Trial of Acceptability, Tolerability and Biochemical Indicators of Hibiscus Sabdariffa L. Drink in Pregnant Women
Phase: NA
Conditions: Pregnant Woman
Sponsor: Victor Eduardo Alcantar Rodríguez

 
 
 
  
 
 
 
 
RESEARCH PROTOCOL  
NCT ID Not yet assigned 
 
“Trial of acceptability, tolerability and biochemical indicators of Hibiscus sabdariffa 
L. drink in pregnant women” 
 
 
 
 
APRIL 2024 
 
 
 
 
 
 
 
 
NATIONAL POLITECHNIC INSTITUTE 
BIOTIC PRODUCTS DEVELOPMENT 
CENTER 
 
AUTONOMOUS UNIVERSITY OF 
BARCELONA 
 
 

1. INTRODUCTION  
When developing foods with potential health benefits, it is necessary to evaluate their 
acceptability and tolerability to determine whether the product will be consumed by the 
intended population. (Yang & Lee, 2018), In addition to ensuring that its consumption does 
not compromise the consumer's health, these tests allow the developer to make the necessary 
modifications to increase the likelihood of consumption. 
 
The acceptability of a product can be studied through surveys or interviews and the way to 
apply these resources is through sensory tests oriented to the consumer, these tests allow to 
obtain information about the aspects that are liked and disliked about the product in question 
(Ramírez-Nava, 2012). To achieve this, sensory analysis is used, which is defined as the 
scientific discipline used to measure, identify and interpret food characteristics perceived by 
the senses of sight, smell, taste, touch and hearing (Lawless & Heymann, 2010). Among 
consumer-oriented tests, there are two main ones: preference tests and acceptance tests. In 
preference tests, consumers are asked to indicate which sample they prefer. While in 
acceptance tests, consumers are presented with the products and asked to indicate their level 
of liking them on a scale that can be 3, 5, 7, and 9 points (Sharif et al., 2017). Acceptance 
tests are often used to compare two or more products or used on a single product to measure 
the level of liking. 
 
Pregnancy is a stage that is marked by physiological changes related to hormones that modify 
sensory perception (Muluh et al., 2024), mainly affecting taste (Choo & Dando, 2017) and 
smell (Haddad-Tóvolli et al., 2023); However, this has been very little studied and most of 
the knowledge on the subject is empirical (Kölble et al., 2001), but it has now begun to be 
studied as the work conducted by Matsuda et al. (2024) who have suggested that oxytocin 
has an effect on the olfactory sensitivity of pregnant women, meanwhile Muluh et al. (2024) 
found moderate evidence of sensory changes during pregnancy, such as a reduction in the 
ability to identify odors and changes in the perception of the intensity of odors and hedonic 
responses to flavors. 
The proposed beverage to be evaluated is made entirely from natural compounds, which in 
theory should not cause gastrointestinal intolerance effects, but to reaffirm this in the 

acceptability test, a gastrointestinal tolerability questionnaire was included evaluating the 
presence or absence of symptoms such as nausea, regurgitation, vomiting, diarrhea, 
constipation and flatulence. (De Luis et al., 2015). 
 
In this work, a phase I pilot test was carried out, which consists of testing on a smaller scale 
the execution logistics planned for a higher-level project and thereby reducing the probability 
of errors in future studies (Díaz-Muñoz et al., 2020). This work was approved by the 
Research Ethics Committee of the Biotic Products Development Center of the National 
Polytechnic Institute.  
 
The study evaluated the gastrointestinal acceptability and tolerability of the proposed 
beverage developed to help control hypertensive disorders associated with pregnancy. 
Indicators such as blood pressure, blood markers of glucose, cholesterol, triglycerides and 
creatinine, and general urine test were also assessed. This trial was conducted on pregnant 
women, because this is the group to which the drink is directed and at this stage physiological 
changes occur that modify the sensory perception of food (Matsuda et al., 2024), This is why 
it is important to conduct it in this group. This trial aims to lay the groundwork for defining 
the logistics used in a double-blind, randomized, controlled clinical trial in which the 
beverage will be given for a longer period of time. This is why it is important to know the 
acceptability of the beverage, in case it is necessary to make changes to the beverage that 
allow for increased consumption (Lawless & Heymann, 2010). 
 
