Effect of Consuming a Cimarrón Bean Extrudate Solution on Platelet Function and Postprandial Biochemical Parameters
Phase: NA
Conditions: Postprandial Glucose, Platelet Activation
Sponsor: University of Talca

 
1 
Clinical Trial Protocol 
NCT Number: NCT07043829 
Protocol ID: PCSAEPF29-2021 
Official Title: Effect of Consuming a Cimarrón Bean Extrudate Solution on Platelet 
Function and Postprandial Biochemical Parameters.  
Sponsor: University of Talca, Chile.  
Principal Investigator: PhD. Iván Francisco Palomo González 
Ethics Approval: Folio 29-2021 (Approved) 
Version Date: June 19th, 2025 
 
1. Background  
The common bean (Phaseolus vulgaris), a legume with high nutritional value, has been 
shown to have beneficial effects on cardiovascular health. Its bioactive components, 
including proteins, polyphenols, and fiber, contribute to the modulation of platelet 
activity. Evidence from previous studies indicates that regular bean consumption may 
reduce inflammatory markers, oxidative stress, and overall cardiovascular risk, 
positioning it as a key functional food for the prevention and management of chronic 
diseases. Incorporating beans into the regular diet could represent a practical strategy 
to improve cardiovascular health and promote healthy eating habits in the general 
population. The primary objective of this trial is to obtain preliminary evidence on the 
acute capacity of the extrudate to reduce thrombotic activity under controlled 
conditions, minimizing potential confounding factors that could happen when using a 
delivery vehicle. 
 
2. Objectives 
 
General Objective 
To analyze the acute effect of consuming a Phaseolus vulgaris L. var. Cimarrón 
extrudate solution on platelet function, postprandial blood glucose, and total plasma 
polyphenol levels.  
 
Specific Objectives 
1. To evaluate the effect of consuming SPVCE (Solution Phaseolus vulgaris L var. 
Cimarrón extrudate) on platelet function, assessed by PFA and platelet 
reactivity.  
2. To determine the change in postprandial blood glucose after consuming SPVCE.  

 
2 
3. To quantify the effect of consuming SPVCE in postprandial total plasma 
polyphenol levels. 
 
3. Study Design 
 Model Description:  A single-arm, open-label, acute, non-randomized interventional 
study in which all participants consumed a single dose of a functional solution 
containing 10 grams of Phaseolus vulgaris L var. Cimarrón extrudate dissolved in water. 
No placebo or comparator group is included. Outcomes are measured before and after 
the intervention within the same participants to assess acute changes in platelet 
function, postprandial glycemia, and total plasma polyphenol levels.  
 
4. Participants 
 
Recruitment: Participants were recruited through posters, flyers, and announcements 
distributed in various university facilities, inviting the community to participate. 
Individuals expressing interest received a supplementary information sheet about the 
study and completed an initial digital survey for preliminary data collection. After 
reviewing the information, any questions regarding the study procedures were 
addressed. Written informed consent was obtained from each participant, and a copy 
was provided for their records. 
 
Inclusion Criteria 
1. Adults 20-59 years old, male or female.  
2. Not taking medications affecting platelet function. 
3. Voluntary participant, willingness to participate, and provide written informed 
consent 
Exclusion Criteria: 
1. Documented food allergies, especially to legumes or beans 
2. Diagnosed with liver, kidney, autoimmune diseases, or severe illnesses such as 
cancer. 
3. Diagnosed gastrointestinal diseases, including inflammatory bowel disease 
(e.g., Crohn’s disease, ulcerative colitis), celiac disease, or any condition 
causing chronic gastrointestinal symptoms such as malabsorption, persistent 
diarrhea, or gastrointestinal bleeding 

 
3 
4. Use medication known to alter platelet function like antiplatelet agents (aspirin, 
clopidogrel); non-steroidal anti-inflammatory drugs (NSAIDs, ibuprofen, 
naproxen); Anticoagulants (Warfarin, heparin, direct oral anticoagulants like 
rivaroxaban or apixaban); Selective serotonin reuptake inhibitors (SSRIs) and 
other drugs known to impact platelet aggregation or hemostasis 
5. Intervention 
Phaseolus vulgaris L. var. Cimarrón Extrudate composition  
Phaseolus vulgaris L. var. Cimarrón was processed using a hammer mill extruder 
equipped with a 6 mm screen, operating at an extrusion temperature of 120 °C, with a 
product exit temperature of 110 °C. Moisture content of the extruded flour was 
determined with a TK100 moisture sensor and measured at 9.5%. Processing was 
conducted at the University of Concepción, Chile. The proximate physicochemical 
characterization and an organoleptic evaluation performed by ANALAB S.A. (Santiago, 
Chile), indicated a beige to light brown coloration, an aroma reminiscent of nuts with a 
toasted note, a mild flavor with slight natural sweetness, and a fine, uniform, floury 
texture, smooth to the touch. The nutritional profile of the extruded flour is summarized 
in Table 1 (proximate and physicochemical analysis) and Table 2 (nutrition facts).  
 
