Efficacy of a Tomato Pomace Extract for Inhibiting Platelet Aggregation
Phase: NA
Conditions: Cardiovascular Diseases
Sponsor: Centro de Estudios en Alimentos Procesados

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Ascending dose tolerance study                             Version 1                                           13 June 2016 
Platelet aggregation study                                   Study protocol 
 
 
2
 
Table of Contents 
 
1. Summary 
2. Scientific background 
3. Ascending dose human tolerance study 
3.1 
Study objectives 
3.2 
Study design 
3.3 
Inclusion criteria 
3.4 
Exclusion criteria 
3.5 
Recruitment strategy 
3.6 
Clinical screening 
3.7 
Preparation of tomato pomace drinks 
3.8 
Intervention procedure 
3.9 
Stop criteria 
3.10 
Participant randomization 
3.11 
Participant payment 
4. Effects of ingesting a tomato pomace extract on platelet aggregation 
4.1 
Study hypothesis 
4.2 
Study objectives 
4.3 
Study design 
4.4 
Inclusion criteria 
4.5 
Exclusion criteria 
4.6 
Recruitment strategy 
4.7 
Clinical screening 
4.8 
Participant randomization 
4.9 
Preparation of tomato pomace drinks 
4.10 
Intervention procedure 
4.11 
Treatment compliance 
4.12 
Participant payments 
4.13 
Assessment of platelet function 
4.14 
Power calculations and statistical analysis 
5. Ethical considerations 
5.1 
Reporting of adverse events 
5.2 
Liability insurance 
5.3 
Informed consent 
5.4 
Participant fasting 
5.5 
Food safety 
5.6 
Blood sampling 
5.7 
Amendments to study protocol 
5.8 
Confidentiality 
6. References 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
  

Ascending dose tolerance study                             Version 1                                           13 June 2016 
Platelet aggregation study                                   Study protocol 
 
 
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Protocol 
 
Efficacy of a tomato pomace extract for inhibiting platelet aggregation 
 
 
1. Summary 
Evidence from human intervention trials and mechanistic studies suggests that 
tomato and tomato based products are associated with a reduction in CVD risk. The 
mechanism by which this protective effect occurs is not clearly understood but 
research has focused on its potential to modulate platelet function. In a single-blind, 
randomized, parallel design human intervention trial (3-groups), we aim to recruit 99 
participants (33/group) to ingest an orange flavoured beverage containing different 
doses of a tomato pomace extract (1.0 and 2.5 g,) or placebo control over a 5-day 
period. The acute and longer term effects of the treatments on platelet aggregation 
will be evaluated at the start and end of the intervention period. Before implementing 
this large scale human intervention trial we will also conduct a preliminary ‘ascending 
dose human tolerance study’ in a small group of participants across a ~ 3 week 
period.   
 
Dr Ivan Palomo is Chief Investigator (CI) for this trial. The trial will be conducted at 
the University of Talca by Adriana Vásquez (Research nurse), Dr Iván Palomo (CI; 
Researcher), Dr Eduardo Fuentes (Researcher) and other members of the research 
group may be required to assist. Statistical analysis of the data will be undertaken by 
Dr Henri Tapp (statistician; Institute of Food Research, UK). Tomato pomace will be 
processed and supplied by Centro de Estudios en Alimentos Procesados (CEAP). 
This study is funded by the Newton Picarte Fund. This fund supports joint projects 
between Chilean and British researchers.  
 
2. Scientific Background 
 
Epidemiological evidence supports the concept that a diet rich in fruit and vegetables 
affords protection against the onset of cardiovascular disease (CVD) [1]. The 
aetiology of CVD is multi-factorial, but it has been recognized for some time that 
platelet function is related to the risk of developing atherosclerosis [2], a precursor to 
CVD. Activated platelets play a key role in the development of atherosclerosis by 
contributing to plaque formation within blood vessels during the early stages of 
atherogenesis. Platelets become activated after binding with von Willibrand (vWF) 
factor and collagen, which are secreted in response to damaged endothelial cells [3]. 
Subsequently, collagen and vWF bind to glycoprotein receptors (GPIIb/IIIa complex) 
situated on the platelet surface [4]. The activated platelets in turn synthesize and 
secrete thromboxane A2 and intracellular ADP which, combined with conformational 
changes in the glycoprotein complex, result in platelet aggregation. Platelet hyper-
aggregation is known to be apparent in disease states such as CVD and several anti-
platelet therapies such as aspirin and glycoprotein IIb/IIIa inhibitors significantly 
reduce the incidence of coronary events [5]. In addition, healthy people (middle aged 
and older) and people under stress can also display impaired platelet activity. 
Optimal platelet function is therefore an important tool in the prevention of CVD and 
dietary solutions may be considered as one approach to maintaining cardiovascular 
health.  
 