 
 
 
 
 
 
 
 
 

2. OBJECTIVES 
2.1. General objective 
Evaluate the acceptability, tolerability, blood pressure and biochemical indicators of a 
beverage with Hibiscus sabdariffa L. in pregnant women. 
2.2. Specific objectives 
- To evaluate the acceptability of the beverage by sensory analysis in a 4-week trial with a 7-
point hedonic scale. 
- Determine the tolerability of the beverage by the presence of gastrointestinal symptoms. 
- Evaluate biochemical indicators, blood pressure and general urine test before and after the 
beverage consumption trial. 
 
3. MATERIAL AND METHODS  
A 4-week phase I pilot trial was conducted in 10 women in their second trimester of 
pregnancy, who consumed a daily dose of H. sabdariffa beverage. Acceptability and 
tolerability were assessed. Blood pressure, urinalysis, and blood biomarkers of glucose, 
cholesterol, triglycerides, and creatinine were also measured. 
 
Figure 1 presents the experimental diagram of the beverage acceptability and tolerability test. 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Experimental diagram of the acceptability and tolerability test of a beverage with 
Hibiscus sabdariffa L. 
 
 
Acceptability and 
tolerability assay 
Week 1 
Week 2 
Week 3 
Week 4 
Acceptability and tolerability interviews  
(Once a week) 
Daily 
beverage 
consumption 
and 
adherence 
Measuring markers (start and end) 
Participants 
selection 
Criteria: 
inclusion, 
exclusion 
Signing 
informed 
consent 

3.1. Ethical considerations 
This research project complies with the International Ethical Guidelines for Research Related 
to Human Health, developed by the Council for International Organizations of Medical 
Sciences (CIOMS) in collaboration with the WHO. 
 
All participants in this trial received dignified treatment and were not discriminated against 
under any circumstances based on their ethnic or national origin, social or economic status, 
religion, opinions, or marital status, in accordance with the Federal Law to Prevent and 
Eliminate Discrimination. Participants were never recruited against their will, nor did they 
suffer any form of disapproval or retaliation if they decided not to participate or withdrew 
from the study. It was emphasized that participation was completely voluntary and that they 
could refuse to participate or withdraw from the research at any time if they so desired. 
 
The results obtained were used solely by the research team. The privacy of the participants, 
as well as the confidentiality of their personal information, was protected. All collected 
samples were identified by the participant's initials and a consecutive number, and only the 
researchers had access to their personal information. This information was kept safe with 
their file and will not be disclosed or published at any time. The results may be published for 
scientific purposes, but personal and identifying data of the participants will not be disclosed 
at any time. The results obtained were analyzed using Past software through pairwise analysis 
to determine statistical differences between the indicators measured before and after 
treatment with a significance level of P <0.05. Descriptive statistics with means, standard 
deviations, and percentages were also applied to characterize the group. 
 
The researchers of this protocol declare that there is no conflict of interest and that the 
objective is to generate knowledge that allows us to evaluate the effect of a beverage with H. 
sabdariffa, which can be used in the prevention and/or treatment of hypertensive disorders 
associated with pregnancy, as well as to determine its degree of acceptability and tolerability. 
 
 
 

3.2. Materials  
The proposed beverage was made with H. sabdariffa calyces from the Temixco cultivar in 
Morelos, and was given to participants as a powder for beverage preparation. The powder 
contained 5.7 g of powdered H. sabdariffa calyces and 5 g of standard sugar. 
 
3.3. Methods 
3.3.1. Microbiological analysis of Hibiscus sabdariffa L. calyces 
Prior to the acceptability and tolerability test of the beverage, a microbiological analysis of 
the powdered calyces of H. sabdariffa was performed, measuring mesophilic counts, total 
coliforms, molds, yeasts, Escherichia coli and Salmonella spp. to ensure the safe 
consumption of the hibiscus. All indicators were within permissible values according to 
Mexican standards. 
 
3.3.2. Participants selection  
Because this was a phase 1 pilot study, where participants were self-controlled, with marker 
measurements taken before starting to consume the beverage and at the end of the 4-week 
treatment period, the sample size was based on convenience. All women who met the 
inclusion criteria and did not meet the exclusion criteria were invited to participate. At least 
ten pregnant women were recruited through the consultation of PhD. Edmundo González 
Vargas, a specialist in obstetrics and gynecology and maternal-fetal medicine at the 
"MEDOMAI" Comprehensive Health Services Clinic located in Cuernavaca, Morelos, 
México. 
 
