The Effect of Palm Tocotrienol Rich Fraction on Alcoholic Fatty Liver Disease (AFLD): A Phase II Clinical Trial
Phase: PHASE2
Conditions: Alcoholic Fatty Liver Disease
Sponsor: Universiti Kebangsaan Malaysia Medical Centre

 
 
Title: The Effect of Palm Tocotrienol Rich Fraction on Alcoholic Fatty Liver Disease 
(AFLD): A Phase II Clinical Trial 
 
Version 2 (Date: 30/11/2023) 
 
Researchers: 
Dr. Siti Norain Azahar (M.D.) 
 
Principal Investigator  
Professor Dr. Nur Azlina Mohd Fahami (DVM, MMed Sc, PhD) (UKM) 
Co-investigators 
Dr. Zaida Zainal (PhD) 
Associate Professor Dr. Chin Kok Yong (PhD) (UKM) 
Investigator (Clinical) 
Dr. Deborah Chew Chia Hsin (MBChb (Aberdeen, UK) MRCP) (UKM) 
 
Industrial Collaborators: 
1. Malaysian Palm Oil Board 
No.6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang Selangor 
 
2. ExcelVite Sdn Bhd 
Lot 56442, 7½ Mile 
Jalan Ipoh/Chemor 31200, Chemor, Perak 
 
3. Hovid Berhad 
121, Jalan Tunku Abdul Rahman, 
30010 Ipoh, Perak. 
 
Research Locations: 
1. Hospital Canselor Tuanku Muhriz Universiti Kebangsaan Malaysia 
2. Department of Pharmacology, Faculty of Medicine, Universiti Kebangsaan Malaysia 
 
 

Protocol Number: PT3_AFLD; Version Number: 02 
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THE EFFECT OF PALM TOCOTRIENOL RICH FRACTION ON ALCOHOLIC 
FATTY LIVER DISEASE (AFLD): A PHASE II CLINICAL TRIA 
ABSTRACT 
Background: Alcohol abuse is a serious public health concern that is associated with many 
diseases, including alcoholic liver disease (ALD). The World Health Organization (WHO) 
reported that the use of alcohol has resulted in more than 3 million deaths every year and 
accounting for 5.9% of all deaths worldwide. As the metabolism of alcohol in individuals 
mainly depends on the liver, long-term and excessive alcohol consumption damages the liver 
leading to alcoholic fatty liver disease (AFLD). Since tocotrienols have been proven for their 
hepatoprotective effects in animal models of NAFLD, the aim of the study is to explore the 
potential role of tocotrienol rich fraction, in patients with AFLD. 
Methodology: 26 patients with AFLD aged 18 to 65 years old, will be randomized into 
treatment (n=13) and placebo group (n=13). The treatment group will be prescribed with palm 
tocotrienol soft gel (200 mg twice daily) while the placebo group will receive refined, bleached, 
and deodorised (RBD) palm olein for six months. The subjects will be answering 
questionnaires related to alcohol consumption and their blood will be collected for the 
determination of liver function test, redox, haematological profile and inflammatory 
biomarkers. Fibroscan and FibroTest will be done twice during the 3-monthly follow-ups. 
Significance: This study will validate the efficacy of tocotrienol in AFLD and its related 
morbidity, thereby producing data for subsequent phases of clinical trials. The findings of this 
study will also enhance the medicinal values of palm tocotrienol and diversify the usage of 
palm-based products in sustaining the Malaysian palm industry. 
General Information 
Protocol title: The Effect of Palm Tocotrienol Rich Fraction on Alcoholic Fatty Liver Disease 
(AFLD): A Phase II Clinical Trial 
Protocol identifying number and date: PT3_AFLD; Version Number: 02; 30/11/2023 
Amendment number(s): Version 2 
Name and address of the sponsors: Malaysian Palm Oil Board 
No.6, Persiaran Institusi, Bandar Baru Bangi, 43000 
Kajang Selangor 
 
ExcelVite Sdn Bhd (Ms. Chan Yuen Teng) 
Lot 56442, 7½ Mile 
Jalan Ipoh/Chemor 
31200, Chemor, Perak 
 
Hovid Berhad (Mr David Ho, Ms Michelle Ho) 
121, Jalan Tunku Abdul Rahman, 
30010 Ipoh, Perak. 

Protocol Number: PT3_AFLD; Version Number: 02 
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Name and title of the person authorized to sign the protocol and the amendment(s): 
Professor Dr. Nur Azlina Mohd Fahami (DVM, MMed Sc, PhD) (UKM) 
 
Name and title of the investigator(s): 
Project Principal Investigator: Professor Dr. Nur Azlina Mohd Fahami (DVM, 
MMed Sc, PhD) (UKM) 
Co-investigators: 
Dr. Zaida Zainal (PhD) 
Associate Professor Dr. Chin Kok Yong (PhD) (UKM) 
Dr. Deborah Chew Chia Hsin (MBChb (Aberdeen, UK) MRCP) (UKM) 
PhD Candidate: Dr. Siti Norain Azahar 
Trial site: 
Address: Hospital Canselor Tuanku Muhriz 
Universiti Kebangsaan Malaysia 
Jalan Yaacob Latif, Bandar Tun Razak, 
56000, Cheras, Kuala Lumpur 
Telephone Number: 03-91455555 
 
Other department(s) and/or institutions involved in the trial: 
Name: Department of Pharmacology 
Address: Faculty of Medicine, Universiti Kebangsaan Malaysia 
Jalan Yaacob Latif, Bandar Tun Razak, 
56000, Cheras, Kuala Lumpur 
 
 
 
PROJECT SYNOPSIS 
Alcoholic misuse has resulted in 3.3 million deaths every year according to the World Health 
Organization (WHO) report on noncommunicable diseases [1]. Meanwhile, the National 
Health and Morbidity Survey (NHMS) 2019 data shows that the prevalence of current drinkers 
among Malaysian adults was 11.8%, of which 17.6% were risky drinkers [2]. Alcoholic liver 
disease has caused an estimated 48% of deaths from cirrhosis. The most common health 
problem associated with chronic alcoholism is alcoholic liver disease (ALD) [1]. Chronic 
alcohol abuse can result in a spectrum of liver injury that ranges from mild fatty infiltration to 
cirrhosis and hepatocellular carcinoma [3]. Fat accumulation in the liver cells is the earliest and 
most predictable response to alcoholic ingestion and is seen in 90% of heavy drinkers. The 
development of necroinflammation and fibrosis (alcoholic hepatitis or steatohepatitis) occurs 
in 10 to 35% of heavy drinkers [4]. It is important to recognize alcoholic hepatitis as patients 
with severe disease have extremely high short-term mortality rates, they can also develop portal 
hypertension in the absence of cirrhosis and the development of alcoholic hepatitis is a well- 
documented precursor for cirrhosis with long term risk 9 times higher than that for patients 
with fatty liver alone [1]. 

Protocol Number: PT3_AFLD; Version Number: 02 
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Pathogenesis of alcoholic hepatitis: 
The liver is the main organ responsible for ethanol metabolism. Ethanol is metabolized by 3 
major systems in the liver: alcohol dehydrogenase (ADH), cytochrome p450 2EI (CYP2EI) 
and finally catalase. ADH and CYP2EI convert ethanol to acetyldehyde which is then 
converted by aldehyde dehydrogenase (ALDH) to acetate. Acetyldehyde is a highly reactive 
and potentially toxic compound that is responsible for the many toxic effects of alcohol such 
as nausea, headaches and flushing. 
Acetyldehyde is also postulated to play an etiologic role in alcoholic liver disease as it forms 
adducts with reactive residues on proteins or small molecules like cysteine [5]. These chemical 
modifications can alter or interfere with normal biological processes resulting in formation of 
substances such as acetyldehyde and malondialdehyde (MAA). The formation of MAA 
antigens during alcohol-induced liver disease likely contributed to the immunologic reaction 
associated with alcohol related liver damage [6]. Acetyldehyde has also been shown to impair 
mitochondrial glutathione transport and to sensitize hepatocytes to TNF-mediated killing. 
Lastly, acetyldehyde distrupts intestinal barrier function leading to endotoxemia and 
proinflammatory cytokine production. 
Oxidative stress in the liver also increases after alcohol consumption. In hepatocytes, CYP2EI 
activity increases after alcohol consumption [7]. The CYP2EI system leaks electrons which 
initiate the oxidative stress [8]. Other sources of oxidative stress are infiltrating inflammatory 
cells in the liver. Oxidative stress in alcoholic liver disease is documented by detection of 
protein oeixation (eg: protein thiol or carbonyl products), lipid oxidation (eg: MAA), DNA 
oxidation (eg: oxodeoxyguanosine) or depletion of antioxidant defences (eg: vitamin E, 
glutathione, thioredoxin) [8]. Therefore, antioxidant supplementation could be a useful adjunct 
in the treatment of alcoholic hepatitis. 
 
