Olive Leaf Extract Effects on Meta-inflammation and Anxiety Symptoms in Women With Excess Weight
Phase: NA
Conditions: Women, Excess Weight, Anxiety Disorders
Sponsor: University of Guadalajara

University of Guadalajara 
Health Sciences University Center 
High Resolution Microscopy Laboratory 
 
 
 
 
 
Olive leaf extract effects on meta-inflammation and 
anxiety symptoms in women with excess weight 
 
 
 
Document date: March 20th, 2024. 
Approval date: April 17th, 2024. 
 

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Abstract 
Background: During anxiety, inflammation and oxidative stress (OS) occur in the brain. OS and inflammation 
create a positive feedback loop that prolongs anxious symptomatology. A low, sustained and similar 
inflammatory process occurs in overweight individuals, called meta-inflammation. The excessive presence of 
weight and the associated meta-inflammation leads to changes in brain architecture at the organic and 
psychological level. Being a woman increases the probability of suffering from anxiety and overweight women 
have double chances of suffering from anxiety disorders due to psychological and physiological characteristics. 
In Guadalajara’s metropolitan area (GMA), 75.9% of women suffer from excessive weight (BMI ≥25 kg/m2). The 
overweight prevalence (≥25-<30 kg/m2) represents 36.7% and obesity’s (≥30 kg/m2) prevalence 39.2% from that 
75.9%; hence, they maintain a higher prevalence of anxiety disorders compared to men. 
Due that excessive weight and anxiety disorders are modifiable risk factors on the rise in our population, different 
innovative ways have emerged to seek to treat them, such as supplementation with natural products and 
functional foods with antioxidant and anti-inflammatory effects, among them, olive leaf extract (OLE); Studies 
report good results of its anxiolytic effect in animal models; in addition, antioxidant and anti-inflammatory effects 
are attributed to it in both animals and humans. 
Objective: Based on this information, the present study aims to evaluate the effects from the supplementation 
of OLE on meta-inflammation and anxiety symptoms in GMA women living with excessive weight, in their 
comorbidities and associated components.  
Methodology: A randomized double-blind placebo clinical trial is proposed. The study groups will be conformed 
by women living with excessive weight from the GMA who meet the following criteria: age ≥18- ≤40 years, body 
mass index (BMI) ≥25- ≤40 kg/m2, blood pressure ≤ 129 mmHg systolic and ≤80 diastolic; anxiety 
symptomatology score 6 to 14 (mild) for the Hamilton Anxiety Scale (HAS). 
70 women at the University Center for Health Sciences (CUCS) who meet the inclusion criteria will be invited to 
participate. Two groups will be randomly formed consisting of 35 women with excessive weight and anxious 
symptoms each (in accordance with the BMI and HAS score, respectively); one will receive cellulose as a 
placebo and the other 750 mg/day of OLE orally. The Beck Anxiety Inventory (BAI) and the HAS will be 
administered to all participants every 30 days for 3 months; On the other hand, adherence to treatment will be 
reviewed on a biweekly basis. To evaluate meta-inflammation, the levels of leptin, IL-6, TNF-α and cortisol in 
serum will be quantified using the ELISA technique; weight, height, age and components associated with 
excessive weight such as percentage of fat and muscle, lipid profile (LDL cholesterol; LDL-c, total cholesterol; 
TC, triglycerides; TG and HDL-cholesterol; HDL-c) and glycosylated hemoglobin will be also evaluated; these 
evaluations will be taken at the beginning and at the end of the study, in order to determine if OLE has effects 
on them. 
Based on the reported literature, in this trial, it’s expected to observe a reduction in levels of proinflammatory 
cytokines, cortisol and leptins; consequently, this decrease would also reflect lower anxiety symptomatology 
scores. Regarding the components associated with excessive weight, a reduction in LDL-c, TC and TG levels is 
likely to be observed. 
Background 
Anxiety and stress 
The term anxiety can be defined as a set of diffuse emotions caused by a situation that is potentially harmful 
with the probability of harm occurring being uncertain. When anxiety is sustained for a long time or is produced 
by non-threatening stimuli, is considered pathological (Daviu, 2019); It is accompanied by nervousness, worry, 
uneasiness, anticipation and mental tension (Bystritsky, 2014). 
Stress is one of the main causes of anxiety and can produce inflammation. It is characterized by a poor 
relationship between the individual and the environment, in which the former is perceived to be surpassed by 

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the latter and its well-being or balance is at risk. Stress can lead to anxiety and anxiety can also be considered 
a type of stressor (Saccaro, 2021). 
Anxiety, inflammation and oxidative stress; molecular mechanisms.  
 