Tomatoes and tomato based products are a rich dietary source of vitamins (C & E), 
carotenoids (β-carotene, lycopene), polyphenols (naringenin, quercetin, kaempferol) 
and folates. Evidence from human intervention trials and mechanistic studies suggest 
that tomato and tomato based products are associated with a reduction in CVD risk. 

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Whilst some reports have attributed these beneficial effects to lycopene [6, 7], others 
report that lycopene may not be associated with CVD risk [8, 9], indicating that other 
components of tomato may be responsible for the cardio-protective effects observed. 
The cardio-protective properties of tomatoes and tomato based products in humans 
include the ability to improve lipid profiles [10-12] as well as induce anti-inflammatory 
[12] and anti-platelet activities [13, 14].  
 
Industrial processing of tomatoes into sauces, purees, pastes and juices result in the 
accumulation of tomato by-products, or pomace. Historically, tomato pomace has 
been viewed as a waste product and is either disposed of or used as animal feed. 
The tomato pomace is about 5% of the processed tomato (by weight) and consists of 
tomato peel and pulp (78%) and crushed seeds (22%). The tomato pomace is 
composed of fibre (60%), sugar (27%), protein (19%), pectin (8 %), fat (6%) and 
minerals (4%). Research already conducted at the University of Talca evaluating the 
in vitro, ex vivo and In vivo effects of aqueous extracts of fresh tomatoes and tomato 
pomace on platelet function, has demonstrated marked decreases in platelet 
aggregation [15-17]. Moreover, the inhibitory effect on platelet aggregation was 
shown to be greater in tomato pomace extracts when compared with fresh tomato 
extracts [15, 17]. This is an important finding because the data indicate that industrial 
processing of tomato into pomace, may not adversely affect the anti-platelet activity 
observed in other studies and may therefore, be an important tool in the prevention of 
CVD.          
 
It is our intention to investigate the acute and longer term effects of ingesting tomato 
pomace extracts on platelet function in humans. Since the tomato pomace extract is 
not ordinarily consumed by the wider population in this processed format, we firstly 
propose to conduct a preliminary ascending dose human tolerance study in a small 
group of people. In short, the purpose of this submission is to seek ethical approval 
to conduct a two phase study.  
 
Phase 1: A preliminary ‘ascending dose’ human tolerance study of ingesting a 
tomato pomace extract; leading onto 
Phase 2: A single-blind, randomized, parallel design, human intervention trial to 
investigate the effects of ingesting different doses of the tomato pomace 
extract on platelet aggregation.  
 
Each phase of the study is described in detail below. 
 
 
3. PHASE 1 – Ascending dose human tolerance study of a tomato pomace 
extract 
 
3.1 Study objectives 
The objective of this single ascending dose study is to assess the safety and 
tolerability of the ingestion of tomato pomace extracts in humans.  
 
3.2 Study Design 
Male participants (n=15) will be recruited to ingest a tomato pomace extract once 
daily for 5-days across three different doses. In week one, five participants will ingest 
1.0 g of extract per day for 5 days. In week two, the next five participants will ingest 
2.5 g of extract per day, for five days. In week three, the next five participants will 
ingest 10 g extract per day, for five days. The 1.0 and 2.5 g doses of extract are the 
same doses we intend to investigate in phase 2. Tolerability of the tomato pomace 
ingestion will be assessed by physical measurements (blood pressure and pulse 
rate) and a gastro-intestinal well-being assessment before ingestion, and then again 

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Platelet aggregation study                                   Study protocol 
 
 
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3-h after ingestion of the tomato pomace extract on each of the 5 treatment days. 
The blood test for full blood count and biochemical profile undertaken at clinical 
screening (section 3.6) will form part of the baseline assessment profile. The same 
blood tests will be repeated 24-h after the last treatment on day 5. Figure 1 is a 
schematic overview of the study. 
 
 
Figure 1. Schematic overview of phase 1 
 
 
 
3.3 Inclusion criteria 
● Apparently healthy men aged between 18 and 26 years 
● BMI >19.5 and < 26.0 
 
3.4 Exclusion criteria 
      ● Known tomato allergy 
 ● Chronic medical conditions requiring active treatment (e.g. Cardiovascular 
    disease, diabetes, asthma) 
● Medically or self-prescribed medication  
● Kidney or liver disease/disorders 
● Gastro-intestinal disease/disorders 
● Smokers  
● Parallel participation in another research project which involves dietary 
   intervention 
● Depressed or elevated blood pressure measurements (<90/50 or 95/55 if  
    symptomatic or ≥160/100 (mmHg) 
● Any person related to or living with any member of the study team 
 
3.5 Recruitment strategy 
The study population will comprise apparently healthy men aged 18 – 26 years. A 
total of 15 participants are required to complete this phase of the study.  
 