 
Diagnosis of alcoholic liver disease: 
AFLD is currently defined by steatosis with a range of hepatic histopathologic changes 
including the presence of inflammatory infiltrates and varying degrees of fibrosis and cirrhosis 
that develop in the presence of chronic alcohol consumption, which is a known trigger factor 
that causes fat accumulation. Despite having comparable pathophysiological spectrum, the 
distinction between AFLD and NAFLD is determined by the amount of alcohol ingested, with 
AFLD being diagnosed based on the patient’s history of regular alcohol usage, defined as >30 
g/day in males and >20 g/day in females (according to clinical practice guidelines from the 
scientific associations for the study of liver diseases) [9, 10]. 
Apart from a reliable history of significant alcohol intake, no single laboratory or imaging 
testing can definitively confirm the diagnosis of ALD. Hence, its diagnosis is based on the 
compilation of clinical findings of hepatomegaly with signs of chronic liver disease, 
radiographic evidence of liver steatosis, fibrosis or cirrhosis, combined with abnormal 
biochemical results suggestive of liver injury [11]. 
Meanwhile, NAFLD is closely linked to obesity, caused by excessive food intake with a high- 
calorie, high-fat diet combined with a sedentary lifestyle [12, 13]. Most often, it is also 
associated with metabolic dysfunction [11, 14]. Obese people are more likely to develop 

Protocol Number: PT3_AFLD; Version Number: 02 
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metabolic syndrome, and nonalcoholic fatty liver disease (NAFLD) has been identified as the 
hepatic manifestation of metabolic syndrome [15]. 
Therefore, in the context of this study, we propose to exclude the subjects who are obese (a 
body mass index (BMI) of 30 kg/ m2 or higher) and with metabolic syndrome (based on the 
harmonised criteria) to minimise the risk of misclassification between AFLD and NAFLD. 
Clinical evidence of decompensated liver disease such as ascites and its severity, hepatic 
encephalopathy; severity according to West Haven criteria, jaundice and coagulopathy must be 
assessed for. Evidence of alcoholic hepatitis as demonstrated by hepatocellular transaminitis of 
AST:ALT > 2:1 due to deficiency of pyridoxal 5’phosphate level in alcoholic patients, raised 
gamma glutamyl transpeptidase (GGT) raised mean corpuscular volume (MCV), raised total 
bilirubin with severely ill patients having coagulopathy as demonstrated by raised international 
normalised ratio (INR) and prothrombin time (PT). Patients with other forms of bleeding 
disorder, as well as those on anticoagulant or antiaggregant treatments are not eligible to 
participate in the trial. Patients who have been taking vitamin E, herbal supplements or other 
investigational products are also excluded. 
Other types of liver disease, such as hepatitis B, hepatitis C and autoimmune hepatitis, 
hereditary hemochromatosis and non-alcoholic fatty liver disease will also need to be excluded. 
Assessment of the presence of advanced fibrosis or cirrhosis can be accessed via Fibroscan as 
well as FibroTest (FT) which is a serum marker used to calculate the degree of liver fibrosis. 
Patients should also be screened for hepatocellular carcinoma (HCC), a complication that can 
occur in alcoholic cirrhosis. Patients who are found to have HCC should be excluded from the 
study. Patients who have other coexisting chronic liver diseases (CLD) will not be eligible for 
recruitment as the presence of the CLD can confound the prognosis of alcoholic hepatitis. Once 
other chronic liver diseases have been ruled out, the severity of alcoholic liver disease will then 
be graded according to Maddrey's discriminant function. 
The pathogenesis of ALFD involves oxidative stress and disruptions of lipid metabolism [7]. 
It also has been demonstrated that the response to hypoxia plays an essential role in the 
development of ALFD [8] and this in turn has been linked to mitochondrial radical oxygen 
species (ROS) generation. Currently, the most effective AFLD treatment is alcohol abstinence 
and there are still few effective pharmacological treatments for patients afflicted with this 
disease. Therefore, new therapies are urgently needed to prevent the progression of AFLD. 
Background Information of the investigational product 
 
Vitamin E are well known for its potent antioxidant capabilities, while studies had also shown 
its positive effects on inflammation. Animal study had also shown the protective effect of palm 
tocotrienol on the liver [16]. The most common homologue of the vitamin E family investigated 
in treating liver disease is alpha tocopherol. Other homologues of vitamin E, namely 
tocotrienols, also exist in isomers designated as alpha, beta, gamma and delta, and they 
differentiate from analogous isomers of tocopherol by the presence of an unsaturated phytyl 
chain (‘tail’) as opposed to a saturated one in tocopherols [17]. Tocotrienols are preferentially 
distributed to the liver [18, 19] and alpha-tocotrienol has been reported to be 40-60 times more 
potent than alpha-tocopherol against lipid peroxidation in rat liver microsomes [20]. 

Protocol Number: PT3_AFLD; Version Number: 02 
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Several clinical studies have been conducted to evaluate the tocotrienol effects on the liver. 
All these studies have demonstrated the hepatoprotective effects of palm tocotrienols in their 
adult subjects. According to Patel et al. (2012) [29], tocotrienol attenuated the time- 
dependent rise in the Model for End Stage Liver Disease (MELD) score vs the alpha- 
tocopherol group in liver transplant subjects with end-stage liver disease. Magosso et al. 
(2013) [30] have demonstrated that in subjects with mild untreated hypercholesterolemia and 
ultrasound-proven non-alcoholic fatty liver (NAFLD), there is a normalization of the hepatic 
echogenic response in the tocotrienol-treated group vs. the placebo group. 
There is also a significant reduction in aspartate aminotransferase (AST), alanine 
aminotransferase (ALT), malondialdehyde (MDA), and C-reactive protein (CRP) among 
subjects with ultrasound-proven non-alcoholic fatty liver (NAFLD) in the tocotrienol group 
vs. the placebo group, as reported by Pervez et al. (2018) [31]. These findings are in 
agreement with the outcomes of following study by Pervez et al. (2020) [21]. In their study 
subjects with NAFLD, tocotrienol significantly reduced AST, ALT, MDA, CRP, fatty liver 
index (FLI), and improved grading of hepatic steatosis on ultrasound, compared to the 
placebo group. 
Tocotrienols are generally safe and are usually well tolerated. By participating in this 
research, it is possible that the treatment given to the subjects may or may not help improve 
their liver function. There is a risk that their condition will not get better and that the 
intervention won't work. The treatment given to them are usually in low doses and are 
unlikely to cause serious side-effects. However, in patients with bleeding disorders, 
tocotrienols might slow blood clotting. Tocotrienols may also interact with "blood thinners" 
such as warfarin and antiaggregants like aspirin, increasing the risk of bleeding. Thus, we 
advise against taking the treatment with these medications. Symptoms like nausea, diarrhea, 
stomach cramps, tiredness, dizziness, and blurred vision may rarely occur in cases of 
overdose. Allergic reactions to this supplement are rare; however, if they develop any 
symptoms of a serious allergic reaction, including rash, itching or swelling (especially of the 
face, tongue, or throat), dizziness, or trouble breathing, they should tell the doctor and get 
medical help right away. Venous blood taking would require a needle to pierce through their 
skin, which might also result in bruising and mild pain. 
This trial will be conducted in compliance with the protocol, GCP and the applicable 
regulatory requirement(s). The population to be studied is adult patients aged 18-65 years, 
with a history of alcohol use disorder, who could comply with alcohol abstinence, and those 
with diagnosis of AFLD with lab evidence of Alcoholic Steatohepatitis (AST:ALT >2.0, 
elevated GGT) who come for follow up in Gastroenterology & Hepatology clinic in Hospital 
Canselor Tuanku Muhriz UKM (HCTM). Additionally, our research subjects were also 
recruited from the general public who responded to our recruitment poster on social media 
platforms. 
 