Anxiety disorder is closely associated with inflammatory processes. The onset of central inflammation in anxiety 
occurs due to disruption of the blood-brain barrier (BBB). Stress decreases tight and adherent junctions; 
increases the production of pro-inflammatory components (TNF-α, IL-6, IL-1β, IL-17A and IL-23) that activate 
microglia, astrocytes and affect endothelial permeability (Welcome, 2020). 
The increase in endothelial permeability of the BBB, the production of pro-inflammatory cytokines and stress 
damage favors the production of lipid peroxides and reactive oxygen species (ROS), generating oxidative stress 
(Welcome, 2020; Salim, 2012). Oxidative stress further promotes the production of proinflammatory cytokines 
by astrocytes and microglia through the activation of the transcription factor NF-kβ (its activation in the 
hippocampus, amygdala and locus coeruleus (LC) is related to anxious behavior) (Salim, 2012; Berríos-
Cárcamo, 2020); cytokines perpetuate the activation of these glial cells and promote the production of oxidative 
stress. Thus, oxidative stress and inflammation generate a positive feedback loop that perpetuates anxious 
behavior (Berríos-Cárcamo, 2020). 
Excessive weight and meta-inflammation 
 
With excessive weight, a chronic, low-intensity, inflammatory process occurs, defined as meta-inflammation 
(Blancas-Flores, 2010). It happens with the increase in adipose tissue and two processes are identified: increase 
in its size (hypertrophy) and quantity (hyperplasia). The inflammatory response begins in adipocytes and 
subsequently they recruit macrophages, mainly of the M1 type, among other immune cells, which favor the 
production of pro-inflammatory molecules and increases the recruitment of more macrophages, producing a 
positive-feedback loop (Blancas-Flores, 2010; Xu, 2013). Among the substances that adipose tissue can release 
are proinflammatory molecules, hormones, acute phase proteins and leptins (Manzur, 2010; Calder, 2011); it is 
capable of producing IL-1, an antagonist of the IL-1 receptor, IL-6, IL-7, IL-18, IL-10 and TNF-α. (Calder, 2011). 
In turn, the increased number of adipocytes favors the deoxygenation of cells furthest from the vascular zone. 
This promotes stress in the endoplasmic reticulum and produces oxidative stress (Blancas-Flores, 2010). Thus, 
similar mechanisms of oxidative stress and inflammation can be known between the external stressors that 
produce anxiety and those present in excessive weight. 
Anxious symptoms and excessive weight in women 
Women are twice as likely to suffer from anxiety (Catuzzi, 2014) and the lifetime prevalence of an anxiety disorder 
is 60% higher in them than in men (Donner, 2013). 
They may present greater hormonal fluctuations that spread anxiety disorders (Hantsoo, 2017); They have 
greater activation of the central amygdala in relation to negative emotions, more sensitivity to threatening stimuli, 
and tend to overestimate threat more than men. This, combined with inflexibility to explore a diversity of possible 
responses to potentially dangerous situations, lays the foundation for suffering from anxiety disorders (Catuzzi, 
2014; Donner, 2013). 
Regarding obesity, according to ENSANUT 2018, the prevalence of excessive weight in the state of Jalisco, in 
women, is 75.9%, where 36.7% correspond to overweight and 39.2% to obesity, respectively. The frequency of 
anxiety diagnosis in obese women is twice that of obese men; Likewise in women, anxiety and depression are 
more likely to reflect increased appetite and weight gain. This is associated with being the result of stress to 
favor the consumption of foods with high palatability (Fulton, 2022). 
Olive leaves and their polyphenolic compounds. 

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Leaves from olive tree, Olea europaea, are rich in phenolic compounds in even greater quantities than the fruit, 
or olive oil (Sabry, 2014). The main phenolic compounds from olive leaves are: oleuropein, tyrosol, 
hydroxytyrosol, ligstroside and caffeic acid (Acar-Tek, 2020); being oleuropein the most important. It can inhibit 
the formation of free radicals and exert antioxidant activity by giving up hydrogen ions (Acar-Tek, 2020; 
Khalatbary, 2013; Sabry, 2014); Also, it is mainly responsible for the anti-inflammatory effect (Qabaha, 2018) 
Among the main effects attributed to olive leaf extract, in studies with animal models, are improvement of glucose 
metabolism, increase in antioxidant function and reduction of oxidative stress and inflammation (Table 1), as 
well as reduction in anxious-type behaviors (Table 2). 
Likewise, studies with humans report lower expression of genes associated with inflammation and a decrease 
in the expression of proinflammatory cytokines, as well as improvement in glucose metabolism. It also has 
hypotensive effects and can lower fat mass as well as plasma levels of TC, LDL-C and TG (Table 3). 
 
