotrienol can prevent the condition.

Cardiovascular and lipid health

Trials on cholesterol homeostasis. | To determine if optimal levels of cholesterol can be maintained in the long run by tocotrienols.

Trials on non-alcoholic fatty liver disease. | To determine if liver damage and destruction can be prevented or delayed by the treatment of tocotrienols.

Suffice to say, the momentum of research has never been stronger, and the gears are set for further discoveries (and perhaps even more health benefits). On that note, we look forward to continuing this effort of ours with future editions of this book, to make sure that what we write remains relevant and accurate, and to keep our readers updated.

# References & Citations

  1. Tocotrienols, the Vitamin E of the 21st Century: its Potential Against Cancer and Other Chronic Diseases. Bharat B. Aggarwal, Chitra Sundaram, Seema Prasad, and Ramaswamy Kannappan. Biochem Pharmacol. 2010 December 1; 80(11): 1613–1631. doi:10.1016/j.bcp.2010.07.043.

  2. Tocotrienols in health and disease: the other half of the natural vitamin E family. Chandan K. Sen, Savita Khanna, and Sashwati Roy. Mol Aspects Med. 2007 ; 28(5-6): 692–728.

  3. A different side of vitamin E. Tan Shiow Chin The Star Online > Health, Sunday January 8, 2012.

  4. Free Radical Recycling and intramembrane mobility in the antioxidant properties of alpha-tocopherol and alpha tocotrienol. Elena Serbinova, Valerian Kagan, Derick Han, Lester Packer. Radical Biology and Medicine, 1991;10:263-275.

  5. Characterization of the potent neuroprotective properties of the natural vitamin E alpha-tocotrienol. Khanna S, Roy S, Parinandi NL, Maurer M, Sen CK. J Neurochem. 2006 September ; 98(5): 1474–1486.

  6. Gamma- and delta-tocotrienols inhibit skin melanin synthesis by suppressing constitutive and UV-induced tyrosinase activation. W. N. Yap, N. Zaiden, C. H. Xu, A. Chen, S. Ong, V. Teo and Y. L. Yap. Pigment Cell Melanoma Res. 23; 688–692.

  7. Palm-tocotrienol rich fraction (TRF) is a more effective inhibitor of LDL oxidation and endothelial cell lipid peroxidation than alpha-tocopherol in vitro. Mutalib, M.S.A., Khaza'ai, H. & Wahle, K.W.J. (2003). Food Res. Int. 36:405-413.

  8. Studies of LDL oxidation following alpha-, gamma-, or delta-tocotrienyl acetate supplementation of hypercholesterolemic humans. O'Byrne, D., Grundy, S., Packer, L., Devaraj, S., Baldenius, K., Hoppe, P. P., Kraemer, K., Jialal, I. & Traber, M. G. (2000). Free Radic. Biol. Med. 29:834-845.

  9. Protective effect of alpha-tocotrienol against free radical-induced impairment of erythrocyte deformability. Begum, A. N. & Terao, J. (2002). Biosci. Biotechnol. Biochem. 66:398-403.

  10. Structural and dynamic membrane properties of alpha-tocopherol and alpha-tocotrienol: implication to the molecular mechanism of their antioxidant potency. Suzuki, Y. J., Tsuchiya, M., Wassall, S. R., Choo, Y. M., Govil, G., Kagan, V. E. & Packer, L. (1993). Biochemistry 32:10692-10699.

  11. Isolation and identification of novel tocotrienols from rice bran with hypocholesterolemic, antioxidant, and antitumor properties. Qureshi, A. A., Mo, H., Packer, L. & Peterson, D. M. (2000). J. Agric. Food Chem. 2000, 48, 3130-3140.

  12. Comparative study on the action of tocopherols and tocotrienols as antioxidant: chemical and physical effects. Yasukazu Yoshida, Etsuo Niki, Noriko Noguchi. Chemistry and Physics of Lipids 123 (2003) 63-75.

  13. The Atlas of Heart Disease and Stroke, Nonserial Publication. Mackay,J., Mensah,G. World Health Organization ISBN-13 9789241562768.

  14. Australian Institute of Health and Welfare. Home>Subject>Risk factors, diseases & death>Health priority areas. http://www.aihw.gov.au/cardiovascular-health-priority-area/.

  15. Is augmentation index a good measure of vascular stiffness in the elderly? Francesco Fantin, Adriana Mattocks, Christopher J Bulpitt, Winston Banya and Chakravarthi Rajkuma. Age Ageing (2007) 36 (1): 43-48. doi: 10.1093/ageing/afl115.

  16. The effects of a tocotrienol-rich fraction on experimentally induced atherosclerosis in the aorta of rabbits. Nafeeza MI, Norzana AG, Jalaluddin HL, Gapor MT. Malays J Pathol. 2001 June 23(1):17-25.