Table 1. Nutritional analysis carried out on Phaseolus vulgaris L. var. “Cimarrón” 
Bioactive Compound 
Total Protein (g/100 g DM-Dumas) 
29.1 
Soluble Protein (mg/mL) 
3.8 
Amino acids (mg/g Protein) 
Gly.His 
55.3 
Thr 
34.3 
Val 
36.9 
Met 
7.4 
Ile 
31.5 
Leu 
58.4 
Phe 
45.3 
Lys 
54.4 
EAAs 
323.4 
Asp 
78.1 
Glu 
115.0 
Ser 
48.1 

 
4 
Arg 
58.7 
Ala 
34.4 
Pro 
34.0 
Tyr 
24.8 
NEAAs 
394.3 
Macro elements (g/10 g DM) 
P 
4.6 
K 
15.8 
Ca 
1.1 
Mg 
1.9 
Micro elements (mg/10g DM) 
Mn 
14.0 
Zn 
45.3 
Cu 
10.3 
Fe 
105.0 
B 
18.0 
Sugar 
RFO (mmol/100g DM) 
5.0 
Sucrose (g/ 100g DM) 
2.3 
Antioxidant capacity 
TPC (mg GA/ 100g DM) 
74.5 
ORAC (umol  TE /100g DM) 
9474.9 
 
Table 2. Nutrition facts- Phaseolus vulgaris L. var. Cimarrón Extrudate 
 
Nutrition Facts 
100 g 
Portion  (10 g) 
Calories (kcal) 
227.9 
22.79 
Total Fat (g) 
0.4 
0.04 
Sodium (mg) 
0.62 
0.062 
Total Carbohydrate  (g) 
44.29 
4.29 
Dietary Fiber  (g) 
17.83 
1.78 
Total Sugars (g) 
6.82 
0.68 
Includes 0g  Added Sugars 
0 
0 
Protein (g) 
29.1 
2.5 
 
 

 
5 
Time line 
Participants were scheduled in advance and instructed to arrive after an overnight fast 
of 10–12 hours. Upon arrival, they completed a digital basic health assessment 
questionnaire covering medical history, current health status, dietary and nutritional 
habits, tobacco and alcohol consumption, education level, and socioeconomic status. 
A phlebotomist inserted an intravenous line to maintain venous access for blood 
collection, and participants remained at rest throughout the intervention. 
1. At time 0 (baseline), blood samples were collected in two tubes containing 3.2% 
sodium citrate (Ez MedLab Co., Zhejiang, China) for baseline platelet function 
analysis (platelet reactivity and PFA) and quantification of total plasma 
polyphenols, and in one serum separator tube (Ez MedLab Co., Zhejiang, China) 
for baseline glucose measurement. 
2. Two hours after SPVCE consumption, an additional serum separator tube was 
collected for postprandial glucose measurement and quantification of total 
plasma polyphenols. 
3. Six hours after SPVCE consumption, blood samples were again collected in two 
sodium citrate tubes for postprandial platelet function analysis (platelet 
reactivity and PFA) and quantification of total plasma polyphenols. 
Preparation and administration details 
 
1. Fasting state: about 10 to 12 hours of nocturnal fasting; the participant can 
drink water while in the fasting state.  
2. Administration of SPVCE: The participant has 10 to 15 minutes to drink the 
SPVCE. The research team made the SPVCE, and the self-team supervised the 
administration.  
 
7. Outcomes 
 
Primary endpoint 
 
- 
Platelet function measured by flow cytometry assay: binding fluorescein 
isothiocyanate–conjugated anti-fibrinogen antibody (FITC) and PE-conjugated 
anti–P-selectin (PE) antibody after stimulation with ADP (0.03-30µM), collagen-
related peptide (CRP; 0.003-3µg/mL), and thrombin receptor activator peptide 
6 (TRAP-6; 0.05-15µM). Measurements performed using a FACSLyric flow 

 
6 
cytometer (BD, San Diego, USA). Analysis performed by Thrombosis Center 
Research, Talca, Chile.  
 