 
 
 
 
 
 
 
 
 
 

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Table 1. Olive leaf extract (OLE) effects in studies with animal models. Among the main effects attributed to it are improvement of glucose metabolism, 
improvement of antioxidant function and reduction of oxidative stress and inflammation. 
 
 
 
 
 
 
Subjects 
Methodology 
Results 
References 
Korean regional mice.  
OLE at 3g, 6g and 30g/kg. 
 
Groupss: 
-Control. 
-Diabetes (D). 
-D+ low dose OLE. 
-D+ mid dose OLE. 
-D+ high dose OLE. 
Decrease in glucose and insulin 
plasma 
levels; 
increase 
in 
antioxidant enzymes and decrease 
in proinflammatory cytokines. 
 
Park, J. H. et al. (2013).  
Wistar rats of 3 months and 20 months 
of age.  
OLE 500 and 1000 mg/kg. 
2 months 
 
Groups: 
-Young rats. 
-Old rats. 
-Old rats + OLE (500mg/kg). 
-Old rats + OLE (1000mg/kg) 
 
Reduction of malonyl aldehyde 
(MDA; oxidative stress) in all aged 
rats with 1000 mg. 
 
Çoban, J. et al. (2014).  
Dark Agouti male rats (2-3 months). 
OLE 
1024mg/kg 
(45.96mg/kg 
oleuropein). 
20 and 30 days. 
Experimental 
autoimmune 
encephalomyelitis (EAE). 
 
Groups: 
-EAE ctrl 20d. 
-EAE+ OLE 20d. 
-EAE ctrl 30d. 
-EAE+OLE 30d. 
Decreased 
TBARS 
(oxidative 
stress) 
and 
increased 
GPx 
expression; 
attenuated 
myelin 
destruction 
and 
increased 
microglia differentiation to M2 
phenotype. 
 
Giacometti, J., & Grubić-Kezele, T. 
(2020).  
Male Wistar rats. 
6 weeks of treatment. 
STZ: streptozotocin. 
MT: metformin (600mg/kg). 
OLE: 200 and 400mg/kg. 
DM: Diabetes Mellitus 
 
Groups: 
-DM 
-DM+MT 
-DM+OLE 200 
-DM+OLE 400 
-Control 
OLE decreased glucose, MDA and 
AGEs levels and increased SOD 
and nitric oxide (NO) levels. 
 
Khattab, H. A. et al. (2020).  
6-week-old Sprague Dawley rats.  
OLE 20, 50 and 100mg/kg.  
Ibuprofen (IBU) 40mg/kg. 
21 days of treatment.  
PTSD: post-traumatic stress. 
 
Grupos: 
-Ctrl. 
-PTSD. 
-PTSD+OLE 20. 
-PTSD+OLE 50. 
-PTSD+OLE 100. 
-PTSD+IBU. 
OLE inhibited the increases in 
CORT and reduced the levels of IL-
1β and TNF-α; increased CREB 
and 
BDNF 
levels 
in 
the 
hippocampus; 
attenuated 
the 
memory deficit. 
 
Lee, B. et al. (2018).  

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Table 2. Olive Leaf Extract (OLE) effects, in animal models, on anxiety-like behavioral disorders. 
Regarding OLE toxicity, in vitro studies with doses of 320, 800, 2000, 5000 ng/mL do not reveal promutagenic 
effects on salmonella typhimurium and E.Coli in the bacterial reverse mutation test. Similarly, concentrations of 
250, 500, 750, 1000 and 1250 µg/mL do not affect the number of aberrant cells and polyploid range in guinea 
pig lung cells in the chromosome aberration test (Romero-Márquez, 2023). 
There was also no genotoxicity in male Wistar rats due to the consumption of olive leaf extract at doses of 100, 
200, 400 and 2000 mg/kg supplemented for 28 days; they did not show signs of toxicity, mortality, behavioral or 
physical alterations, or abnormalities in the liver or kidney. In another study in which 300, 600 and up to 1,000 
mg/kg were given for three months, similar results were observed. Daily consumption of 250 mg by elderly 
women for 1 year also did not reveal adverse effects (Romero-Márquez, 2023; Acar-Tek, 2020; Dekanski, 2014; 
Filip, 2015). 
 