  17. Arterial Compliance and Vitamin E Blood Levels with a Self-Emulsifying Preparation of Tocotrienol Rich Vitamin E. Aida Hanum Ghulam Rasool, Abd. Rashid Abd. Rahman, Kah Hay Yuen, and Abdul Rahim Wong. Arch Pharm Res Vol 31, No 9, 1212-1217, 2008 DOI 10.1007/s12272-001-1291-5.

  18. Nitric oxide synthase activity in blood vessels of spontaneously hypertensive rats: antioxidant protection by gamma-tocotrienol. Newaz, M. A., Yousefipour, Z., Nawal, N. & Adeeb, N. (2003).J. Physiol. Pharmacol. 54:319-327.

  19. Cholesterol intake and plasma cholesterol: an update. Namara DJ. J Am Coll Nutr 1997 Dec; 16(6):530-4.

  20. The structure of an inhibitor of cholesterol biosynthesis isolated from barley. Qureshi AA, Burger WC, Peterson DM, Elson CE. J Biol Chem. 1986 August 15;261(23):10544-50.

  21. Dietary tocotrienols reduce concentrations of plasma cholesterol, apolipoprotein B, thromboxane B2, and platelet factor 4 in pigs with inherited hyperlipidemias. Qureshi AA, Qureshi N, Hasler-Rapacz JO, Weber FE, Chaudhary V, Crenshaw TD, Gapor A, Ong AS, Chong YH, Peterson D. American Journal of Clinical Nutrition. 1991a;53(4 Suppl):1042S–1046S.

  22. Novel tocotrienols of rice bran suppresses cholesterogenesis in hereditary hypercholesterolemic swine. Qureshi AA, Peterson DM, Hasler-Rapacz JO, Rapacz J. J Nutr. 2001 February; 131(2):223-30.

  23. Tocotrienols confer resistance to ischemia in hypercholesterolemic hearts: insight with genomics.  Das S,  Mukherjee S,  Lekli I,  Gurusamy N,  Bardhan J,  Raychoudhury U,  Chakravarty R,  Banerji S,  Knowlton AA,  Das DK. Mol Cell Biochem (2012) 360:35–45. DOI 10.1007/s11010-011-1041-9.

  24. Palm tocotrienols protect ApoE +/- mice from diet-induced atheroma formation. Black TM, Wang P, Maeda N, Coleman RA. J Nutr. 2000 October 130(10):2420-6.

  25. Effect of either gamma-tocotrienol or a tocotrienol mixture on the plasma lipid profile in hamsters. Raederstorff, D., Elste, V., Aebischer, C. & Weber, P. (2002). Ann. Nutr. Metab. 46:17-23.

  26. Suppression of 7, 12-dimethylbenz[α]anthracene-induced carcinogenesis and hypercholesterolaemia in rats by tocotrienol-rich fraction isolated from rice bran oil. Iqbal, J., Minhajuddin, M. & Beg, Z. H. (2003). Eur. J. Cancer Prev. 12:447-453.

  27. Tocotrienol rich fraction supplementation improved lipid profile and oxidative status in healthy older adults: A randomized controlled study. Siok-Fong Chin, Johari Ibahim, Suzana Makpol, Noor Aini Abdul Hamid, Azian Abdul Latiff, Zaiton Zakaria,Musalmah Mazlan, Yasmin Anum Mohd Yusof, Aminuddin Abdul Karim and Wan Zurinah Wan Ngah. Nutrition & Metabolism 2011, 8:42. http://www.nutritionandmetabolism.com/content/8/1/42.

  28. Effect of a palm-oil-vitamin E concentrate on the serum and lipoprotein lipids in humans. Tan DT, Khor HT, Low WH, Ali A, Gapor A. American Journal of Clinical Nutrition 1991 April 53(4 Suppl):1027S-1030S.

  29. Synergistic effect of tocotrienol-rich fraction (TRF (25)) of rice bran and lovastatin on lipid parameters in hypercholesterolemic humans. Qureshi AA, Sami SA, Salser WA, Khan FA. J Nutr Biochem. . 2001 June 12(6):318-329.

  30. Lowering of serum cholesterol in hypercholesterolemic humans by tocotrienols (palmvitee). Qureshi AA, Qureshi N, Wright JJ, Shen Z, Kramer G, Gapor A, Chong YH, DeWitt G, Ong A, Peterson DM. American Journal of Clinical Nutrition 1991 April 53(4 Suppl):1021S-1026S.

  31. Dose-dependent suppression of serum cholesterol by tocotrienol-rich fraction (TRF25) of rice bran in hypercholesterolemic humans. Qureshi AA, Sami SA, Salser WA, Khan FA. Atherosclerosis. 2002 March ;161(1):199-207.