 
Research Gap 
Concerns about the high prevalence of AFLD globally and the not fully understood reasons 
for its progression to more severe forms of liver diseases have led to the initiation of this 
interventional, double-blind placebo-controlled study. The aim of the investigation was to 

Protocol Number: PT3_AFLD; Version Number: 02 
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evaluate the activity of tocotrienols in normalising hepatic echogenic response in adults with 
AFLD. 
Currently, there are no effective treatment for alcoholic fatty liver disease apart from alcohol 
abstinence and making some lifestyle changes to control or reverse the fat build-up in the liver. 
An antioxidant and anti-inflammatory agent are speculated to enhance the healing of liver after 
alcohol abstinence, but this concept has not been effectively demonstrated in a clinical trial. 
Therefore, new therapies are urgently needed to prevent the progression of AFLD. Tocotrienol 
are well established for its potent antioxidant capabilities, while evidence had also shown its 
positive effects on inflammation. Thus, this study will validate the efficacy of tocotrienol in 
AFDL and its related morbidity, thereby producing data for subsequent phases of clinical trials. 
The findings of this study will also enhance the medicinal values of palm tocotrienol and 
diversify the usage of palm-based products in sustaining the Malaysian palm industry. 
SCIENTIFIC IMPACT 
The health benefits of TRF in relation to liver health have been reported in vitro, in vivo and 
even human clinical studies. However, these clinical studies were conducted on subjects with 
liver cirrhosis and non-alcoholic fatty liver disease (NAFLD). To date, no data is available on 
human intervention studies involving TRF in alcoholic fatty liver disease (AFLD). It’s 
important to carry out this clinical trial to find out if TRF would show the similar protective 
effect on subjects with AFLD, as it did with NAFLD. 
In addition, the findings from this study could serve as the basis for future clinical studies 
related to liver diseases. 
 
 
SOCIAL IMPACT 
This project outcome will give a significant impact to social and economy, locally and 
worldwide. Alcohol consumption is a major risk factor for chronic disease. Based on 58 studies 
from 17 Global Burden of Diseases (GBD) regions, alcohol use disorders accounted for 9.6% 
(7.7%-11.18%) of age-standardized disability-adjusted life years (DALYs) worldwide in 2010 
[26]. Alcohol-induced liver cirrhosis was responsible for 0.9% of all global deaths and 47.9% 
of all liver cirrhosis deaths in 2010 [27]. 
Palm Oil is the largest agricultural contributor to Malaysia's gross domestic product (GDP) 
with a total of RM44. 8 billion or 3.8 per cent of the GDP contribution in 2017 [28]. Malaysia 
is the largest tocotrienols manufacturer and the second largest palm oil producer in the world. 
The beneficial effect of Palm TRF in AFLD would encourage the local and global usage of 
Palm TRF and Palm Oil (the richest source of TRF) in pharmaceutical, dietary supplement, and 
functional food and drinks. 
Meantime, there is no drug or treatment for AFLD at the moment. Palm TRF can be the 
potential adjuvant therapy to help the AFLD subjects in potentially maintaining healthy liver 
function. This project is also expected to produce a postgraduate student who will develop a 
strong background in clinical trial. 

Protocol Number: PT3_AFLD; Version Number: 02 
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Nath B, Levin I, Csak T, Petrasek J, Mueller C, Kodys K, et al. Hepatocyte-specific 
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9. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and 
management of non-alcoholic fatty liver disease: practice Guideline by the American 
Association for the Study of Liver Diseases, American College of Gastroenterology, and the 
American Gastroenterological Association. Hepatology. (2012) 55:2005–23. 
10. European Association for the Study of the Liver, European Association for the Study of 
Diabetes, European Association for the Study of Obesity. EASL-EASD-EASO clinical practice 
guidelines for the management of non-alcoholic fatty liver disease. J Hepatol. (2016) 64:1388– 
402. 
11. Sharma, P., & Arora, A. (2020). Clinical presentation of alcoholic liver disease and non- 
alcoholic fatty liver disease: spectrum and diagnosis. Translational Gastroenterology and 
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12. Sullivan S. Implications of diet on nonalcoholic fatty liver disease. Curr Opin 
Gastroenterol 2010; 26: 160-164 
13 Zelber-Sagi S, Nitzan-Kaluski D, Goldsmith R, Webb M, Blendis L, Halpern Z, Oren R. 
Long term nutritional intake and the risk for non-alcoholic fatty liver disease (NAFLD): a 
population based study. J Hepatol 2007; 47: 711-717 

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14. Cotter TG, Rinella M. Nonalcoholic fatty liver disease 2020: the state of the disease. 
Gastroenterology. (2020) 158:1851–64. 
15. Marchesini G, Bugianesi E, Forlani G, Cerrelli F, Lenzi M, Manini R, Natale S, Vanni E, 
Villanova N, Melchionda N, Rizzetto M. Nonalcoholic fatty liver, steatohepatitis, and the 
metabolic syndrome. Hepatology 2003; 37: 917-923 
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Nur Azlina Mohd Fahami & Muharani Tajudin. 2011. Kesan suplimentasi tokotrienol 
dan tokoferol ke atas perubahan status oksidatif hepar dan aktiviti enzim antioksidan tikus 
aruhan stres (Effect of tocotrienol and tocopherol on oxidative status and antioxidant enzymes 
activities in stress-induced rat’s liver). Sains Malaysiana 40(5): 481-487. 
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Ahsan H, Ahad A, Siddiqui WA. A review of characterization of tocotrienols from plant 
oils and foods. J Chem Biol. 2015 Jan 20;8(2):45-59. 
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Lluis JM, Colell A, García–Ruiz C, Kaplowitz N, Fernández–Checa JC. Acetaldehyde 
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Serbinova E, Kagan V, Han D, Packer L. Free radical recycling and intramembrane 
mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol. Free Radic 
Biol Med. 1991;10(5):263–275. 
21. Pervez et al. Delta-tocotrienol supplementation improves biochemical markers of 
hepatocellular injury and steatosis in patients with nonalcoholic fatty liver disease: A 
randomized, placebo-controlled trial. Complementary Therapies in Medicine 52 (2020) 102494 
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Whiteford HA, Degenhardt L, Rehm J, Baxter AJ, Ferrari AJ, Erskine HE, Charlson 
FJ, Norman RE, Flaxman AD, Johns N, et al. Global burden of disease attributable to mental 
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Rehm J, Samokhvalov AV, Shield KD. Global burden of alcoholic liver diseases. 
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29. 
Patel, V., et al. Oral Tocotrienols Are Transported to Human Tissues and Delay the 
Progression of the Model for End-Stage Liver Disease Score in Patients. The Journal of 
Nutrition. 2012; 142(3): 513–519. 
30. 
Magosso, E., et al. Tocotrienols for normalisation of hepatic echogenic response in 
nonalcoholic fatty liver: a randomised placebo-controlled clinical trial. Nutrition Journal. 2013; 
12(1):166. 
31. 
Pervez et al. Effects of Delta-tocotrienol Supplementation on Liver Enzymes, 
Inflammation, Oxidative stress and Hepatic Steatosis in Patients with Nonalcoholic Fatty Liver 
Disease. Turk J Gastroenterol. 2018; 29(2):170-176. 
 
 
STUDY OBJECTIVES 
General: 
To determine the effects of palm tocotrienol rich fraction (TRF) on the progression of alcoholic 
fatty liver disease. 
Specific: 
1. 
To determine the effects of TRF on fatty liver index and FibroScan score of patients 
with alcoholic fatty liver disease. 
2. 
To determine the effects of TRF on the liver enzyme profile of patients with alcoholic 
fatty liver disease. 
3. 
To determine the effects of TRF on metabolic status of patients with alcoholic fatty 
liver disease. 
4. 
To determine the effect of TRF on inflammatory cytokines and redox status of patients 
with alcoholic fatty liver disease. 
HYPOTHESIS 
1. 
TRF reduces fatty liver index and improves FibroScan score of patients with alcoholic 
fatty liver disease. 
2. 
TRF normalises the liver enzyme profile of patients with alcoholic fatty liver disease. 
3. 
TRF normalises the metabolic profile of the patients with alcoholic fatty liver disease. 
4. 
TRF normalises the cytokines level and redox status of patients with alcoholic fatty 
liver disease. 