Secondary endpoints 
 
1. Platelet Function (Closure Time): Measured by PFA-200 (Collagen+ADP 
cartridge, Siemens Healthcare, Marburg, Germany) at 0 h and 6 h on whole 
blood. Analysis performed by Thrombosis Center Research, Talca, Chile.  
2. Baseline and postprandial Glucose: Serum glucose measured by GOD-POD 
(glucose oxidase-peroxidase) enzymatic colorimetric method using BS300 
Mindray (Shenzhen, China) at time 0h (baseline) and 2 h postprandial. Analysis 
performed by Clinic laboratory Talca, Chile.  
3. Plasma total polyphenols concentration: Prussian blue colorimetric method, 
measured in plasma at 0 h, 2 h, and 6 h.  Analysis performed by Thrombosis 
Center Research, Talca, Chile.  
 
Safety endpoints: Adverse events, vital signs, and routine clinical laboratory safety 
tests. Any clinically relevant abnormality will be recorded and reported per the ethics 
committee’s requirements.  
 
Sample handling:  specify tubes and processing:  
 
1. Flow cytometry platelet reactivity: Whole blood anticoagulated with 3.2% 
sodium citrate processed according to Thrombosis Center SOPs, centrifuged at 
1200 rpm for 12 minutes at T° ambient, obtaining platelet-rich plasma (PRP). 
Agonist time-to-stimulation: 10 minutes at ambient temperature and staining 
20 minutes at ambient temperature 
2. PFA-200: Whole blood anticoagulated with 3.2% sodium citrate processed to 
Thrombosis Center SOPs in recommended cartridge tube tested within 
manufacturer’s timeframe: 30 minutes after sampling at 15-25 °C.  
3. Glucose: Serum separator tubes, centrifuge at 3000 rpm for 8 minutes at 
ambient temperature, and analyze per Clinic Lab. Talca SOP.   
4. Total polyphenols: Whole blood anticoagulated with 3.2% sodium citrate 
processed to Thrombosis Center SOPs, centrifuged at 3000 rpm for 8 minutes 
at ambient temperature, obtaining platelet-poor plasma (PPP), aliquot and 
store at -80°C until assay if not run immediately.  
 

 
7 
Laboratories: Thrombosis Center Research, University of Talca (Platelet function 
assays and total polyphenols), and Clinic Laboratory Talca (glucose).  
 
Visit schedule & sample timing  
 
1. Day 0 (Treatment): Participants arrived in fasting conditions (³8 hours).  
2. Baseline samples at T0h: PFA-200, flow cytometry (reactivity), serum glucose, 
plasma total polyphenols. Administer 10 g extrudate in 500 ml water 
(supervised). 
3. Post-dose samples: T2h-serum glucose, plasma total polyphenols; T6h-PFA-
200 Flow cytometry, plasma total polyphenols. Record AEs and vital signs 
throughout.  
 
Discharge/follow-up: The participant has signed a request for sample storage and 
subsequent use of the samples in future research. Depending on the option selected 
(Acceptance or Rejection), the samples will be stored accordingly, labeled with the 
corresponding code, or discarded following the biological sample disposal protocol of 
the University of Talca, Chile. 
 
8. Sample size and Statistical considerations  
 
Planned enrollment: 30 participants 
Statistical software: GraphPad Prism 10.1.1. 
Statistical Plan: Paired analysis comparing pre- and post-dose measures, two-sided 
p <0.05. More details in the SAP appendix.  
 
9. Criteria for Withholding and Stopping 
 
Withholding Criteria (no intervention on study day): 
The investigational product will not be administered, and the participant’s study visit 
will be suspended for the remainder of that scheduled 8-hour session if any of the 
following are present at baseline (pre-dose) or detected during the immediate pre-
administration checks: 
1. Acute illness or fever on the study day. 
2. Fasting blood glucose ≥ 126 mg/dL or ≤ 60 mg/dL at baseline measurement. 
3. Blood pressure ≥ 160/100 mmHg at baseline assessment. 