  32. Effect of citrus flavonoids and tocotrienols on serum cholesterol levels in hypercholesterolemic subjects. Roza JM, Xian-Liu Z, Guthrie N. Altern Ther Health Med. 2007 November 13(6):44-8.

  33. Tocotrienols regulate cholesterol production in mammalian cells by posttranscriptional suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Parker RA, Pearce BC, Clark RW, Gordon DA, Wright JJK. J Biol Chem 1993, 268:11230-11238.

  34. Hypocholesterolemic activity of synthetic and natural tocotrienols. Pearce BC, Parker RA, Deason ME, Qureshi AA, Wright JJ. J Med Chem. 1992 October t 2; 35(20):3595-606.

  35. Tocotrienol is the most effective vitamin E for reducing endothelial expression of adhesion molecules and adhesion to monocytes. Theriault, A., Chao, J. T., Gapor, A., Chao, J. T. & Gapor, A. Atherosclerosis 160 (2002):21-30.

  36. Inhibition of THP-1 cell adhesion to endothelial cells by alpha-tocopherol and alpha-tocotrienol is dependent on intracellular concentration of the antioxidants. Noguchi, N., Hanyu, R., Nonaka, A., Okimoto, Y. & Kodama, T. (2003). Free Radic. Biol. Med. 34:1614-1620.

  37. Inhibitory effect of delta-tocotrienol, a HMG-CoA reductase inhibitor, on monocyte-endothelial cell adhesion. Chao, J. T., Gapor, A. & Theriault, A. (2002). J. Nutr. Sci. Vitaminol. 48:332-337.

  38. Tocotrienols-induced inhibition of platelet thrombus formation and platelet aggregation in stenosed canine coronary arteries. Asaf A Qureshi, Charles W Karpen, Nilofer Qureshi, Christopher J Papasian, David C Morrison and John D Folts. Lipids in Health and Disease 2011, 10:58. http://www.lipidworld.com/content/10/1/58.

  39. Caveolin and Proteasome in, Tocotrienol Mediated Myocardial Protection. Manika Das, Samarjit Das, Ping Wang, Saul R. Powell and Dipak, K. Das. Cell Physiol Biochem 2008;22:287-294.

  40. Diabetes-Induced Accelerated Atherosclerosis in Swine. Ross G. Gerrity, Rama Natarajan, Jerry L. Nadler, and Troy Kimsey. DIABETES, VOL. 50, JULY 2001.

  41. Tocotrienols-rich diet decreases advanced glycosylation end-products in non-diabetic rats and improves glycemic control in streptozotocin-induced diabetic rats. W.M. Wan Nazaimoon, B.A. Khalid. Malays J Pathol, 24 (2002), pp. 77-82.

  42. The effects of palm oil tocotrienol-rich fraction supplementation on biochemical parameters, oxidative stress and the vascular wall of streptozotocin-induced diabetic rats. S.B. Budin, F. Othman, S.R. Louis, M.A. Bakar, S. Das, J. Mohamed. Clinics (Sao Paulo), 64 (2009), pp. 235-244.

  43. Vitamin E tocotrienols improve insulin sensitivity through activating peroxisome proliferator-activated receptors. F. Fang, Z. Kang, C. Wong. Mol Nutr Food Res, 54 (2010), pp. 345-352.

  44. A rice bran oil diet increases LDL-receptor and HMG-CoA reductase mRNA expressions and insulin sensitivity in rats with streptozotocin/nicotinamide-induced type 2 diabetes. Chen CW, Cheng HH. J Nutr. 2006;136(6):1472–6.

  45. Rice bran extract prevents the elevation of plasma peroxylipid in KKAy diabetic mice. Y. Kanaya, T. Doi, H. Sasaki, A. Fujita, S. Matsuno, K. Okamoto et al. Diabetes Res Clin Pract, 66 (Suppl. 1) (2004), pp. S157-S160.

  46. Risk of developing dementia in people with diabetes and mild cognitive impairment.  Latha Velayudhan. The British Journal of Psychiatry (2010) 196: 36-40 doi:10.1192/bjp.bp.109.067942.

  47. Attenuation of diabetic nephropathy by tocotrienol: involvement of NFκB signalling pathway. A. Kuhad, K. Chopra. Life Sci, 84 (2009), pp. 296-301.

  48. Suppression of NF-kappabeta signaling pathway by tocotrienol can prevent diabetes associated cognitive deficits. A. Kuhad, M. Bishnoi, V. Tiwari, K. Chopra. Pharmacol Biochem Behav, 92 (2009), pp. 251-259.

  49. A Regenerative Antioxidant Protocol of Vitamin E and α-Lipoic Acid Ameliorates Cardiovascular and Metabolic Changes in Fructose-Fed Rats. Jatin Patel, Nur AzimMatnor, Abishek Iyer, and Lindsay Brown, Hindawi Publishing Corporation. Evidence-Based Complementary and Alternative Medicine Volume 2011, Article ID 120801, 8 pages doi:10.1155/2011/120801.