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METHODOLOGY (STUDY DESIGN, WORK PLAN, MILESTONES) 
This study would be a randomized, double-blind, placebo-controlled, phase 2 Study on the 
Effect of Palm Tocotrienol Rich Fraction on Alcoholic Fatty Liver Disease (AFLD). Patients 
who come for follow up in Gastroenterology & Hepatology clinic in PPUKM, with diagnosis 
of AFLD would be recruited in the study. Secondary data obtained from Casemix unit would 
also be screened to look for potential patients for the study. 
Primary Outcome Measures: 
1.Change from baseline in Liver Enzyme Profile (Aspartate Aminotransferase [AST], Alanine 
Aminotransferase [ALT], and Gamma-Glutamyl Transferase [GGT]) at 3 and 6 months. 
Measured in units per liter (U/L) and reported as absolute and percentage change. Lower values 
indicate improved liver function.  
2.Between-group difference in Liver Enzyme Profile (AST, ALT, GGT) at 3 and 6 months 
(Tocotrienol-Rich Fraction vs placebo). Measured in U/L. Lower values indicate improvement.  
3.Change from baseline in Fatty Liver Index (FLI) at 3 and 6 months. Unitless score (range 0–
100). Higher scores indicate greater hepatic steatosis (worse outcome). Reported as absolute 
and percentage change.  
4.Between-group difference in Fatty Liver Index (FLI) at 3 and 6 months (Tocotrienol-Rich 
Fraction vs placebo). Range 0–100. Higher scores indicate worse steatosis.  
5.Change from baseline in Liver Stiffness Measurement using Transient Elastography 
(FibroScan® score) at 3 and 6 months. Measured in kilopascals (kPa; typical range 2–75). 
Higher values indicate greater fibrosis. Reported as absolute and percentage change.  
6.Between-group difference in Liver Stiffness Measurement (FibroScan® score) at 3 and 6 
months (Tocotrienol-Rich Fraction vs placebo). Measured in kPa. Higher values indicate worse 
fibrosis. 
 
*Aspartate aminotransferase (AST) is usually used to detect liver damage. It is often ordered 
in conjunction with other liver enzymes, alanine aminotransferase (ALT) and gamma-glutamyl 
transpeptidase (GGT), or as part of a liver panel or comprehensive metabolic panel (CMP) to 
screen for and/or help diagnose liver disorders. The changes in these liver enzymes can also be 
used to monitor the compliance of patients toward alcohol abstinence. 
 
Secondary Outcome Measures: 
 
1. Change From Baseline in Plasma Cytokine Levels (Anti-inflammatory Effect of 
Tocotrienols) at baseline, 3 months, and 6 months. 
•Cytokines measured: Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), Interleukin-
1 beta (IL-1β) in plasma; Method: Multiplex Enzyme-Linked Immunosorbent Assay (ELISA); 
Unit: picograms per milliliter (pg/mL); Typical range: IL-6 (0–50 pg/mL), TNF-α (0–100 
pg/mL), IL-1β (0–50 pg/mL); Higher values indicate greater inflammation (worse outcome). 
 
 

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2. Change From Baseline in Metabolic Profile (Fasting Blood Glucose [FBG] and Lipid Panel) 
at baseline, 3 months, and 6 months. 
•Parameters measured: FBG, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-
C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG); Unit: milligrams per 
deciliter (mg/dL); Typical ranges: Fasting glucose (70–100 mg/dL), TC (125–200 mg/dL), 
LDL-C (0–130 mg/dL), HDL-C (40–60 mg/dL), TG (0–150 mg/dL); Higher values indicate 
worse metabolic profile. 
 
 
SAMPLE SIZE CALCULATION 
 
Sample size calculation was based on the comparison of two means derived from the effects of 
tocotrienol on fatty liver index (FLI) in patients with non-alcoholic fatty liver disease (NAFLD) 
(21). This is because the effectiveness of tocotrienol in alcoholic hepatitis has not been 
evaluated previously. 
Formula of calculating sample size in clinical trial (22): 
n = [(Zα/2 + Zβ)² × {2(ó)²}]/ (μ1 - μ2)² 
 
Where: 
n = sample size required in each group 
μ1 = mean of FLI for tocotrienol group = 12.82 μ2 = mean of FLI for placebo group = 3.86 
ó = standard deviation = 7.0 
Zα: This depends on level of significance, for 5% this is 1.96 Zβ: This depends on power, for 
80% this is 0.84 
 
n = [(1.96 + 0.84)² × {2(7.0)²}]/ (12.82 – 3.86)² 
 
The sample size calculated is 10 per group. Taking into account 20% of subjects might be lost 
to follow up, the number of each group is adjusted to 13. Hence, the total sample size required 
for this study is 26 individuals. 
 
 
 
 
 
 
 

Protocol Number: PT3_AFLD; Version Number: 02 
13 
 
 
SCREENING 
Primarily we will be recruiting adult patients who fulfil all the inclusion criteria of this study, 
who come for follow up in Gastroenterology & Hepatology clinic in Hospital Canselor Tuanku 
Muhriz UKM (HCTM). Additionally, our research subjects were also recruited from the 
general public who responded to our recruitment poster in the hospital and on social media 
platforms. Subjects will answer a questionnaire on their demographic and lifestyle details, 
medical and medication history. Their alcohol consumption behaviour will be surveyed using 
the AUDIT questionnaire. A qualified physician will perform physical examination on the 
subjects. The AUDIT questionnaire (23) is a 10-item screening tool used to identify individuals 
with harmful drinking behaviour. A score of 8 or more indicates harmful or hazardous drinking 
behaviour. In addition, patients will be screened for clinical (signs and symptoms of alcoholic 
liver disease such as parotid swelling, hepatomegaly and clinical evidence of cirrhosis) and 
biochemical evidence of alcoholic liver disease (transaminitis, AST:AST ratio>2.0, raised 
gamma glutamyl transferase (GGT), mean corpuscular volume (MCV) and total bilirubin). 
Body anthropometry 
Body anthropometry will be performed on every visit. Height of the subjects will be measured 
using a stadiometer. Body weight of the subjects will be measured using a weighing scale. 
Body composition of the subjects will be analysed using a 4-point bioimpedance machine. 
Body mass index of the subjects will be calculated as per the convention. 
 
 
Blood collection 
Blood collection will be performed on every visit. Subjects will be requested to fast for 8 
hours before blood withdrawal. Venepuncture will be performed by a qualified phlebotomist. 
5 ml of fasting blood will be drawn in the morning. 
 
 
 
 
 
 
 

Protocol Number: PT3_AFLD; Version Number: 02 
14 
 
 
Determination of plasma tocotrienol level (HPLC) 
A portion of the blood will be collected using heparin tubes. Plasma will be extracted and 
aliquoted into 200 µL. The plasma will be stored in - 70°C until analysis. Upon analysis, the 
plasma will be thawed, added with 50 ul 95% ethanol containing 10 ug/mL bytylated 
hydroxytoluene, and vortexed vigorously for 5 seconds. Then, 1 mL absolute alcohol will be 
added to the mixture, which will be remixed before being centrifuged at 1500 g for 15 minutes 
at 18°C. The pellet will be removed, and the supernatant will be added with 3 mL hexane and 
mixed vigorously for 5 minutes. The samples will be centrifuged again at 1500 g for 15 minutes 
at 18°C. The upper layer (2.5 mL) of the supernatant will be removed, placed in new Eppendorf 
tubes and vacuum evaporated for 40 minutes. The dried residues will be reconstituted in 100 
uL HPLC grade hexane, vortexed, and passed through a 0.45 uM filter to remove residual 
particles. The samples will be collected using an amber vial and analysed by an HPLC machine 
(Shidmadzu RF-10A XL with fluorescence detector) at the excitation wavelength of 294 nm 
and the emission wavelength of 330 nm. The tocotrienol isomers and alpha- tocopherols will 
be separated on a 250 mm x 4.6 mm, 5 um silica column and eluted with a mobile phase of 
99:1 (v/v) hexane-isopropanol at 1.5 mL per minutes flow rate. The identity of each compound 
will be confirmed by co-elution with spiked standard obtained from Excelvite Sdn. Bhd. The 
peaks will be quantified and integrated using Shimadzu Workstation software. 
Biochemical assay 
Full blood count, liver profile (AST, ALT, GGT), lipid profile and fasting glucose test will be 
sent to an accredited laboratory for analysis using automated biochemistry analyser. HepatitisB 
and C status, as well as antibodies for autoimmune hepatitis will be assayed using an automated 
immunoassay machine. A portal of the blood will be centrifuged to isolate serum for 
acetyldehyde, malondialdehyde and inflammatory cytokines. These parameters will be assayed 
using enzyme-linked immunoassay based on manufacturer protocols. 
Ultrasound 
Ultrasound of the hepatobiliary system will be performed during initial recruitment to rule out 
the presence of hepatocellular carcinoma (HCC) which can occur in patients with alcoholic 
liver cirrhosis. During the screening an ultrasound probe will be placed in the patients’ right 
hypochondrium to assess for any abnormal lesions representative of HCC. It is a non-invasive 
procedure which takes approximately 20 minutes. Patients with hepatocellular carcinoma will 
be excluded from the study. 
Fibroscan 
This is a non-invasive test to measure liver stiffness, kPa (indicator of liver fibrosis) and to 
detect degree of fat accumulation (CAP range value of 100-400 dB/m). Liver stiffness of > 
8kPa indicates advanced fibrosis while CAP of > 263 indicates fatty liver. During the screening, 
the patient will lie down on the examination bed with his/her right hand behind 

Protocol Number: PT3_AFLD; Version Number: 02 
15 
 
 
his/her head. Investigator will place the probe between the right ribs of the patient. 
Measurements will be recorded into the machine (FibroScan® 502 Touch). The screening is 
quick, easy and non-invasive and usually takes less than 15 minutes. 