Recruitment of participants to this study will be achieved through poster and E-mail 
advertisements (annex 1) placed across the University of Talca, inviting anyone who 
is interested in receiving information about the study to contact named researchers. 
Those expressing an interest will be provided with a participant information sheet 
(annex 2). After reading the information sheet, those wishing to take part in the study 
will be invited to the research facility for an informal meeting with a member of the 
research team who will explain the study fully, focusing on the participant’s 

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Platelet aggregation study                                   Study protocol 
 
 
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involvement. Participants will be encouraged to ask questions at this stage prior to 
making any commitment. At the end of the meeting, participants will be given ample 
opportunity to consider whether they wish to take part in the study. If, following the 
consideration period they wish to participate in the study, they will be asked to 
contact a member of the research team to arrange an appointment at the research 
facility for the pre-study clinical screening.  
 
3.6 Clinical screening 
Participants will attend the research facility following a 10-hour overnight fast but will 
be advised that they should drink water during the fasting period. On arrival at the 
research facility a member of the research team will go through the consent form 
(annex 3) with the participant and answer any questions that may arise at this stage. 
He will then be asked to sign a consent form agreeing to participate in the study. A 
copy of this form will be given to the participant to keep. A research nurse will then 
complete a basic health screening questionnaire (annex 4), measure and record 
blood pressure, pulse, height and weight and calculate body mass index. A 10 mL 
blood sample will be obtained for full blood count (hemogram) and biochemical 
profile. The biochemical profile will include: glucose, urea nitrogen, urea, total 
cholesterol, uric acid, total proteins, albumin, globulins, total bilirubin, transaminase  
GOT, GPT transaminase, GGT, lactate dehydrogenase, alkaline phosphatase, total 
calcium and phosphorous. The results of the full blood count and biochemical profile 
will be reviewed by a medical doctor (Álvaro Valdéz MD and personnel of the 
Laboratorio Talca, Centro Diagnóstico Diagonal). In the event that the medical doctor 
deems the results of the blood test to be indicative of a health problem (that may 
compromise the well-being of the participant should he take part), then he will be 
excluded from the study. In these circumstances, the participant will be advised to 
contact a medical practitioner of his choice for further treatment/investigation.  
 
Those participants meeting the basic inclusion/exclusion criteria (assessed using the 
health screening questionnaire) and with normal results of the clinical laboratory tests 
(full blood count, biochemical profile) will be eligible to participate in this study. 
Participation will occur no later than 2 weeks after clinical screening. 
 
3.7 Preparation of tomato pomace drinks. 
Tomato pomace will be processed and supplied by CEAP. In brief, chopped fresh 
tomatoes are heated to produce a pulp. The pulp is passed through a ‘pulper’ and a 
‘finisher’ before being extracted through a turbo press to produce a pomace. The 
tomato pomace is concentrated to dryness by evaporation. The pomace is then 
extracted and dried to form a powder extract. A certificate of analysis for pesticide 
and microbial safety tests on the final product is provided by CEAP (annex 5).  
 
Immediately prior to ingestion, ‘drinks’ containing the appropriate mass of tomato 
pomace extract will be prepared at the research facility as described below. The water 
used to dilute the extract will be flavoured using a commercially available orange 
flavoured powder (Kool aid) which will be diluted according to the manufacturer’s 
instructions before adding to the tomato pomace extract.  Flavoured water is 
necessary because this is how we intend to blind the participants to the test product 
in phase 2. 
 
1) Low dose pomace extract – 1 g extract will be accurately weighed before 
dissolving in flavoured water to a final mass of 100 g 
2) Medium dose pomace extract – 2.5 g extract will be accurately weighed before 
dissolving in flavoured water to a final mass of 100 g 
3) High dose pomace extract  - 10 g extract will be accurately weighed before 
dissolving in flavoured water to a final mass of 100g 

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Platelet aggregation study                                   Study protocol 
 
 
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3.8 Intervention procedure 
Participants may maintain normal dietary habits during the 5-day intervention period 
but will be instructed to refrain from drinking any alcohol. The format for phase 1 is 
intended to reflect the phase 2 study and is described below.  
 