  50. Protective Effect of Vit E Analogs against Carbon Tetrachloride-Induced Fatty Liver in Rats. Rieko Yachi, Osamu Igarashi and Chikako Kiyose. J. Clin. Biochem. Nutr., 47, 148–154, September 2010.

  51. The E solution to liver disease. Lim Wey Wen. starhealth@thestar.com.my Wednesday September 29, 2010.

  52. Oral Tocotrienols Are Transported to Human Tissues and Delay the Progression of the Model for End-Stage Liver Disease Score in Patients. Patel V, Rink C, Gordillo GM, Khanna S, Gnyawali U, Roy S, Shneker B, Ganesh K, Phillips G, More JL, Sarkar A, Kirkpatrick R, Elkhammas EA, Klatte E, Miller M, Firstenberg MS, Chiocca EA, Nesaretnam K, Sen CK. J Nutr. 2012 Feb 1.

  53. Tocotrienol: the natural vitamin E to defend the nervous system? Sen CK, Khanna S, Roy S. Ann N Y Acad Sci. 2004 December 1031:127-42 (2004).

  54. Nanomolar vitamin E α-tocotrienol inhibits glutamate-induced activation of phospholipase A2 and causes neuroprotection. Savita Khanna, Narasimham L. Parinandi, Sainath R. Kotha, Sashwati Roy, Cameron Rink, Douglas Bibus, and Chandan K. Sen. J Neurochem. 2010 March ; 112(5): 1249–1260. doi:10.1111/j.1471-4159.2009.06550.x.

  55. Molecular basis of vitamin E action. Tocotrienol potently inhibits glutamate-induced pp60(c-Src) kinase activation and death of HT4 neuronal cells. Sen, C. K., Khanna, S., Roy, S. & Packer, L. (2000). J. Biol. Chem. 275:13049-13055.

  56. Molecular basis of vitamin E action: tocotrienol modulates 12-lipoxygenase, a key mediator of glutamate-induced neurodegeneration. Khanna, S., Roy, S., Ryu, H., Bahadduri, P., Swaan, P. W., Ratan, R. R. & Sen, C. K. (2003). J. Biol. Chem. 278:43508-43515.

  57. Neuroprotective properties of the natural vitamin E alpha-tocotrienol. Khanna S, Roy S, Slivka A, Craft TK, Chaki S, Rink C, Notestine MA, DeVries AC, Parinandi NL, Sen CK. Stroke. 2005b;36(10):2258–64.

  58. Tocotrienol vitamin E protects against preclinical canine ischemic stroke by inducing arteriogenesis. Cameron Rink, Greg Christoforidis, Savita Khanna, Laura Peterson, Yojan Patel, Suchin Khanna, Amir Abduljalil, Okan Irfanoglu, Raghu Machiraju, Valerie K Bergdall and Chandan K Sen. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 2218–2230.

  59. Protection for brain cells. New Straits Times, 11 March 2012.

  60. Palm Oil–Derived Natural Vitamin E α-Tocotrienol in Brain Health and Disease. Chandan K. Sen, Cameron Rink, Savita Khanna. J Am Coll Nutr. 2010 June; 29(3 Suppl): 314S–323S.

  61. Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke. Paul R, Zhang ZG, Eliceiri BP, Jiang Q, Boccia AD, Zhang RL, Chopp M, Cheresh DA. Nat Med. 2001;7(2):222–7.

  62. Role of oxygen free radicals in cancer development. Dreher D, Junod AF. Eur J Cancer, 1996 Jan; 32A(1):30-8.

  63. Oxidative Damage and Cancer. Terry D. Oberley. Am J Pathol. 2002 February; 160(2): 403–408.

  64. Induction of cytotoxicity in human lung adenocarcinoma cells by 6-O-carboxypropyl-alpha-tocotrienol, a redox-silent derivative of alpha-tocotrienol. Yano Y, Satoh H, Fukumoto K, Kumadaki I, Ichikawa T, Yamada K, Hagiwara K, Yano T. Int J Cancer. 2005;115(5):839–46.

  65. Suppression of mevalonate pathway activities by dietary isoprenoids: protective roles in cancer and cardiovascular disease. Elson CE. J Nutr. 1995 June 125(6 Suppl):1666S-1672S.

  66. DNA chip analysis of comprehensive food function: Inhibition of angiogenesis and telomerase activity with unsaturated vitamin E, tocotrienol. Nakagawa K, Eitsuka T, Inokuchi H, Miyazawa T. Biofactors. 2004;21(1–4):5–10.

  67. Isoprenoids suppress the growth of murine B16 melanomas in vitro and in vivo. He L, Mo H, Hadisusilo S, Qureshi AA, Elson CE. Journal of Nutrition. 1997;127(5):668–74.