Protocol Number: PT3_AFLD; Version Number: 02 
16 
 
 
ALGORITHM OF DIAGNOSIS OF ALCOHOLIC HEPATITIS: 
Alcohol misuse disorder: 
Regular alcohol consumption > 20g/day in female and >30g/day in males 
Clinical parameters: 
Ascites (Degree) 
Hepatic encephalopathy (West haven criteria) 
Biochemistry: 
Raised ALT and AST ratio AST: ALT>2.0 
Raised mean corpuscular volume (MCV) 
Raised gamma glutamyl transpeptidase (GGT) 
Raised total bilirubin 
Platelets 
Coagulopathy: Deranged INR and PT 
Oxidative markers: 
Acetyldehyde 
Malondialdehyde 
Exclusion of: 
Obese patients (a BMI of a BMI of 30 kg/m2 or more) and with metabolic syndrome 
Patients with bleeding disorders, and on anticoagulant or antiaggregant treatments 
Hepatitis B (HBsAg) 
Hepatitis C (Anti-HCV) 
Autoimmune hepatitis (ANA, AMA, ASMA, IgG) 
NAFLD (BMI, fasting glucose, blood pressure, fasting total cholesterol, triglyceride 
and HDL) 
Hereditary hemochromatosis (Ferritin, Tsat) 
Severe alcoholic hepatitis (Maddrey’s discriminant score) 
Assessment of advanced fibrosis/cirrhosis: 
Fibroscan, FibroTest (FT) 
Radiology: 
Ultrasound of the hepatobiliary system TRO hepatocellular carcinoma 

Protocol Number: PT3_AFLD; Version Number: 02 
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Randomisation and Treatment 
The treatment assigned to participants for 6 months was either mixed tocotrienols 200 mg twice 
daily or placebo. A treatment period of 6 months will be adopted because only patients with 
mild alcohol steatohepatitis are recruited, whose condition would resolve within months with 
alcohol abstinence alone. The content in each capsule of the mixed tocotrienols preparation 
was 61.5 mg, 112.8 mg and 25.7 mg for alpha-, gamma- and delta-tocotrienol, respectively and 
61.1 mg of alpha-tocopherol. Both placebo and tocotrienols, were soft gel capsules and were 
similar in terms of colour, size, shape and surface texture. Participants were randomised using 
a computer-generated random allocation sequence. A permuted block design was employed. 
Each block of specified size contained an allocation ratio of 1:1 (placebo:tocotrienols). The 
Treatment with tocotrienol-rich fraction (TRF) 
Reassessment of parameters of alcoholic hepatitis post tocotrienol: 
Clinical Parameters: 
Ascites (Degree) 
Hepatic encephalopathy (West haven criteria) 
Biochemistry: 
ALT, AST, and GGT 
Mean corpuscular volume (MCV) 
Gamma glutamyl transpeptidase (GGT) 
Total bilirubin 
Platelets INR and PT 
Oxidative markers: 
Acetyldehyde Malondialdehyde 
Assessment of fibrosis and cirrhosis: 
Fibroscan, Fibrotest 

Protocol Number: PT3_AFLD; Version Number: 02 
18 
 
 
random allocation sequence would select the next block and determine the next allocations. 
Sequence was not made known to researchers who enrolled participants. For the purpose of 
assigning participants to interventions, a subject number was used. The researcher who 
generated the random allocation sequence and assigned participants was blinded to subjects’ 
clinical data and was independent from the persons who enrolled participants. The mixed 
tocotrienols preparation and the placebo capsules were purchased from Hovid (Ipoh, Malaysia). 
Researchers and volunteers were blinded to the assigned treatment. 
Source and composition of tocotrienol 
Tocotrienol mixture derived from palm oil will be used. It will be sponsored by Hovid Sdn Bhd 
(Ipoh, Malaysia). The composition of the tocotrienol mixture is 24.7% α-tocotrienol, 4.5% β- 
tocotrienol, 36.9% γ-tocotrienol, 12.0% σ-tocotrieno l and 21.6% α-tocopherol. It is formulated 
with a self-emulsifying system (SES) to enhance absorption of tocotrienol. 
The gelatin sheet used in the manufacturing of tocotrienol soft gel capsules is prepared using 
gelatin derived from a bovine source. 
Dosing of tocotrienol 
Animal studies reported that a dose of 60 mg/kg tocotrienol could be effective in maintaining 
liver health [16]. To extrapolate this to human equivalent dose, body surface conversion (24) 
is used: 
Human equivalent dose (HED) 
= [Km rat/Km human] x [dose in rat] x [expected body weight of humans] 
= [6 / 37] x [60 mg/kg] x [60 kg] 
= 584 mg ≈ 600 mg 
However, this calculation does not consider the bioavailability-enhancing effect of SES. A 
previous study showed that the use of SES enhanced the bioavailability of tocotrienol by 4- 
fold [25]. Notwithstanding that, the effects of SES on the tissue distribution of tocotrienol, 
especially in the musculoskeletal system are not well-known. Therefore, we propose to use 400 
mg (a decrease of 33.3% from the original calculated dose) in the trial subjects. 
It will be packaged into two soft gels, each containing 200 mg palm tocotrienol. One soft gel 
will be taken orally, daily after breakfast and dinner to complete the 400 mg daily dose. The 
treatment period will be 6 months. The placebo will consist of equivolume refined, bleached, 
and deodorised (RBD) palm olein. Both tocotrienol and placebo capsules will be manufactured 
in accordance with any applicable GMP, and is coded and labeled in a manner that protects the 
blinding. In addition, the labeling should comply with applicable regulatory requirement(s). 
 
 
Dispense of TRF 
Since the study activity for each subject will last for six months, either TRF or placebo with 
identical appearance and packaging will be dispensed to the subjects twice, which is every three 
months (during baseline and visit 1). There is a +7-day allowable window period for visit 1 as 
some subjects might not be able to turn up on the exact visit date. Softgel counting will be 

Protocol Number: PT3_AFLD; Version Number: 02 
19 
 
 
performed at visit 1 and 2 to determine the compliance of the subjects. Subjects will be 
requested to consume the softgel after meal. 
“Stopping rules" or "discontinuation criteria 
The participation of the trial subjects will be terminated if they cannot adhere to alcohol 
abstinence and cannot comply with the treatment regimes. Failure to adhere is a serious 
problem that not only affects the subjects but also the overall conduct and outcome of the 
research. They will also be stopped from participating in situations where the risks outweigh 
the anticipated benefits, in cases of severe allergic reactions or any adverse events that occurred 
in individual trial subjects. Subjects in these trials should be particularly closely monitored and 
should be withdrawn if they appear to be unduly distressed. 
Accountability procedures for TRF product and placebo 
We will take the responsibility for TRF product and placebo accountability at the trial site. 
The records of the product's delivery to the trial site, the inventory at the site, the use by each 
subject, and the return to the sponsor or alternative disposition of unused product will be 
maintained. These records should include dates, quantities, batch/serial numbers, expiration 
dates and the unique code numbers assigned to the TRF and placebo products and to trial 
subjects. We will maintain the records that document adequately that the subjects were 
provided the doses specified by the protocol and reconcile all investigational product received 
from the sponsor. The products will be stored in accordance with applicable regulatory 
requirements, and will be ensured that they are used only in accordance with the approved 
protocol. We will explain the correct use of the products to each subject and will check, at 
intervals appropriate for the trial, that each subject is following the instructions properly. 
Maintenance of trial treatment randomization codes and procedures for breaking code 
We will follow the trial's randomization procedures, and will ensure that the code is broken 
only in accordance with the protocol. Given that the trial is blinded, we will promptly 
document and explain to the sponsor any premature unblinding (e.g., accidental unblinding, 
unblinding due to a serious adverse event) of the investigational product. We will have a 
written procedure for rapidly identifying a ‘blinded’ IP in case of an emergency. The 
procedure will be secure, readily available at all times during the trial, and not allow breaks 
of the blinding to go undetected. 
Data recorded directly on the CRFs is to be considered the source data. 
We will maintain adequate and accurate source documents and trial records that include all 
pertinent observations on each of the site’s trial subjects. Source data should be attributable, 
legible, contemporaneous, original, accurate, and complete. Changes to source data should be 
traceable, should not obscure the original entry, and should be explained if necessary (via an 
audit trail). We will ensure the accuracy, completeness, legibility, and timeliness of the data 
reported to the sponsor in the CRFs and in all required reports. Data reported on the CRF, that 
are derived from source documents, should be consistent with the source documents or the 
discrepancies should be explained. Any change or correction to a CRF will be dated, initialed, 
and explained (if necessary) and will not obscure the original entry (i.e. an audit trail should be 
maintained); this applies to both written and electronic changes or corrections. Records of the 
changes and corrections will be retained. 