Week 1: 5 participants will start on the lowest dose of tomato pomace extract being 
tested (1.0 g). Participants will arrive at the research facility on the morning of each of 
the 5 treatment days after fasting from 10pm the night before. During the fasting 
period, the participant may drink as much water as he needs. At the start of each 
treatment day, the research nurse will record pulse rate and blood pressure and 
complete a gastro-intestinal well-being questionnaire (Annex 6). The participant will 
then ingest the tomato pomace extract. 3-h post ingestion of the extract, the research 
nurse will once again record pulse rate and blood pressure and complete another 
gastro-intestinal well-being questionnaire after which the participant may eat and 
drink as normal. The participant will then be free to leave the research facility with 
instructions to return the following morning to repeat the procedure. 24-h after the 
final treatment on day 5, another 10 mL fasted blood sample will be obtained for full 
blood count and biochemical assessment as described in the clinical screening 
section of this protocol (section 3.6).  
 
Week 2: If no problems occur after 5-days ingestion at the lowest dose tested (see 
section 3.9 for criteria) then the next 5 participants will ingest the next incremental 
dose (2.5 g). The same procedure described for week 1 above, will be followed. 
 
Week 3:  If no problems occur in week 2 (section 3.9), then the last 5 participants will 
ingest the highest dose of extract (10 g). The same procedure described for week 1 
above will be followed. 
 
3.9 Stop criteria for dose escalation 
Blood tests will be assessed (by a medical doctor) for abnormal variations at the end 
of the intervention period by comparison with the test results obtained at clinical 
screening. Similarly the CI will evaluate the results from the gastro-intestinal well- 
being questionnaires. Table 1 is the STOP criteria in the event that abnormal 
variations in blood test results and/or gastro-intestinal problems occur.    
 
       Table 1 Stop criteria for phase 1 
 
Dose 
extract 
(g) 
 
Previous dose 
outcome 
Abnormal 
at current 
dosing 
Next step 
Abnormal 
at re-test 
Next step 
1.0 
N/A 
  ≤ 2 (a) 
 
≥ 3 
Escalate to next dose 
 
Replace and re-test 
n/a 
 
  0-1(b) 
 
≥ 2 
n/a 
 
Escalate to next dose 
 
STOP 

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Platelet aggregation study                                   Study protocol 
 
 
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      n/a - Not applicable; STOP - the dose-escalation study should be terminated.   
 
 
If evaluation of the data indicates that the pomace extract is well tolerated at the 
highest dose tested, we will move onto the phase two efficacy trial.  
 
Dr Ivan Palomo (CI) will assume responsibility for the dose escalation procedures 
and the decision to move into the phase two efficacy trial. 
 
3.10 
Participant randomization 
Participants in this ascending dose human tolerance study will ingest 1, 2.5 or 10 g 
tomato pomace incorporated into a drink, over a 5 day period.  Each treatment will be 
assigned a letter (A-C). Once all 15 participants have been recruited they will be 
block randomized to treatment by the computer programme randomization.com. The 
participants will be blinded to the treatment dose. 
 
3.11 Participant payment 
Each participant will receive an inconvenience allowance of 15,000 Chilean Peso 
upon completion of the study or pro-rata for non-completion. 
 
 
4. PHASE 2 – The Effects of ingesting a tomato pomace extract on platelet 
aggregation 
 
Upon successful completion of the preliminary ascending human dose tolerance 
study we will conduct a parallel design human intervention trial to investigate the 
effects of ingesting a tomato pomace extract on platelet aggregation. 
 
4.1 Study hypothesis 
 
Daily ingestion of a tomato pomace extract for 5 days will significantly reduce platelet 
aggregation in humans compared with a placebo control and in a dose dependent 
manner.  
 
 
 
 
 
 
 
 
2.5 
All normal 
 
 
 
 
 
1-2 abnormal (a) 
 
 
 
≥ 3 abnormal (b) 
≤ 2 
 
≥ 3 
 
 
 
≤ 2 
 
≥ 3 
 
≥ 2 
 
Escalate to next dose 
 
Replace and re-test 
 
 
 
Escalate to next dose 
 
STOP 
 
STOP 
n/a 
 
0-1 
 
≥ 2 
 
n/a 
 
n/a 
 
n/a 
n/a 
 
Escalate to next dose 
 
STOP 
 
n/a 
 
n/a 
 
n/a 
 
 
 
10 
 
 
 
 
Any 
 
 
 
 
 
 
0 
 
1-2 
 
 
 