Participants were selected according to the following criteria: 
Inclusion criteria 
- 
Pregnant women aged 18 to 35 
- 
Those in their second trimester of pregnancy (14 – 28 gestation weeks) 
- 
Blood pressure <140/90 mmHg 
Exclusion criteria 
- 
Multiple or high-risk pregnancy 
- 
Having suffered from a hypertensive disorder in previous pregnancies 

- 
Having chronic non-communicable diseases such as diabetes mellitus, cardiovascular 
disease, cancer, and chronic kidney disease 
- 
Body Mass Index ≥ 32 kg/m² 
 
Elimination criteria 
- 
Develop any pregnancy complications that prevent you from consuming the beverage 
and/or attending the measurements 
- 
Deciding of your own volition to withdraw from the study 
 
The patient records were reviewed to select patients who met the criteria for pregnancy weeks 
and age. They were contacted by telephone, where their medical history was taken to confirm 
that they met the remaining criteria. The procedures to be performed and how to prepare the 
beverage were explained in detail. In this conversation, the candidates were asked about their 
interest in participating and the date they should arrive to sign the informed consent form, 
receive the powdered doses to prepare the beverage along with its preparation instructions, 
and perform blood pressure measurements, biochemical indicators, and provide a urine 
sample. 
 
3.3.3. Acceptability and beverage consumption adherence 
Beverage acceptance was assessed through a sensory analysis once a week for 4 weeks using 
a 7-point hedonic scale, considering the characteristics of color, odor, flavor, mouthfeel, 
aftertaste, and overall appearance. The scoring system was as follows: 1 = I dislike it a lot, 2 
= I dislike it moderately, 3 = I dislike it a little, 4 = I neither like it nor dislike it, 5 = I like it 
a little, 6 = I like it moderately, and 7 = I like it a lot. To conduct this assessment, participants 
were sent a Google Form via WhatsApp. This form also included a section to indicate any 
other sensations they perceived when consuming the beverage, as well as their percentage of 
daily consumption of the beverage, and thus assess attachment. 
 
Beverage acceptance is assessed using the hedonic scale, and the score is evaluated whether 
the score is maintained over the duration of the trial. The feedback provided by participants 
allows adjustments to improve beverage acceptance, if necessary. 

3.3.4. Gastrointestinal tolerability due to consumption of the beverage. 
Once a week for the 4 weeks of the trial, participants completed a gastrointestinal tolerability 
assessment form as proposed by De Luis et al. (2015), where participants indicated the 
presence or absence of gastrointestinal symptoms such as nausea, regurgitation, vomiting, 
constipation, diarrhea, flatulence, and abdominal pain. These symptoms were assessed using 
5 points where: 0=absent symptom, 1=symptom present but not bothersome, 2=annoying 
symptom but does not interfere with daily activities or sleep, 3=a symptom present and 
bothersome but does interfere with daily activities and sleep, and 4=a symptom present that 
requires medical attention.  
 
3.3.5. Blood pressure, urinalysis and biochemical markers 
Blood pressure, urinalysis, and biochemical indicators of serum glucose, cholesterol, 
triglycerides, and creatinine were measured before the start of the trial and after the four 
weeks of consumption of the beverage were completed, to compare these parameters before 
and after treatment. The measurements and blood samples were taken by trained and 
experienced personnel. Blood pressure was measured using a noninvasive technique using 
the Korotkoff auscultation method. 
 
Blood samples were obtained by puncture in the antecubital region of the participants' non-
dominant arm, using sterile, disposable materials. Blood was placed in anticoagulant-free 
tubes for clinical chemistry, extracting 6 ml of blood. These tubes were kept refrigerated until 
the determinations were performed in the Biomarker Measurement Laboratory of the Biotic 
Product Development Center by personnel from the Department of Nutrition and Functional 
Foods, who are trained and experienced in sample collection and processing. Blood 
chemistry was measured using the Spinlab series 5-5312 semi-automated turbidimetry 
equipment for clinical chemistry. 
 
The biological materials were used only for the aforementioned tests and were kept 
refrigerated until their disposal as Biologically Infectious Hazardous Waste (BIHW) in 
accordance with Mexican Official Standard NOM-087-SEMARNAT-SSA1-2002. The 

results of the biochemical indicators were reported in writing to the participants, who 
received free medical and nutritional guidance, if necessary. 
 
The general urine test was performed by the Jacobo Curiel external clinical analysis 
laboratory in Cuernavaca, Morelos. Participants submitted their samples to the "MEDOMAI" 
clinic on the indicated date, and the samples were then transferred to the external laboratory. 
 