Protocol Number: PT3_AFLD; Version Number: 02 
20 
 
 
We will maintain the trial documents as specified in Essential Documents for the Conduct of a 
Clinical Trial in the Malaysian Guideline for Good Clinical Practice and as required by the 
applicable regulatory requirement(s). Measures that prevent accidental or premature 
destruction of these documents will be taken. All the essential documents will be retained until 
at least 2 years or for a longer period, if required by the applicable regulatory requirements. We 
will make available for direct access all requested trial-related records, upon request of the 
monitor, auditor, IRB/IEC, or regulatory authority. 
 
 
SELECTION AND WITHDRAWAL OF SUBJECTS 
Inclusion criteria: 
1. 
Patients with history of alcoholic use disorder with clinical and biochemical evidence 
of alcoholic steatohepatitis. 
2. 
Patients with Maddrey's discriminant function ≤ 32, and do not require the treatment of 
corticosteroid therapy or pentoxifylline. 
3. 
Patients aged 18 to 65 
4. 
Patients who could comply with alcohol abstinence. 
Exclusion criteria: 
1. 
Severe alcoholic hepatitis defined as Maddrey’s discriminant function >32 
2. 
Patients with other concomitant liver diseases: 
a. Hepatitis B 
b. Hepatitis C 
c. Non-alcoholic fatty liver disease (NAFLD) 
d. Autoimmune hepatitis (AIH) 
e. Hereditary hemochromatosis 
 
3. 
Patients who are obese (a BMI of 30 kg/ m2 or more) and with metabolic syndromes 
4. 
Patients with bleeding disorders and who have been on anticoagulant or antiaggregant 
treatments 
 
5. 
Patients who have been on corticosteroid therapy or pentoxifylline for alcoholic 
hepatitis 
6. 
Patients with hepatocellular carcinoma 
7. 
Pregnant patients 
8. 
Patients who are breastfeeding 
9. 
Patients with Childs C liver cirrhosis 
10. 
Patients who have pyridoxine allergy or history 

Protocol Number: PT3_AFLD; Version Number: 02 
21 
 
 
11. 
Patients who are judged by investigator that participation of the study is difficult due to 
disease as follow; hepatic cirrhosis, Wilson's disease, malignant tumor, serious metabolic 
disease, severe renal disease, severe pulmonary disease, severe cardiovascular disease, severe 
nervous disease/psychiatric disorder, muscle disease and etc 
12. 
Patients taking vitamin E, herbal supplements, or other investigational products within 
90 days prior to the participation in the study. 
13. 
Patients who have been taken any medications that could affect the treatment: 
hypoglycemic agents, colchicine, penicillamine, corticosteroids, ursodeoxycholic acid, 
pentoxifylline, long-term use of NSAIDs, statins, neuroleptics, anticonvulsant medications, 
high-dose acetaminophen(>=2.5g/day) 
14. 
Patients who have received treatment that may affect liver function within 1 month 
prior to the participation in the study 
15. 
Patients who could not comply with alcohol abstinence. 
16. 
Patient who considered ineligible for participation in the study as Investigator's 
judgment 
Note: Patients enrolled in this study MUST abstain from alcohol consumption throughout the 
study period 
Withdrawal criteria 
During the course of the trial a subject may choose to withdraw early from the trial treatment 
at any time. This may happen for a number of reasons, including but not limited to: 
• The occurrence of what the participant perceives as an intolerable adverse event (AE). 
• Inability to comply with trial procedures 
• Participant decision 
Subjects may choose to stop their participation, and it will not affect their treatment at UKM- 
affiliated hospitals and clinics in any way. Without losing any of their rights as patients, they 
will not be penalised and can still continue with the standard treatment available and for follow- 
up visits at UKMMC. The data and samples already collected would not be used in the final 
study analysis and would be discarded. 
In addition, we may discontinue a participant from the trial treatment at any time if we consider 
it necessary for any reason including, but not limited to: 
• Pregnancy 
• Ineligibility (either arising during the trial or retrospectively having been overlooked at 
screening) 
• Significant non-compliance with treatment regimen or trial requirements 
• An adverse event which requires discontinuation of the trial medication or results in 
inability to continue to comply with trial procedures 

Protocol Number: PT3_AFLD; Version Number: 02 
22 
 
 
• Disease progression which requires discontinuation of the trial medication or results in 
inability to continue to comply with trial procedures 
We have adjusted our sample size calculation for this study by considering the risk of dropout 
among the subjects. Hence, the withdrawn subjects will not be replaced while ensuring that the 
minimal sample size of this study is achieved. The type of withdrawal and reason for 
withdrawal will be recorded in the CRF. If the participant is withdrawn due to an adverse event, 
we will arrange them for follow-up visits at our clinic until the adverse event has resolved or 
stabilised. 
Treatment of Subjects 
The research takes place over six months in total. It will be necessary for each subject to come 
to the clinic at HCTM for an initial screening and three-monthly follow-ups. During these 
visits, they will undergo a routine screening which involves blood collection procedure, a liver 
fibrosis scan, and a dual-energy absorptiometry scan and they will be informed of the results 
of the tests conducted. They will also be given either the test supplement or the placebo for the 
management of AFLD. They will be required to take 2 softgels containing 200 mg TRF or 
placebo orally, each after breakfast (one softgel) or dinner (one softgel). The assignment of 
TRF and placebo will be random, and the physician and enumerator attending you will not be 
aware of your assigned treatment. At the end of six months, all data will be collected and 
analysed, and the research will be completed. 
Identifying specific barriers for each subject and adopting suitable techniques to overcome 
them will be necessary to improve the subject’s compliance and medication adherence. Health 
care professionals involved in this study such as physicians and nurses have significant role in 
their daily practice to improve subject medication adherence. We will give medication 
reminders via text or email to our study subjects. The subjects s may keep a diary, or they can 
bring all unused or part-used medication/vials and packaging from used medication at each 
follow-up visits for soft gel capsule counting and to discuss their compliance. Another method 
is to keep track of their medications with a "pill card" or "medication calendar." The subjects 
will be required to bring their "pill cards" to every appointment, and/or we may ask them to 
keep a monthly calendar on which they will mark each day when they took their capsules. 
Subjects’ non-adherence to alcohol abstinence during the duration of this study might also pose 
a risk to this study. We will try to minimise this risk by closely monitoring all study subjects 
to detect any withdrawal symptoms and offering them comprehensive care and the necessary 
psychological counseling or pharmacotherapy. 
Adverse Events (AEs), Serious Adverse Events (SAEs), and Suspected Unexpected 
Serious Adverse Reactions (SUSARs) 
While the possibility of having some unwanted side effects and unexpected events related to 
the tocotrienol supplementation and the treatment procedures is very low, the enrolled subjects 
should still be aware of the possibility. We will try to decrease the chances of this event 
occurring through close monitoring of all subjects but if something unexpected happens, we 
will provide them with proper care and appropriate treatment. We may use some other 
medicines to decrease the symptoms of the side effects or reactions. Or we may stop the use of 
this supplement and end their participation. If this is necessary, we will discuss it with the 