≥ 3 
Proceed to phase 2 
 
Replace and re-test 
 
 
 
STOP 
n/a 
 
0 
 
1-2 
 
n/a 
n/a 
 
Proceed to phase 2 
 
STOP 
 
n/a 
 

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Platelet aggregation study                                   Study protocol 
 
 
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4.2 Study Objectives 
 
1) To determine the acute effect of ingesting two different doses of a tomato 
pomace extract on platelet aggregation 
2) To determine the longer term effect of ingesting two different doses of a 
tomato pomace extract on platelet aggregation 
 
4.3 Study design 
A single blind, parallel design (3-groups), placebo controlled trial will be conducted to 
assess the acute and longer term effects of ingesting a tomato pomace extract on 
platelet aggregation. Participants will be randomized to receive either: a) 1.0 g 
tomato pomace extract delivered in flavoured water b) 2.5 g tomato pomace extract 
delivered in flavoured water or c) Flavoured water (placebo control). A total of 99 
people are required to complete (n=33/group). Each group will ingest either a 
treatment or placebo once daily, for 5-days. For 14-days preceding the baseline 
assessment day and for the 5-day period of intervention, participants will completely 
exclude from the diet some foods/beverages that are known to affect platelet function 
(tomato and all tomato based products, alcohol, cocoa and cocoa products and 
green tea). To assess the acute effects of the intervention on platelet aggregation, a 
blood sample will be obtained before ingestion (D15; 0-h) and post ingestion (D15; 3-
h) of the treatment. To assess the longer term effects, further blood samples will be 
taken at the end of the intervention period (D19; 0 and 3-h). Figure 2 is a schematic 
overview of phase 2. 
 
 
Figure 2. Schematic overview of phase 2 
 
 
 
4.4 Inclusion criteria 
● Apparently healthy men aged between 18 and 26 years 
● BMI >19.5 and <26.0 
 
Recruitment / eligibility assessment
/randomization to treatment 
Day 15 
 
Restricted diet 
  
Tomato pomace or placebo once daily
 
Day 0 
Day 19  
 Fasted b lood sample for 
platelet aggregation (t=0h) 
      Ingestion of tomato pomace  
  
   Fasted blood sample for 
platelet aggregation (t=3h) 
   
  
 
 
  Fasted blood sample for 
platelet aggregation  (t=0h) 
    Ingestion of tomato pomace  
   
  Fasted blood sample for 
platelet aggregation (t=3h) 
  
Day 16  
Day 17 
Day 18 
Run-in phase 

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Platelet aggregation study                                   Study protocol 
 
 
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● Platelet aggregation response corresponding to ≥ 65% 
 
4.5 Exclusion criteria 
●Known tomato allergy 
●Chronic medical conditions requiring active treatment (e.g. cardiovascular disease, 
   diabetes, asthma) 
● Gastro-intestinal disease/disorders 
● Smokers 
● Medically prescribed medication known to affect platelet function 
● Self-prescribed medication known to affect platelet function (e.g. aspirin and 
   non-steroidal anti-inflammatory drugs) unless participant is willing to give up. 
● Bleeding disorders (e.g. haemophilia) 
● Dietary supplements judged to affect study outcome 
● Parallel participation in another research project which involves dietary intervention 
● Blood donation within 16 weeks prior to the study 
● Depressed or elevated blood pressure measurements (<90/50 or 95/50 if  
   symptomatic or ≥ 160/100 mmHg) 
● Any person related to or living with any member of the study team 
 
4.6 Recruitment strategy 
The study population will comprise apparently healthy men aged 18 – 26 years. A 
total of 99 participants are required to complete this trial. However, recruitment for 
this study will only cease once we are confident that the required number of 
participants will complete the study. 
 
Recruitment of participants to this trial will be achieved through poster and E-mail 
advertisements (annex 7) placed across the University of Talca, inviting anyone who 
is interested in receiving information about the study to contact named researchers. 
Those expressing an interest will be provided with a participant information sheet 
(annex 8). If insufficient numbers of people are recruited from the University of Talca 
we may place advertisements in the local media.  
 
After reading the information sheet, those interested in taking part will be invited to 
the research facility for an informal meeting with a member of the research team who 
will explain the study fully, focusing on the participant’s involvement. Participants will 
be encouraged to ask questions at this stage prior to making any commitment. At the 
end of the meeting, participants will be given ample opportunity to consider whether 
they wish to take part in the study. If, following the consideration period they wish to 
participate in the study, they will be asked to contact a member of the research team 
to arrange an appointment at the research facility for the pre-study clinical screening.  
 