The researchers of this protocol declare that there is no conflict of interest and that the 
objective is to generate knowledge that allows us to evaluate the effect of a beverage with H. 
sabdariffa, which can be used in the prevention and/or treatment of hypertensive disorders 
associated with pregnancy, as well as to determine its degree of gastrointestinal acceptability 
and tolerability. 
4. SCHEDULE OF ACTIVITIES 
Activity 
2024 
March  
April  
May  
June 
July 
Information 
meeting 
with 
potential participants 
X 
X 
 
 
 
Interviews to verify inclusion 
criteria 
 
X 
 
 
 
Participants selection  
 
X 
 
 
 
Measurement 
of 
blood 
pressure 
and 
biochemical 
indicators before the test. 
 
X 
 
 
 
Beverage 
acceptability 
and 
tolerability test 
 
 
X 
 
 
Measurement 
of 
blood 
pressure 
and 
biochemical 
indicators after the test 
 
 
X 
 
 
Analysis of results 
 
 
X 
X 
 
Preparation and submission of 
the report to the Research 
Ethics Committee 
 
 
 
 
X 
 
 
 
 
 

5. BIBLIOGRAPHY 
Choo, E., & Dando, R. (2017). The impact of pregnancy on taste function. Chemical 
senses, 42(4), 279-286. 
De Luis, A. D., Izaola, O., Castro, A., Martin, M., Torres, B., López Gomez, J. J., & Blanco 
Naveira, M. (2015). Tolerance and acceptance study of a fiber-enriched high-protein 
enteral formula. Nutrición Hospitalaria, 31(1), 326-333. 
Díaz-Muñoz, G. (2020). Pilot study methodology. Revista Chilena de Radiología, 26(3), 
100-104. 
Haddad-Tóvolli, R., & Claret, M. (2023). Metabolic and feeding adjustments during 
pregnancy. Nature Reviews Endocrinology, 19(10), 564-580. 
Kölble, N., Hummel, T., von Mering, R., Huch, A., & Huch, R. (2001). Gustatory and 
olfactory function in the first trimester of pregnancy. European Journal of Obstetrics 
& Gynecology and Reproductive Biology, 99(2), 179-183. 
Lawless, H. T., & Heymann, H. (2010). Sensory evaluation of food: principles and practices. 
Springer Science & Business Media. 
Matsuda, K. I., Takahashi, T., Morishita, S., & Tanaka, M. (2024). Histological analysis of 
neuronal changes in the olfactory cortex during pregnancy. Heliyon, 10(5), 1-12. 
Muluh, E. A. E., McCormack, J. C., Mo, Y., Garratt, M., & Peng, M. (2024). Gustatory and 
olfactory shifts during pregnancy and the postpartum period: A systematic review and 
meta-analysis. Physiology & behavior, 273, 114388. 
Ramírez-Navas, J. S. (2012). Sensory analysis: consumer-oriented tests. ReCiTeIA, 12 (1), 
84-103. 
Sharif, M. K., Butt, M. S., Sharif, H. R., & Nasir, M. (2017). Sensory evaluation and 
consumer acceptability. Handbook of Food Science and Technology, 10, 362-386. 
Yang, J., & Lee, J. (2018). Korean consumers’ acceptability of commercial food products 
and usage of the 9‐point hedonic scale. Journal of Sensory Studies, 33(6), 1-12. 
 