Protocol Number: PT3_AFLD; Version Number: 02 
23 
 
 
subjects, and they will always be consulted before we move on to the next step. Given that the 
subjects must adhere to alcohol abstinence throughout the course of this study, if they 
experience any withdrawal symptoms, we will give them supportive care and medications as 
part of the treatment regimens used in alcohol withdrawal states to minimise the symptoms and 
prevent complications. 
Our qualified physician, who is a co-investigator for the trial, will be responsible for all trial- 
related medical decisions. During and following a subject's participation in this trial, we will 
ensure that adequate medical care is provided to a subject for any adverse events, including 
clinically significant laboratory values, related to the trial. We will inform a subject when 
medical care is needed for intercurrent illness (es) of which we become aware. We will also 
recommend the subjects to inform their primary physician about their participation in this trial 
if the subject has a primary physician and if the subject agrees to the primary physician being 
informed, while fully respecting the subject's rights. 
Reporting Procedures for Adverse Events 
Adverse events and/or laboratory abnormalities identified as critical to safety evaluations will 
be reported to the sponsor according to the reporting requirements and within the specified 
time periods. 
All AEs occurring during the trial that are observed by the Investigator or reported by the trial 
subject will be reported on the trial case report form (CRF), whether or not attributed to trial 
medication. The following information will be reported on the CRF: description, date of onset 
and end date, severity, assessment of relatedness to trial medication, other suspect drug or 
device and action taken. Follow-up information should be provided as necessary. 
The severity of events will be assessed on the following scale: 1 = mild, 2 = moderate, 3 = 
severe. 
Non-serious AEs considered related to the trial medication as judged by a medically qualified 
investigator or the Sponsor will be followed up either until resolution, or the event is considered 
stable. 
It will be left to the clinical judgment of our medical officer to decide whether or not an AE is 
of sufficient severity to require the participant’s removal from treatment. A participant may 
also voluntarily withdraw from treatment due to what he or she perceives as an intolerable AE. 
If either of these occurs, the participant will be given appropriate care under medical 
supervision until symptoms cease, or the condition becomes stable. 
Reporting Procedures for Serious Adverse Events 
All SAEs must be reported on the SAE Reporting Form to the Sponsor or delegate immediately 
or within 24 hours of Site Study Team becoming aware of the event being defined as serious. 
The immediate reports will be followed promptly by detailed, written reports. The immediate 
and follow-up reports should identify subjects by unique code numbers assigned to the trial 
subjects rather than by the subjects' names, personal identification numbers, and/or addresses, 
in accordance with the applicable regulatory requirement(s) related to the reporting of 
unexpected serious adverse drug reactions to the regulatory authority(ies) and the IRB/IEC. 

Protocol Number: PT3_AFLD; Version Number: 02 
24 
 
 
Review of SAEs must be timely, taking into account the reporting timeline for a potential 
SUSAR. SAEs that are reported late must be accompanied by an explanation for this. 
 
 
SUSAR Reporting 
All SUSARs will be reported by the sponsor delegate to the relevant Regulatory Authority and 
to the REC and other parties as applicable. For fatal and life-threatening SUSARS, this will be 
done no later than 7 calendar days after the Sponsor or delegate is first aware of the reaction. 
Any additional relevant information will be reported within 8 calendar days of the initial report. 
All other SUSARs will be reported within 15 calendar days. 
Treatment codes will be un-blinded for specific participants. Additionally, principal 
investigator will be informed of all SUSARs for the relevant IMP for all studies with the same 
sponsor, whether or not the event occurred in the current trial. 
 
 
STATISTICAL ANALYSIS 
Normality of the data will be assessed through Shapiro-Wilk test. Since the study adopts a time 
x group design, mixed-design ANOVA will be used to compare the effects across time (within 
group) and between treatment groups. Small effect analysis will be used as the pairwise post 
hoc analysis. If the data is skewed, the data will be analysed with Kruskal Wallis test and Mann 
Whitney U-test with Bonferroni correction for type-1 error. All analyses will be performed 
using Statistical Package for Social Sciences version 23 (IBM, Armonk, USA). The data will 
be presented as mean (standard deviation). A p-value less than 0.05 is considered statistical 
significance. 
 
 
Direct Access to Source Data/Documents 
Direct access to the subjects’ original medical records and any data pertaining to the research 
study will not be shared with or given to anyone. Such access is only authorised to the research 
team, the monitor(s), the auditor(s), the IRB/IEC including the REC UKM, and the regulatory 
authority(ies) for verification of clinical trial procedures and/or data without violating the 
confidentiality of the subject, to the extent permitted by the applicable laws and regulations. 
We will permit trial related monitoring, audits, IRB/IEC review, and regulatory inspection(s), 
providing direct access to source data/documents. 
 
 
Quality Control and Quality Assurance 
The trial will be conducted in accordance with the current approved protocol, GCP, relevant 
regulations and standard operating procedures. A risk assessment and monitoring plan will be 
prepared before the study opens and will be reviewed as necessary over the course of the trial 
to reflect significant changes to the protocol or outcomes of monitoring activities. 

Protocol Number: PT3_AFLD; Version Number: 02 
25 
 
 
Ethical and Regulatory Considerations 
We will ensure that this trial is conducted in accordance with the principles of the Declaration 
of Helsinki, with relevant regulations and with Good Clinical Practice. 
This research protocol was approved by the Research Ethics Committee, from the Research 
and Ethics Review Board, UKMMC (reference number: UKM PPI/111/8/JEP-2021-694) 
Other Ethical Considerations 
The tocotrienol preparation used in this trial were encapsulated in soft gel capsules. The main 
component of these capsules sheet is gelatin, derived from a bovine source, which may be 
culturally unacceptable for certain groups of people. 
 
 
Data Handling and Record Keeping 
All trial data will be entered on to paper CRFs and/or an electronic data entry system which 
have been validated by the standard operating procedures. 
The participants will be identified by a unique trial specific number and/or code in any 
database. The name and any other identifying detail will not be included in any trial data 
electronic file. 
If identifiable personal data may be transferred during or after the study, it is necessary to assure 
against the risks that are presented by this processing, i.e., risks of accidental or unlawful 
destruction, loss, alteration, unauthorised disclosure of, or access to the personal data 
transferred and stored or otherwise processed by the recipient. This is true for paper based and 
electronic data transfers. 
If some samples collected in the study are intended for further use beyond the study, the consent 
form will need to be retained for the life of the sample to meet the regulatory requirements. 
If participants are given the option to be approached for future research, it is necessary to retain 
the consent form as the basis for retention of details and future approach. Those contact details 
should be held securely, separately from the research data, and kept updated. 
Compliance with the relevant sponsor organisation’s data policy should be ensured. 
 
 
FINANCE AND INSURANCE 
Funding 
This study is being conducted with a UKM Research Grant, co-sponsored by the Malaysian 
Palm Oil Board, ExcelVite Sdn. Bhd and Hovid Sdn. Bhd. 
Insurance 
This study has yet to receive its trial insurance. However, we have started the purchasing 
process and are still waiting for the details and updates from the insurance agency. 

Protocol Number: PT3_AFLD; Version Number: 02 
26 
 
 
Publication Policy 
The publication policy which cover authorship, acknowledgements, and review procedures for 
scientific publications should comply to department or institution policy, or trial agreement. 
We will ensure that the publication policy stated here is consistent with any contract applicable 
to the trial. We will inform the trial subjects that the data from this study will be made into a 
report, which may be published. If the trial results are published, the subject's identity will 
remain confidential. 

Protocol Number: PT3_AFLD; Version Number: 02 
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50 patients will undergo screening in response to newsletter, recruitment poster 
and database from local hospital. 
 