4.7 Clinical screening 
Clinical screening for this trial will be conducted as described in section 3.6. The only 
addition will be the assessment of platelet aggregation. This will be assessed by the 
research team and those participants not meeting the platelet aggregation response 
range defined in section 4.4 will be excluded from the trial.  
 
4.8 
Participant randomization 
Participants in this parallel design study will ingest 1.0 or 2.5 g tomato pomace 
extract incorporated into a drink or a placebo control drink, over a 5 day period.  Each 
treatment will be assigned a letter (A-C). Each time a participant is successfully 
recruited onto the study, he will be assigned the next allocated treatment. 
Randomization to treatment will be assigned by the computer programme 
randomization.com. The participants will be blinded to the treatments. 
 

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Platelet aggregation study                                   Study protocol 
 
 
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4.9 Preparation of tomato pomace drinks 
The tomato pomace that will be used in phase 2 is that described in section 3.7.  
Immediately prior to ingestion, ‘drinks’ containing the appropriate mass of tomato 
pomace extract will be prepared at the research facility as follows: 
 
Low dose pomace extract – 1.0 g powder will be accurately weighed before 
dissolving in flavoured water to a final mass of 100 g 
High dose pomace extract – 2.5 g powder will be accurately weighed before 
dissolving in flavoured water to a final mass of 100 g 
Placebo control   - 100 g flavoured water 
 
Flavoured water is necessary to ensure that the participants are blinded to the test 
product consumed.  
 
4.10 
Intervention procedure 
The test arm will comprise a 5 day intervention period which will be preceded by a 
run-in phase of 14 days. For standardisation purposes all trial procedures between 
days 15 – 19 will be conducted in the morning time following an overnight fast.  A test 
arm is described below.  
 
Days 1-14 – During the 14-day ‘run-in’ period, participants will be instructed to avoid 
foods and beverages known to significantly contribute to anti-platelet activity. A 14-
day period is thought to be sufficient as this comprises the average lifespan of 
platelets (~ 10 days) and allows for equilibration to dietary change. The foods and 
beverages to be avoided include tomato and all tomato based products, alcohol, 
cocoa and cocoa based products and green tea. In the event that a participant self-
prescribes medication for minor illness during this time period, he will be instructed to 
contact a member of the research team who will assess the impact of the medication 
on platelet function and decide whether or not the run-in period should be re-
scheduled.  
 
Day 15 – Participants will arrive at the research facility on the morning of day 15 
having fasted from 10 pm the night before. Prior to conducting any clinical 
procedures the participant will be asked some basic health related questions (Annex 
9) to establish whether or not any changes in medical conditions/medications etc. 
have occurred since their last visit and that they are well enough to proceed with the 
study day assessment. A baseline (t=0h) fasted blood sample (15 mL) will be 
collected by venepuncture. Once collected, participants will ingest the allocated 
treatment drink. A further blood sample will be obtained 3-h post ingestion (15 mL) to 
assess the acute effects of tomato pomace ingestion on platelet aggregation. At the 
end of the assessment period participants will be free to eat and drink ad libitum.  
 
Days 16 – 18 - Participants will return to the research facility each morning having 
fasted from 10 pm the night before. The allocated treatment will be prepared fresh 
each morning by a member of the research team and ingested by the participant 
whilst in the research facility. On departure, the participants will be instructed to 
continue avoiding the foods and beverages known to significantly contribute to 
platelet activity. 
 
Day 19 – Participants will return to the research facility on the morning of day 19 
having fasted from 10 pm the night before. Prior to conducting any clinical 
procedures the participant will again be asked some basic health related questions 
(Annex 9) for the reasons already described. A blood sample (15 mL) will be 
collected by venepuncture. The participant will once again ingest the allocated 

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treatment drink and a further fasted blood sample (15 mL) collected 3-h later.  This 
completes participation in the trial. 
 
In the unlikely event that a participant is unable to attend the pre-scheduled 
assessment days (e.g. minor illness) they will be given the opportunity to repeat the 
treatment at a later date. The total blood volume collected from each participant is 85 
ml (incl. screening sample).   
 
4.11 Treatment compliance 
Each participant will ingest the allocated treatment at the research facility. 
 
4.12 Participant payments 
Each participant will receive an inconvenience allowance of 15,000 Chilean Peso 
upon completion of the study or pro-rata for non-completion. The inconvenience 
payment has been calculated as follows: 2500 Chilean Peso per study blood sample 
(x4 samples=10,000) and 5,000 Chilean Peso for compliance with dietary 
restrictions. 
 