6. Ethics Committee Approval 

EDUCACION 
 
C 
TAO 
Oí EOl .AClwN 
VB 
A 
1 Instituto Polltécnico Nacional 
"Lil Técnica al Servicio de ti! Patria'" 
Biotic Products Development 
Center
Research Ethics Committee 
CONBIOETICA-17-CEl-001-20170207 
Affair: Dictum 2024-1 CEI-CEPROBI. 
100 Aniversario del CECyT "Gonzalo Vázquez Vela" 
60 Aniversario del Centro Cultural "Jaime Torres Bodet" 
50 Aniversario de la ESIME Unidad Culhuacán, 
ESIA Unidad Tecamachalco y de la Escuela Superior de Turismo 
40 Aniversario del CIIEMAO, CEPROBI y del CITEDI 
PhD. PERLA OSORIO DÍAZ 
PRINCIPAL INVESTIGATOR 
BIOTIC PRODUCTS DEVELOPMENT CENTER 
In relation to your request for re-evaluationand preparation of the opinion, on the following 
research protocol: 
"ACCEPTABILITY, TOLERABILITY AND BIOCHEMICAL INDICATORS OF HIBISCUS SABDARIFFA L. 
DRINK IN PREGNANT WOMEN" 
Discussed at the first extraordinary meeting of the CEI-CEPROBI on April 8th, 2024. 
Le informamos que con base en los artículos 41 Bis, el artículo 98 de la Ley General de Salud, 
la Norma, Oficial Mexicana NOM-012-SSA3-2012 y la Guía nacional para la integración y 
el funcionamiento de los Comités de Ética en Investigación, que establecen los criterios para 
la ejecución de proyectos de investigación para la salud en seres humanos; su proyecto 
de investigación ha sido evaluado por este comité, resultando: 
Approved
Therefore, you may begin the corresponding work with the commitment to address the minor 
modifications indicated in the appendix. We inform you that this opinion is valid for one year 
from its receipt. In accordance with the internal procedures of the Research Ethics 
Committee, and based on the regulations of the General Health Law on Research (Art. 116), 
we request that you send this committee reports on the progress of your project, such as: the 
start of collection work, modifications, amendments, interruptions during development and/or 
definitive suspension, as well as a report on the completion of the research (Annex 5). 
C. Ja •
• va Sánchez 
President of the CEPROBI 
Research Ethics Committee
Ctra. Yautepec-Jojutla, Km.6, calle CEPROBI No. 8, Col. San Isidro, Yautepec, Morelos. México Z.C. 62731. 
Phone (735) 3942020 Tel. {735) 3941896, (55) 57296000 Ext. 82500, 82560, 82505. 
E-mail: ceprobi@ipn.mx Web site: www.ceprobi.ipn.mx 
204 
Felipe Carrillo 
PUERTO 

1 
Biotic Products Development 
Center
Research Ethics Committee 
CONBIOETICA-17-CEl-001-20170207 
Instituto Politécnico Naciooal 
"U Técnia al SerYic.io IN bl Patria• 
ANNEX 
100 Aniversario del CECyT "Gonzalo Vázquez Vela" 
60 Aniversario del Centro Cultural "Jaime Torres Bodet" 
50 Aniversario de la ESIME Unidad Culhuacán, 
ESIA Unidad Tecamachalco y de la Escuela Superior de Turismo 
40 Aniversario del CHEMAD, CEPROBI y del CITEDI 
1. From the protocol:
a) Align the title with the study objective. It should match the statements in the three 
reviewed documents (protocol, consent form, and study summary). Ensure that it 
addresses the study variables, both dependent and independent. It was suggested to 
simplify it with the following: "Evaluation of blood pressure, tolerability, and 
acceptability of Hibiscus sabdariffa L. in pregnant women."
b) Clarify the research design and its scope, based on the maturity of the project, 
recognizing, if applicable, that before implementing the study with the appropriate 
design and sample, an evaluation of the instruments, resources to be employed, and the 
feasibility of the designed logistics is intended.
c) With the current design, and in order to expand the sample size, it is suggested, if 
appropriate for the research group, that a control group with regular consumption of 
Jamaican be included. This would accurately demonstrate the comparability or efficacy 
of the product.
d) Add available quality information of the product (technical sheet): average anthocyanin 
content (ds), variety to be used, origin of the material and the minimum physical-
chemical specifications for its adequate characterization..
e) It is recommended to include the training of human resources in postgraduate studies in 
the contributions section of the study.
f) In any case, any changes or modifications to the original protocol must be noted and 
notified to the committee.
2. Informed consent.
a) Standardize information in the protocol and consent forms regarding benefits for 
participants (nutritional, medical, or both) and duration (whether it will be only during 
the study or until the end of the pregnancy).
3. Summary of previous studies.
b) A systematic review and clinical trial strategy should be undertaken to justify and 
clarify the sample size and selection with greater evidence. The review should consider 
the type of subjects, recruitment strategy and inclusion, exclusion, and elimination 
criteria, the number and size of groups, the method and quantity of the product being 
evaluated, and the effect size and precision. It is suggested that searches be conducted 
in databases such as the Cochrane Library and PubMed.
Ctra. Yautepec-Jojutla, Km.6, calle CEPROBI No. 8, Col. San Isidro, Yautepec, Morelos. México Z.C. 62731. 
Phone(735) 3942020 Tel. (735) 3941896, (55) 57296000 Ext. 82500, 82560, 82505. 
E-mail: ceprobi@ipn.mx  Web site: www.ceprobi.ipn.mx 
Felipe Carrillo 
PUERTO