 
 
 
 
*Regular alcohol consumption of 
>20g/day in females & >30g/day 
in males 
 
 
 
Include: 
➢ 
Patients aged 18-65 years old 
➢ 
Patients with history of alcohol use disorder, with lab evidence of Alcoholic 
Steatohepatitis (AST:ALT >2.0, elevated GGT) 
➢ 
Patients with MDF <32, which do not require corticosteroid therapy or pentoxifylline 
➢ 
Patients who could comply with alcohol abstinence 
 
Randomisation 
 
 
TRF (400 mg) + alcohol abstinence 
n=13 
*Palm TRF and Placebo soft gel capsules (identical in appearance) are supplied by Hovid 
Sdn Bhd 
*They will be relabeled and blinded into two groups. 
*Treatment duration: 6 months
 
 
Placebo + alcohol abstinence 
n=13 
 
Patient’s visits at 0,3,6 months 
➢ 
Body anthropometry 
➢ 
Biochemical assay 
➢ 
Fibroscan 
➢ 
HPLC 
 
 
 
Primary Outcomes 
Secondary Outcomes 
 
 
Figure 1: Flow chart of the summary of the study intervention 
 
*Screening process: 
➢ 
History taking: Patient’s demographic, 
lifestyles, medical & medication 
history 
➢ 
AUDIT Questionnaire 
➢ 
Physical examination 
➢ 
Biochemical assay 
➢ 
Ultrasound (hepatobiliary system) 
Exclude: 
➢ 
Patients who could not comply with 
alcohol abstinence 
➢ 
Patients who are obese (a BMI of 30 
kg/m2 or more) and with metabolic 
syndrome 
➢ 
Patients with severe alcoholic hepatitis: 
Maddrey’s Discriminant Function 
(MDF) >32 
➢ 
Patients with other concomitant liver 
diseases: Hepatitis B & C, NAFLD, 
Autoimmune Hepatitis 
➢ 
Patients with hepatocellular carcinoma 
(HCC) 
➢ 
Patients with bleeding disorders and 
who have been on anticoagulant & 
antiaggregant treatments 
➢ 
Patients who have been taken 
medication: Long term use of NSAIDs, 
corticosteroids, pentoxifylline 
➢ 
Patients taking vitamin E, herbal 
supplements or other investigational 
products 
Changes in ALT, 
AST, & GGT 
Changes in Fatty Liver Index 
(FLI), and FibroScan score 
Changes in 
Tocotrienol level 
Inflammatory 
markers 
Redox 
status 
Metabolic 
status 

Protocol Number: PT3_AFLD; Version Number: 02 
28 
 
 
 
GANTT CHART 
 
 
Year 
Year 1 
Year 2 
Year 3 
Semester 
Semester 1 
Semester 2 
Semester 3 
Semester 4 
Semester 5 
Semester 6 
Activities 
(in Month) 
April- 
June 
July- 
Aug 
Sept 
Oct 
Nov 
Dec- 
Jan 
Feb- 
March 
April- 
May 
June- 
July 
Aug- 
Sept 
Oct 
Nov 
Dec- 
Jan 
Feb- 
March 
April- 
June 
July- 
Aug 
Sept 
Oct 
Nov 
Dec- 
Jan 
Feb- 
March 
Literature 
review 
Research 
proposal 
writing 
Presentation 
for Ethics 
approval 
Obtained 
Ethics 
approval 
Preparation 
and 
submission of 
documents 
for CTX 
application 
Presentation 
of research 
proposal at 
the 
department 
Data 
retrieval 
from the 
Casemix unit 
and A&E 
department 
to look for 
potential 
AFLD 
patients 

Protocol Number: PT3_AFLD; Version Number: 02 
29 
 
 
 
 
Revision of 
sample size 
calculation 
Preparation 
and 
dissemination 
of 
recruitment 
materials 
(poster/flyer, 
bunting, 
Facebook 
page, 
Facebook 
ads, etc) 
Recruitment 
of subjects 
Tocotrienol 
and placebo 
samples 
arrival 
Progress 
report 
presentation 
at the 
department 
Subjects’ 
Visit 1 
Subjects’ 
visit 2 
Subjects’ 
visit 3 
Final data 
collection 
Data analysis 
Final report 
writing 
Final (result) 
presentation 
at the 
department 
Submission 
of final 
manuscript 

Protocol Number: PT3_AFLD; Version Number: 02 
30 
 
 
INFRASTRUCTURE OF TRIAL 
 
Facility 
Site 
Subject screening, and consultation 
Laboratory of Pharmacology Department, 
Faculty of Medicine 
Body anthropometry measurements 
Laboratory of Pharmacology Department, 
Faculty of Medicine 
Dual-energy X-ray absorptiometry 
Laboratory of Pharmacology Department, 
Faculty of Medicine 
Milliplex reader 
Prima Nexus Sdn. Bhd 
ELISA reader 
Laboratory of Anatomy Department, 
Faculty of Medicine 
Automated blood biochemistry analyser 
Pathlab Sdn. Bhd 
High performance liquid chromatography 
Laboratory of Pharmacology Department, 
Faculty of Medicine 
Fibroscan 
Hospital Canselor Tuanku Mukriz 
 
 
 
DETAILED BUDGET 
 
Description 
Price/unit 
(RM) 
Unit 
Total price 
(RM) 
Research materials/ Consumables 
Tocovid SupraBio™ 200mg capsules: 
6 months x RM 4 x 2 x 13 subjects 
18720 
1 
18720 
Placebo capsules: 
6 months x RM 1 x 2 x 13 subjects 
4680 
1 
4680 
Multiplex immunoassay kits for biomarkers: 
3 sampling points for all subjects (26 subjects) 
Human Cytokines Profiling 
Selected Platform: M 
Sample type: Serum / Plasma 
sCD40L, EGF, FGF-2, Flt-3 ligand, Fractalkine, 
G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-1α, 
IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, 
IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL- 
13, IL-15, IL-17A, IP-10, MCP-1, MCP-3, MDC 
(CCL22), MIP-1α, MIP-1β, PDGF-AB/BB, 
RANTES, TGF-α, TNF-α, TNF-β, VEGF, 
Eotaxin/CCL11, PDGF-AA 
2160 
26 
56160 
Immunoassay kits: 
3 sampling points for all subjects (26 subjects). 
6 kits × 3 sampling points (Each sample = RM 80) 
Technical triplicate 
1250 
26 
32500 

Protocol Number: PT3_AFLD; Version Number: 02 
31 
 
 
 
Acetyldehyde, 
malondialdehyde, 
total 
antioxidant capacity, total superoxide dismutase, 
glutathione peroxidase, glutathione reductase 
HPLC Analysis: 
3 sampling points for all subjects (26 subjects). 
(RM 150 per Analysis) 
450 
26 
11700 
Plastics consumables - Blood tubes (heparin, 
EDTA, plain tubes), gloves, syringes and surgical 
mask, eppendorf tubes and tips 
• Cyrovials, 1.5 ml microcentrifuge tubes 
• 15 ml and 50 ml centrifuge tube 
• 10 ul, 100 ul, 200 ul and 1,000 ul pipette 
tips 
8000 
1 
8000 
Cleaning consumables 
• Glove, hand soap, cleaning liquid, wipe, 
trash bag 
5000 
1 
5000 
Stationary 
• All stationaries needed for the whole 
period of trial 
• Photocopy 
of 
questionnaires, 
advertisements, case report forms and 
subject diaries 
2000 
1 
2000 
Total 
138760 
Services 
Clinician Fee 
200 
26 
5200 
Phlebotomy: 
5 sessions × 5 sampling points x 2 phlebotomists 
5600 
1 
5600 
Blood biochemistry: 
26 samples × 3 sampling points x RM 300 
• 
Full Blood Count 
o Differential 
count: 
polymorphs, 
lymphocytes, monocytes, eosinophils, 
basophils 
o Absolute 
count: 
polymorphs, 
lymphocytes, monocytes, eosinophils, 
basophils, ESR and platelet count 
o Film: RBC, WBC, platelet 
• 
Blood Glucose 
• 
Lipid Profile (total cholesterol, LDL, 
HDL, triglycerides, non-HDL cholesterol) 
• 
Liver function: bilirubin, ALP, AST, 
ALT, GGT, albumin, total protein 
• 
INR and PT 
630 
26 
16380 
Fibroscan: 
3 sampling points x RM350 
550 
26 
14300 
Clinic Registration: 
3 Times x RM 25 each 
75 
26 
1950 

Protocol Number: PT3_AFLD; Version Number: 02 
32 
 
 
 
CRO service: 
• Pre-study set-up activities, trial master file 
– RM 10,000 
• Protocol set-up, SOP optimization, SOP 
revision and review – RM 10,000 
• Study familiarization, in house briefing, 
site initiation visit, site monitoring visit, 
site closeout visit – RM 15,000 
• Project management according to the 
project timeline 
(from site initiation visit until site closeout 
visit) and monitoring activities for 24 
months – RM 25,000 
60000 
1 
60000 
Full body Dexa Scan 
Lipid scan 
3 sampling points for all subjects (26 subjects) 
650 
26 
16900 
Insurance 
20000 
1 
20000 
Statistician 
10000 
1 
10000 
Questionnaire platform 
60 
26 
1560 
Total 
151890 
Manpower 
Study coordinator/student (RM2300/month) x 24 
months 
55200 
1 
55200 
Total 
55200 
Miscellaneous 
Publication Fee 
8000 
3 
24000 
Conference Registration 
5000 
2 
10000 
Total 
34000 
SUBTOTAL (RM) 
379850 
UKM & ExcelVite Sdn Bhd (134640+172240) 
181948 
GRANT TOTAL (MPOB) 
197902