4.13 Assessment of platelet function 
Whole blood will be collected by venepuncture directly into 3.2% sodium citrate tubes 
(9:1 v/v) after discarding the first 2.0mL of the draw. Tubes will be centrifuged at 240 
x g for 10 mins to obtain platelet-rich plasma (PRP). After aliquoting a portion of the 
PRP for platelet count (hematologic counter) the tubes will be further centrifuged at 
650 x g for 10 mins to obtain platelet-poor plasma (PPP). The PRP will be adjusted to 
200 x 109 platelets/ L with the PPP.  
 
Aliquots of the adjusted PRP (480 µL) will be pre-incubated (370C; 3 min) before the 
addition of the platelet agonists ADP (final conc. 2 and 4 µmol/L) and collagen (final 
conc. 0.5 and 1 µg/mL). Platelet aggregation will be measured by light transmission 
according to Born and Cross using a Lumi-aggregometer. The result of platelet 
aggregation (% maximal amplitude) will be determined using the software 
AGGRO/LINK.  
 
4.14 Power calculations & statistical analysis 
The primary outcome measure for this study is platelet aggregation. The sample size 
has been powered on data from a previous study reported by O’Kennedy et al [13], 
which is a study not too dissimilar to this one. In this trial, the effect size in platelet 
response between treatment (18 g tomato extract syrup) and control after stimulation 
of platelets with collagen was 17.4% with a standard deviation of 19.5%. Assuming a 
similar maximum difference between the control and the 2.5 g dose of tomato 
pomace extract, and half this difference for the 1.0 g dose of tomato pomace extract 
then to gain sufficient evidence to reject the null hypothesis of no differences 
between the 3 groups at the 5% significance level with 90% power would require 33 
participants per group to complete the trial. The data will be analysed using one-way 
between subjects analysis of variance or its non-parametric equivalent (Kruskal–
Wallis test by ranks). Post hoc comparisons between groups will use Tukey’s honest 
significant difference (HSD) multiple comparison procedure. 
 
5. Ethical considerations (Phases 1 and 2) 
 
5.1 Reporting of adverse event/reactions and serious adverse events 
Adverse events are defined as any undesirable experience occurring to a subject 
during the study, whether or not considered related to the tomato pomace extract. An 
adverse reaction is defined as all noxious and unintended responses to the tomato 
pomace extract related to any of the doses tested. The phrase responses to the 

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tomato pomace extract means that a causal relationship between the product and an 
adverse event is at least a reasonable possibility, i.e. the relationship cannot be ruled 
out. All adverse events and reactions reported by the subject or observed by the CI 
or his staff will be recorded (Annex 10). A serious adverse event (SAE) is defined as 
any untoward medical occurrence or effect that at any dose results in death, is life 
threatening (at the time of the event), requires hospitalization or results in persistent 
or significant disability or incapacity. SAE’s must be reported by the CI immediately to 
the University of Talca ethics committee and the study sponsor (CEAP). In this 
instance the trial will be immediately suspended or terminated.  
 
5.2 Liability claim 
CEAP accepts responsibility for carrying out the tests, and as such will only consider 
claims of participants for any damage suffered as a result of a guilty performance of 
CEAP. Claims arising out of participant negligence shall not be considered. Please 
note that CEAP will not fund any legal costs arising from an action unless awarded 
by a court. 
 
5.3 Informed consent 
All participants will give written informed consent before they can participate in the 
trial. Consent will be considered informed on the basis of written documentation 
(participant information sheets; annexes 2 and 8) and verbal communication 
describing the purpose of the trial, relevant trial procedures and foreseeable risks 
and benefits.  Informed consent will be obtained before any study specific procedures 
are conducted. It will be made clear to each participant that he is free to withdraw his 
consent at any time and without reason.  
 
5.4 Participant fasting 
This study requires participants to be fasted on assessment days. To reduce the 
length of the fasting period study measurements will be conducted in the morning. 
Participants will be able to drink ad libitum during the fasting period. At clinical 
screening, a blood sample will be collected for blood glucose analysis to exclude 
undiagnosed diabetic participants.      
 
5.5 Food safety 
Tomato pomace extracts will be properly stored at dry conditions and controlled room 
temperature prior to participant ingestion. Moreover, project staff will prepare drink 
samples according to the Chilean Food Health Regulations of the Ministry of Health 
to assuring food safety and hygiene standards. 
 
5.6 Blood sampling  
In this trial blood samples will be collected by venepuncture. It is possible that this 
may cause a local hematoma or bruise to occur. The procedure will be conducted by 
an experienced research nurse and this will minimize the risk. 
 