  68. Gamma tocotrienol inhibits pancreatic tumors and sensitizes them to gemcitabine treatment by modulating the inflammatory microenvironment. Ajaikumar B. Kunnumakkara, Bokyung Sung, Jayaraj Ravindran, Parmeswaran, Diagaradjane, Amit Deorukhkar, Sanjit Dey, Cemile Koca, Vivek R. Yadav, ZhiminTong, Juri G. Gelovani, Sushovan Guha, Sunil Krishnan, and Bharat B. Aggarwal. Cancer Res. 2010 Nov 1;70(21):8695-705. Epub 2010 Sep 23.

  69. Gamma-tocotrienol inhibits nuclear factor-kappaB signaling pathway through inhibition of receptor-interacting protein and TAK1 leading to suppression of antiapoptotic gene products and potentiation of apoptosis. Ahn KS, Sethi G, Krishnan K, Aggarwal BB. J Biol Chem. 2007;282(1):809–20.

  70. Role of caspase-8 activation in mediating vitamin E-induced apoptosis in murine mammary cancer cells. Shah S, Gapor A, Sylvester PW. Nutr Cancer. 2003;45(2):236–46.

  71. Telomerase therapeutics for cancer: challenges and new directions. Shay JW, Wright WE. Nat Rev Drug Discov. 2006;5(7):577–84.

  72. Anti-angiogenic activity of tocotrienol. Inokuchi H, Hirokane H, Tsuzuki T, Nakagawa K, Igarashi M, Miyazawa T. Biosci Biotechnol Biochem. 2003;67(7):1623–7.

  73. Inhibitory effect of tocotrienol on eukaryotic DNA polymerase ʎ and angiogenesis. Yoshiyuki Mizushina, Kiyotaka Nakagawa, Akira Shibata, Yasutoshi Awata. Biochemical and Biophysical Research Communications 339 (2006) 949–955.

  74. Anti-angiogenic Potential of Tocotrienol in vitro. T. Miyazawa, H. Inokuchi, H. Hirokane, T. Tsuzuki, K. Nakagawa, and M. Igarashi. Biochemistry (Moscow), Vol. 69, No. 1, 2004, pp. 67-69. Translated from Biokhimiya, Vol. 69, No. 1, 2004, pp. 85-88.

  75. Inhibitory effects of gamma-tocotrienol on invasion and metastasis of human gastric adenocarcinoma SGC-7901 cells. H.K. Liu, Q. Wang, Y. Li, W.G. Sun, J.R. Liu, Y.M. Yang et al. J Nutr Biochem, 21 (2010), pp. 206-213.

  76. Evidence of γ-Tocotrienol as an Apoptosis-Inducing, Invasion-Suppressing, and Chemotherapy Drug-Sensitizing Agent in Human Melanoma Cells. Piek Ngoh Chang, Wei Ney Yap, Davy Tak Wing Lee, M. T. Ling, Y. C. Wong & Yee Leng Yap (2009). Nutrition and Cancer, 61:3, 357-366.

  77. d-Delta-tocotrienol-mediated suppression of the proliferation of human PANC-1, MIA PaCa-2, and BxPC-3 pancreatic carcinoma cells. Hussein D, Mo H. Pancreas 2009;38:e124–e136. [PubMed: 19346993].

  78. Vitamin E Tocotrienol Shows Increased Cancer Cell Apoptosis Without Toxicity In Phase 1 Pancreatic Cancer Trial. Medical News Today, 31 May 2011. http://www.medicalnewstoday.com/releases/226908.php.

  79.  World Cancer Report.  International Agency for Research on Cancer. 2008.  http://globocan.iarc.fr/factsheets/populations/factsheet.asp?uno=900. Retrieved 2011-02-26.

  80. A comparison of tocopherol and tocotrienol for the chemoprevention of chemically induced rat mammary tumors. Gould MN, Haag JD, Kennan WS, Tanner MA, Elson CE. American Journal of Clinical Nutrition. 1991;53(4 Suppl):1068S–1070S.

  81. Tocotrienol-rich fraction from palm oil affects gene expression in tumors resulting from MCF-7 cell inoculation in athymic mice. Nesaretnam K, Ambra R, Selvaduray KR, Radhakrishnan A, Reimann K, Razak G, Virgili F. Lipids. 2004;39(5):459–67.

  82. Effect of tocotrienols on the growth of a human breast cancer cell line in culture.  Nesaretnam K,  Guthrie N,  Chambers AF,  Carroll KK. Lipids. 1995 Dec;30(12):1139-43.

  83. Tocotrienols inhibit the growth of human breast cancer cells irrespective of estrogen receptor status.  Nesaretnam K,  Stephen R,  Dils R,  Darbre P. Lipids. 1998 May;33(5):461-9.