 
8 
4. Self-reported moderate/severe gastrointestinal symptoms within 24 hours 
before the visit (e.g., vomiting, severe diarrhea, uncontrolled nausea). 
5. Any acute condition deemed by the investigator to increase participant risk or 
compromise data integrity. 
Action: If withholding criteria are met, the product will not be given during that 8-hour 
session. The study physician documents the reason, provides appropriate care/advice, 
and notifies the principal investigator. Rescheduling of the visit may occur only 
according to the trial’s rescheduling policy (if allowed by the protocol); otherwise, the 
visit is recorded as not completed. 
Stopping Criteria (Immediate cessation during session): 
The study intervention will be stopped immediately for an individual participant during 
the 8-hour session if any of the following occur: 
1. Any serious adverse event (SAE) possibly, probably, or related to the product 
(e.g., anaphylaxis, respiratory distress, hospitalization). 
2. Acute clinically significant change in monitored clinical parameters judged to 
be related to the product (for example: new onset chest pain, severe 
hypotension or hypertension not responsive to initial measures, severe 
arrhythmia). 
3. New laboratory or point-of-care result during the visit indicating acute organ 
injury (e.g., markedly abnormal glucose) that the investigator deems unsafe to 
continue. 
4. Severe or worsening gastrointestinal reaction during the session (e.g., 
persistent vomiting, dehydration) that requires medical intervention. 
5. Participant requests to stop or withdraw consent at any time. 
Action: On stopping, immediate medical evaluation is provided, the event is 
documented in the case report form, the principal investigator is notified, and 
appropriate medical referral (including emergency care) is arranged if needed. The 
participant’s further participation in subsequent visits will be decided according to the 
protocol and safety assessment. 
Additional operational details  
1. Who decides: the study physician and/or principal investigator make 
withholding/stopping decisions. Site staff follow their instructions. 

 
9 
2. Monitoring during the 8-hour visit: vital signs and symptom checks per protocol 
schedule; any trigger must be recorded with time and action taken. 
3. Reporting: all SAEs and stopping events are reported per the trial safety 
reporting plan and institutional requirements. 
4. Re-enrollment/rescheduling: any decision to allow the participant to return for 
another 8-hour session must follow the protocol’s eligibility and rescheduling 
rules and occur only after medical clearance. 
10. Data management and Ethics 
Records will be kept confidential by the responsible researcher. Participant names or 
identifiers will not be used. Blood samples will be labeled with codes and will not be 
linked to personal data. Identity and health information will not be disclosed and will 
remain confidential. Study approved by the University of Talca Scientific Ethic 
Committee (Folio 29-2021).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
10 
 
 
 
 
Statistical Analysis Plan (SAP) 
NCT Number: NCT07043829 
Protocol ID: PCSAEPF29-2021 
Official Title: Effect of Consuming a Cimarrón Bean Extrudate Solution on Platelet 
Function and Postprandial Biochemical Parameters.  
Sponsor: University of Talca, Chile.  
Principal Investigator: PhD. Iván Francisco Palomo González 
Ethics Approval: Folio 29-2021 (Approved) 
Version Date: June 19th, 2025 
 
1. Introduction: This Statistical Analysis Plan (SAP) outlines the methods and 
procedures for analyzing the data from the clinical trial investigating the acute effects 
of a Cimarrón Bean Extrudate solution on platelet function and postprandial 
biochemical parameters. 
 
2. Analysis Populations 
- 
Per-protocol (pp) population: Includes all participants who complete the 8-
hour observation period and have valid measurements for the outcomes of 
interest without major protocol deviations (e.g., incomplete fasting, incomplete 
product ingestion, prohibited medication use).  Sensitivity analyses will be 
conducted in the PP population to verify robustness. 
- 
Available Case Analysis: An analysis based on all available data pairs for each 
outcome; participants with missing paired data will be excluded from that 
specific analysis. 
3. Software: All statistical analyses will be performed using GraphPad Prism version 
10.  
4. General Analysis Design 
- 
The planned sample size is n = 30. 
- 
Measurements were obtained at baseline (0 h, pre-ingestion) and at 2 h and 6 h 
after consumption of the extrudate solution.  