5.7 Amendments to study protocol 
Amendments are changes made to the research after a favorable opinion has been 
given by the University of Talca ethics committee. Any amendments to the study 
protocol will be notified to the University of Talca ethics committee for approval prior 
to implementation.  
 
5.8 Confidentiality 
Participants will be assigned a unique code which will be used to anonymize all 
biological samples and data arising from this trial. Anonymized information/data will 
be kept in locked cabinets at the CEAP research facility and processed and stored on 
a computer system.  Anonymized study results will be transferred to the Institute of 

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Food Research (UK) for statistical analysis. Only the researchers named in the 
approved documentation will be able to link the code with the participant. 
 
All personal information will be kept confidential and known only to the Chief 
Investigator and study personnel at the University of Talca. Personal information will 
be kept separately to coded information in locked cabinets at the CEAP research 
facility for a period of 5 years.  
 
6. References 
 
1. 
Law, M.R. and J.K. Morris, By how much does fruit and vegetable 
consumption reduce the risk of ischaemic heart disease? European journal of 
clinical nutrition, 1998. 52(8): p. 549-56. 
2. 
Michelson, A.D., Platelets. 3rd ed. 2013, London ; Waltham, MA: Academic 
Press. xliv, 1353 p. 
3. 
Ruggeri, Z.M., Platelets in atherothrombosis. Nature medicine, 2002. 8(11): 
p. 1227-34. 
4. 
Davi, G. and C. Patrono, Platelet activation and atherothrombosis. N Engl J 
Med, 2007. 357(24): p. 2482-94. 
5. 
Hennekens, C.H., Aspirin in the treatment and prevention of cardiovascular 
disease. Annu Rev Public Health, 1997. 18: p. 37-49. 
6. 
Rao, A.V., Lycopene, tomatoes, and the prevention of coronary heart disease. 
Exp Biol Med (Maywood), 2002. 227(10): p. 908-13. 
7. 
Willcox, J.K., G.L. Catignani, and S. Lazarus, Tomatoes and cardiovascular 
health. Critical reviews in food science and nutrition, 2003. 43(1): p. 1-18. 
8. 
Sesso, H.D., et al., Dietary lycopene, tomato-based food products and 
cardiovascular disease in women. J Nutr, 2003. 133(7): p. 2336-41. 
9. 
Burton-Freeman, B. and H.D. Sesso, Whole food versus supplement: 
comparing 
the 
clinical 
evidence 
of 
tomato 
intake 
and 
lycopene 
supplementation on cardiovascular risk factors. Adv Nutr, 2014. 5(5): p. 457-
85. 
10. 
Cuevas-Ramos, D., et al., Effect of tomato consumption on high-density 
lipoprotein cholesterol level: a randomized, single-blinded, controlled clinical 
trial. Diabetes Metab Syndr Obes, 2013. 6: p. 263-73. 
11. 
Silaste, M.L., et al., Tomato juice decreases LDL cholesterol levels and 
increases LDL resistance to oxidation. Br J Nutr, 2007. 98(6): p. 1251-8. 
12. 
Valderas-Martinez, P., et al., Tomato Sauce Enriched with Olive Oil Exerts 
Greater Effects on Cardiovascular Disease Risk Factors than Raw Tomato 
and Tomato Sauce: A Randomized Trial. Nutrients, 2016. 8(3). 
13. 
O'Kennedy, N., et al., Effects of tomato extract on platelet function: a double-
blinded crossover study in healthy humans. Am J Clin Nutr, 2006. 84(3): p. 
561-9. 
14. 
O'Kennedy, N., et al., Effects of antiplatelet components of tomato extract on 
platelet function in vitro and ex vivo: a time-course cannulation study in 
healthy humans. Am J Clin Nutr, 2006. 84(3): p. 570-9. 
15. 
Fuentes, E., et al., Effect of tomato industrial processing on phenolic profile 
and antiplatelet activity. Molecules, 2013. 18(9): p. 11526-36. 
16. 
Fuentes, E., et al., Protective Mechanisms of S. lycopersicum Aqueous 
Fraction (Nucleosides and Flavonoids) on Platelet Activation and Thrombus 
Formation: In Vitro, Ex Vivo and In Vivo Studies. Evid Based Complement 
Alternat Med, 2013. 2013: p. 609714. 

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17. 
Rodriguez-Azua, R., et al., Effect of tomato industrial processing (different 
hybrids, paste, and pomace) on inhibition of platelet function in vitro, ex vivo, 
and in vivo. J Med Food, 2014. 17(4): p. 505-11.