  84. Inhibition of proliferation of estrogen receptor-negative MDA-MB-435 and -positive MCF-7 human breast cancer cells by palm oil tocotrienols and tamoxifen, alone and in combination. Guthrie N, Gapor A, Chambers AF, Carroll KK. Journal of Nutrition. 1997;127(3):544S–548S.

  85. Antiproliferative and apoptotic effects of tocopherols and tocotrienols on preneoplastic and neoplastic mouse mammary epithelial cells. McIntyre, B. S., Briski, K. P., Gapor, A. & Sylvester, P. W. (2000). Proc. Soc. Exp. Biol. Med. 224:292-301 / Lipids. 2000b;35(2):171–80.

  86. Disruption of mitochondria during tocotrienol-induced apoptosis in MDA-MB-231 human breast cancer cells. Takahashi, K. & Loo, G. (2004). Biochem. Pharmacol. 67:315-324.

  87. Intracellular mechanisms mediating tocotrienol-induced apoptosis in neoplastic mammary epithelial cells. Sylvester PW, Shah S. Asia Pac J Clin Nutr. 2005;14(4):366-73.

  88. Combined Treatment of γ-Tocotrienol with Statins Induce Mammary Tumor Cell Cycle Arrest in G1. Vikram B. Wali, Sunitha V. Bachawal and Paul W. Sylvester. Exp Biol Med June 2009 vol. 234 no. 6 639-650. doi: 10.3181/0810-RM-300.

  89. Enhanced antiproliferative and apoptotic response to combined treatment of g-tocotrienol with erlotinib or gefitinib in mammary tumor cells. Sunitha V Bachawal, Vikram B Wali, Paul W Sylvester. BMC Cancer 2010, 10:84. http://www.biomedcentral.com/1471-2407/10/84.

  90. Synergistic anticancer effects of combined γ-tocotrienol and celecoxib treatment are associated with suppression in Akt and NFκB signalling. Amit B. Shirode and Paul W. Sylvester. Biomed Pharmacother. 2010 May; 64(5): 327–332. doi:10.1016/j.biopha.2009.09.018.

  91. Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells. Shah S, Sylvester PW. Exp Biol Med (Maywood) 2004;229(8):745–55.

  92. A novel mechanism of natural vitamin E tocotrienol activity: involvement of ERβ signal transduction. Raffaella Comitato, Kalanithi Nesaretnam, Guido Leoni, Roberto Ambra, Raffaella Canali, Alessandro Bolli, Maria Marino, and Fabio Virgili. Am J Physiol Endocrinol Metab, 2009 Jun 2.

  93. γ-Tocotrienol Inhibits Neoplastic Mammary Epithelial Cell Proliferation by Decreasing Akt and Nuclear Factor K B Activity. Sumit J. Shah and Paul W. Sylvester. Experimental Biology and Medicine 2005, 230:235-241.

  94. Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells. Srivastava JK, Gupta S. Biochem Biophys Res Commun. 2006;346(2):447–53.

  95. In vivo evidence of gamma-tocotrienol as a chemosensitizer in the treatment of hormone-refractory prostate cancer. W.N. Yap, N. Zaiden, S.Y. Luk, D.T.W. Lee, M.T. Ling, Y.C. Wong, Y.L. Yap. Pharmacology. 2010;85(4):248-58. Epub 2010 Apr 7.

  96. http://www.medicinenet.com/liver_cancer.

  97. Tumor suppressive effects of tocotrienol in vivo and in vitro. Wada S, Satomi Y, Murakoshi M, Noguchi N, Yoshikawa T, Nishino H. Cancer Lett. 2005;229(2):181–91.

  98. Effect of tocotrienols on hepatocarcinogenesis induced by 2-acetylaminofluorene in rats. Ngah WZ, Jarien Z, San MM, Marzuki A, Top GM, Shamaan NA, Kadir KA. American Journal of Clinical Nutrition. 1991;53(4 Suppl):1076S–1081S.

  99. Long-term administration of tocotrienols and tumor-marker enzyme activities during hepatocarcinogenesis in rats. Rahmat A, Ngah WZ, Shamaan NA, Gapor A, Abdul Kadir K. Nutrition. 1993;9(3):229–32.

  100. Induction of apoptosis by tocotrienol in rat hepatoma dRLh-84 cells. Sakai M, Okabe M, Yamasaki M, Tachibana H, Yamada K. Anticancer Res. 2004;24(3a):1683–8.

  101. Effects of tocotrienols on cell viability and apoptosis in normal murine liver cells (BNL CL.2) and liver cancer cells (BNL 1ME A.7R.1), in vitro. Har CH, Keong CK. Asia Pac J Clin Nutr. 2005;14(4):374–80.

  102. Apoptosis induction by gamma-tocotrienol in human hepatoma Hep3B cells. Sakai M, Okabe M, Tachibana H, Yamada K. Journal of Nutritional Biochemistry 17 (2006) 672– 676.