 
11 
- 
Continuous variables will be expressed as mean ± standard deviation (SD) when 
approximately normally distributed, or median [interquartile range, IQR] when 
non-normal.  
- 
Categorical variables will be presented as frequency and percentage. For each 
time point, the mean/median and change from baseline will be reported. 
5. Assessment of Normality and Homogeneity 
Normality of data will be evaluated using the Shapiro–Wilk test, and homogeneity of 
variances will be assessed using Levene’s test where applicable. All statistical tests 
will be two-sided with the significance level set at p < 0.05. 
6. Primary Endpoint 
- Platelet Function (Flow Cytometry – Dose–Response Analysis): For each activation 
marker (P-selectin and fibrinogen) and agonist (ADP, TRAP-6, CRP), dose–response 
curves will be generated using non-linear regression to determine EC₅₀, maximum 
response, minimum response, and response span per participant. Comparisons of 
these curve-derived parameters between baseline (0 h) and 6 h post-ingestion will be 
performed. 
The analysis will proceed as follows: For curve-derived parameters (EC₅₀, maximum, 
minimum, and response span), normality of paired differences between baseline and 
6 h will be assessed using the Shapiro–Wilk test. If data are normally distributed, paired 
t-tests will be used for comparisons; if non-normal, Wilcoxon signed-rank tests will be 
applied. For the dose–response curves themselves, a two-way ANOVA will be 
performed to evaluate the effects of treatment (baseline vs. 6 h) and agonist 
concentration. If assumptions for parametric analysis are met, Sidak’s multiple 
comparisons post hoc test will be applied. If assumptions are not met and the analysis 
is non-parametric, a corresponding non-parametric alternative to two-way ANOVA will 
be used, followed by Dunn’s multiple comparisons post hoc test. Data will be 
presented as mean ± SD for normally distributed data and median [IQR] for non-normal 
data 
7. Secondary Endpoints 
1. Platelet Function (Closure Time – PFA-200): Paired differences between 
baseline (0 h) and 6 h will be analyzed after testing for normality with the 

 
12 
Shapiro–Wilk test. Normally distributed data will be compared using a paired t-
test, and non-normal data using the Wilcoxon signed-rank test. 
2. Baseline and Postprandial Glucose: Differences between 0 h and 2 h will be 
analyzed using the same normality testing and paired statistical approach (t-
test or Wilcoxon). 
3. Plasma Total Polyphenols Concentration: Repeated measures at 0 h, 2 h, and 
6 h will be analyzed using one-way repeated measures ANOVA if normality 
assumptions are met, followed by an appropriate post hoc test (e.g., Tukey’s or 
Sidak’s) for parametric data. If non-parametric, the Friedman test will be used, 
followed by Dunn’s multiple comparisons post hoc. 
8. Missing Data Handling 
- 
The primary analysis will be conducted as an available-case analysis: only 
complete pairs of baseline and post-dose measurements will be included for 
paired comparisons, excluding any missing pairs. The number of pairs included 
will be reported for each analysis. 
- 
If missing data in the primary variable is ≤ 5%, only available-case analysis will 
be performed without imputation. 
- 
If missing data exceeds 5% or is suspected not to be missing completely at 
random, sensitivity analyses will be conducted using appropriate methods such 
as: 
- 
Linear mixed models for repeated measures (assuming Missing At Random), or 
- 
Multiple imputation (e.g., predictive mean matching) and comparison with 
available-case results. 
- 
Participants lacking estimable parameters for a speciﬁc variable will be 
excluded from analyses involving that parameter; actual sample sizes will be 
reported accordingly. 
9.  Multiple Comparisons Correction 
No formal correction for multiple comparisons will be applied. The primary endpoint 
analyses are limited to a small set of predefined parameters (EC₅₀, max, min, span) for 
key activation markers and agonists. Secondary endpoint analyses will be considered 
exploratory. Given the focused nature of the primary analyses and the sample size, 
results will be interpreted with caution regarding multiplicity. Where applicable, post 
hoc tests controlling type I error (e.g., Sidak’s or Dunn’s tests) will be used following 
ANOVA or non-parametric analyses. 

 
13 
10. Safety and Adverse Events 
Gastrointestinal symptoms and any adverse events will be summarized as frequencies 
and percentages. Where applicable, events will be reported by post-ingestion time 
point. 
11. Sample Size and Power 
The planned sample size of n = 30 is based on feasibility and prior experience in acute 
postprandial studies. With n = 30 for paired comparisons, the study has approximately 
80% power to detect a medium effect size (Cohen’s d ≈ 0.5) at α = 0.05 (paired t-test). 
12. Presentation of Results 
Results will be presented in tables and figures. Tables will include descriptive statistics 
per time point, changes from baseline with 95% confidence intervals, and adjusted p-
values where applicable. Figures will present time–response profiles, dose–response 
curves, and bar plots of key comparisons.