  103. Tocotrienol-rich fraction of palm oil activates p53, modulates Bax/Bcl2 ratio and induces apoptosis independent of cell cycle association. Agarwal MK, Agarwal ML, Athar M, Gupta S. Cell Cycle. 2004;3(2):205–11.

  104. Down-regulation of telomerase activity in DLD-1 human colorectal adenocarcinoma cells by tocotrienol. Takahiro Eitsuka, Kiyotaka Nakagawa, Teruo Miyazawa. Biochemical and Biophysical Research Communications, Volume 348, Issue 1, 15 September 2006, Pages 170-175, ISSN 0006-291X, 10.1016/j.bbrc.2006.07.029.

  105. http://www.medicinenet.com/skin_cancer/article.htm.

  106. Effect of Delta-Tocotrienol on Melanin Content and Enzymes for Melanin Synthesis in Mouse Melanoma Cells. Akihiro Michihara, Saki Ogawa, Yohei Kamizaki, and Kenji Akasaki. Biol. Pharm. Bull. 33(9) 1471—1476 (2010).

  107. Allergic contact dermatitis from kojic acid. Serra-Baldrich, E., Tribô, M. J. and Camarasa, J. G. (1998). Contact Dermatitis 39: 86–87. doi: 10.1111/j.1600-0536.1998.tb05843.x.

  108. Mutation Research, Genetic Toxicology and Environmental Mutagenesis. June 2005, pages 133-1450 and Toxicological Sciences, September 2004, pages 43-49).

  109. Diet-derived and topically applied tocotrienols accumulate in skin and protect the tissue against ultraviolet light-induced oxidative stress. Maret G Traber, Maurizio Podda, Christine Weber, Jens Thiele, Michalis Rallis, Lester Packer. Asia Pacific J Clin Nutr (1997) 6(1): 63-67.

  110. Selective uptake of dietary tocotrienols into rat skin. Ikeda S, Niwa T, Yamashita K. J Nutr Sci Vitaminol (Tokyo). 2000 Jun;46(3):141-3.

  111. Dietary Tocotrienol Reduces UVB-Induced Skin Damages and Sesamin Enhances Tocotrienol Effects in Hairless Mice. Yasushi Yamada, Mariko Obayashi, Tomoko Ishikawa, Yoshinobu Kiso, Yoshiko Ono, Kanae Yamashita. J Nutr Sci Vitaminol, 54, 117-123, 2008.

  112. Palm Tocotrienols Rich Fraction in Topical Application. Presented by En. Zafaarizal Aldrin and Rosnah Ismail (MPOB), in the Palm International Nutra-Cosmeceutical Conference 2009.

  113. Vitamin E-ffects. Tan Shiow Chin. starhealth@thestar.com.my Sunday March 25, 2012. Cosmetic benefit of the supplement health food combined astaxanthin and tocotrienol on human skin. Yamashita, E (2002). Food Style 21 6(6):112-117.

  114. Tocotrienol-Rich Fraction Prevents Cell Cycle Arrest and Elongates Telomere Length in Senescent Human Diploid Fibroblasts. SuzanaMakpol, LinaWati Durani. Journal of Biomedicine and Biotechnology, Volume 2011, Article ID 506171, 11 pages doi:10.1155/2011/506171.

  115. Oxidative stress and aging: beyond correlation. Golden, T. R., Hinerfeld, D. A. & Melov, S. (2002). Aging Cell 1:117-123.

  116. Extension of life-span with superoxide dismutase/catalase mimetics. Melov S, Ravenscroft J, Malik S, Gill MS, Walker DW, Clayton PE, Wallace DC, Malfroy B, Doctrow SR, Lithgow GJ (2000). Science 289, 1567–1569.

  117. Effects of tocotrienols on life span and protein carbonylation in Caenorhabditis elegans. Adachi, H. & Ishii, N. (2000). J. Gerontol. A Biol. Sci. Med. Sci. 55:B280-B285.

  118. Gamma-Tocotrienol prevents oxidative stress-induced telomere shortening in human fibroblasts derived from different aged individuals. Suzana Makpol, Azrina Zainal Abidin, Khalilah Sairin, Musalmah Mazlan, Gapor Md. Top and Wan Zurinah Wan Ngah. Oxidative Medicine and Cellular Longevity, 3:1 35-43; January/February 2010; © 2010 Landes Bioscience.

  119. Disposition and metabolism of topically administered α-tocopherol acetate: A common ingredient of commercially available sunscreens and cosmetic. Alberts, DS. Nutr Cancer. 26, 193-201 (1996).

  120. γ-Tocotrienol Ameliorates Intestinal Radiation Injury and Reduces Vascular Oxidative Stress after Total-Body Irradiation by an HMG-CoA Reductase-Dependent Mechanism. Maaike Berbéea, Qiang Fua, Marjan Boermaa, Junru Wanga, K. Sree Kumarb, and Martin Hauer-Jensena. Radiat Res. 2009 May ; 171(5): 596–605. doi:10.1667/RR1632.1.

  121. Tocotrienols: Twenty Years of Dazzling Cardiovascular and Cancer Research. Byron J. Richards, CCN. NewsWithViews.com, February 15, 2011.

  122. δ-Tocotrienol and quercetin reduce serum levels of nitric oxide and lipid parameters in female chickens. Asaf A Qureshi, Julia C Reis, Nilofer Qureshi, Christopher J Papasian, David C Morrison, Daniel M Schaefer. Lipids in Health and Disease 2011, 10:39, http://www.lipidworld.com/content/10/1/39.

  123. Tocotrienols inhibit lipopolysaccharide-induced pro-inflammatory cytokines in macrophages of female mice. Asaf A Qureshi, Julia C Reis, Christopher J Papasian, David C Morrison, Nilofer Qureshi. Lipids in Health and Disease 2010, 9:143.

  124. Tocotrienol-rich fraction of palm oil exhibits anti-inflammatory property by suppressing the expression of inflammatory mediators in human monocytic cells. S.J. Wu, P.L. Liu, L.T. Ng. Mol Nutr Food Res, 52 (2008), pp. 921-929.

  125. Long-chain carboxychromanols, metabolites of vitamin E, are potent inhibitors of cyclooxygenases. Q. Jiang, X. Yin, M.A. Lill, M.L. Danielson, H. Freiser, J. Huang. Proc Natl Acad Sci USA, 105 (2008), pp. 20464-20469.

  126. Tocotrienol offers better protection than tocopherol from free radical-induced damage of rat bone. N.S. Ahmad, B.A. Khalid, D.A. Luke, S. Ima Nirwana. Clin Exp Pharmacol Physiol, 32 (2005), pp. 761-770.

  127. Molecular Mechanisms of vitamin E transport. Maret G. Traber, Hiroyuki Arai. Annu. Rev. Nutr. 1999. 19:343–55.

  128. Affinity for alpha-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. Hosomi A, Arita M, Sato Y, Kiyose C, Ueda T, Igarashi O, Arai H, Inoue K. FEBS Letters. 1997;409(1):105–8.

  129. Delivery of orally supplemented alpha-tocotrienol to vital organs of rats and tocopherol-transport protein deficient mice. Khanna S, Patel V, Rink C, Roy S, Sen CK. Free Radic Biol Med. 2005a; 39(10):1310–9.

  130. Postprandial levels of the natural vitamin e tocotrienol in human circulation. Pramod Khosla, Viren Patel, Janice M.Whinter, Savita Khanna, Marina Rakhkovskaya, Sashwati Roy, Chandra Sen. ANTIOXIDANTS & REDOX SIGNALING , Volume 8, Numbers 5 & 6, 2006.

  131. Supplementation with 3 compositionally different tocotrienol supplements does not improve cardiovascular disease risk factors in men and women with hypercholesterolemia. Mustad, V. A., Smith, C. A., Ruey, P. P., Edens, N. K. & DeMichele, S. J. (2002). Am. J. Clin. Nutr. 76:1237-1243.

  132. Pharmacokinetics and bioavailability of α-, γ- and δ-tocotrienols under different food status. S. P. Yap, K. H. Yuen and J. W. Wong. J Pharm Pharmacol. 2001;53:67–71. doi: 10.1211/0022357011775208.

  133. Effect of Mixed-Tocotrienols in Hypercholesterolemic Subjects. Kah Hay Yuen, Jia Woei Wong, Ai Beoy Lim, Bee Hong Ng, Wai Peng Choy. Functional Foods in Health and Disease 2011, 3, 106-117.

  134. Influence of route of administration on the absorption and disposition of α-, γ- and δ-tocotrienols in rats. Siew Ping Yap, Kah Hay Yuen and Ai Beoy Lim. Journal of Pharmacy and Pharmacology,  Volume 55, Issue 1, pages 53–58, January 2003.

  135. Dose-response impact of various tocotrienols on serum lipid parameters in 5-week-old female chickens. Yu SG, Thomas AM, Gapor A, Tan B, Qureshi N, Qureshi AA. Lipids. 2006;41(5):453–61.

  136. Tocotrienols: constitutional effects in aging and disease. Schaffer S, Müller WE, Eckert GP. J Nutr. 2005 February 135(2):151-4.

  137. The therapeutic impacts of tocotrienols in type 2 diabetic patients with Hyperlipidemia. Simant Baliarsingh, Zafarul H. Bega, Jamal Ahmad. Atherosclerosis Volume 182, Issue 2, October 2005, Pages 367–374.