 
Subjects 
Methodology 
Results 
References 
Wistar rats. 
All groups except Ctrl and sham 
had Parkinson's induced through 6-
hydroxydopamine. 
-SS: Saline solution. 
 
Groups: 
-Ctrl. 
-Sham 
(SS). 
-Parkinson 
(SS). 
-OLE 
50mg/kg. 
-OLE 
100mg/kg. 
-OLE 150mg/kg. 
OLE reduces anxiety-like behavior 
in rats administered 6-OHDA. 
 
Hosseini, S., & Hajizadeh, A. 
(2015).  
Male Sprague Dawley rats.  
OLE: 20, 50 and 70mg/kg. 
Fluoxetine (FLX): 10mg/kg. 
PTSD: post-traumatic stress. 
SS: saline solution. 
 
Groups: 
-SS. 
-PTSD. 
-PTSD+OLE20. 
-PTSD+OLE50. 
-PTSD+OLE70. 
-PTSD+FLX. 
OLE reduced anxiety-like behavior 
and CORT levels. The expression 
of 5-HT (serotonin) and NP-Y were 
increased by it. 
 
Lee, B. et al. (2018).  
Male Wistar rats. 
Lead (Pb): 250mg/l of water. 
EHO: 0.1%(w/v). 
 
Groups: 
-Ctrl. 
-Pb. 
-Pb+OLE. 
OLE 
reduced 
blood 
and 
hippocampal Pb and caspase 3 
levels, also, DNA fragmentation. It 
inhibited TNFα, IL-1β and reversed 
anxiety-like behaviour. 
 
Seddik, L., et al. (2011). 
Male mice. 
Cortisol (Cort): 40mg/kg. 
FLX: 20mg/kg. 
Oleuropein (OE): 8, 16, 32 mg/kg.  
FLX or OE was injected 30min prior 
Cort. 
SS: saline solution 
 
Groups:  
-SS 
-Cort+SS. 
-Cort+FLX. 
-Cort+OE 
8. 
-Cort+OE 
16. 
-Cort+OE 
32. 
 
OE reduced anxious-like behavior; 
increased 
GSH 
(antioxidant 
function) and reduced oxidative 
stress; 
attenuated 
the 
Cort-
induced decrease in 5-HT and 
dopamine. 
 
Badr, A. M., et al. (2020).  

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Subjects 
Methodology 
Results 
References 
N=29 men. 
OLE: 
20ml/day 
(121mg 
oleuropein/day 
and 
6.4mg 
hydroxytyrosol/day). 
 
Double-blind, 
placebo-
randomized 
control 
trial 
(RCT).  
Groups: 
-Placebo. 
-EHO. 
8 weeks. 
Less expression from essential 
genes in inflammation (OSM, 
Cox-2, IL-8), IKKB, Myd88 and 
the phospholipase A pathway. 
 
Boss, et al (2016).  
N=32 (22 women and 10 men). 
OLE: 
20g 
of 
leaves 
(100m 
oleuropein) per day. 
Placebo: gluten-free starch. 
URI: upper respiratory infection. 
 
RCT 
Groups: 
-Placebo. 
-OLE. 
 
2 months. 
Shorter duration of URIs 28%, 
mainly in women.  
It had no effect on the incidence. 
 
Somerville, V., et al. (2019).  
N=46 overweight men.  
Pol: 51mg oleuropein and 9.7 
hydroxytyrosol. 
 
RCT. 
Groups: 
-Placebo. 
-Pol. 
12 weeks of tx, 6 weeks off 
and 12 weeks of crossover 
tx. 
 
Insulin sensitivity improved by 
15%, with no changes in TNF-α 
and C-reactive protein (pCr). 
 
De Bock, M. et al. (2013).  
61 subjects 24-72 years old. 
OLE: 20ml/day (136mg oleuropein 
and 6.4mg hydroxytyrosol). 
TC: total cholesterol. 
TAG: triglycerides. 
LDL-c: LDL cholesterol. 
 
RCT. 
Groups: 
-Placebo. 
-OLE. 
6 weeks tx, 4 weeks off and 
6 weeks of crossover tx. 
 
OLE decreased systolic and 
diastolic pressure during the 
day; as well as plasma levels of 
TC, LDL-c, TG and IL-8. 
 
Lockyer, S., et al. (2017).  
N=18 (9 women and 9 men). 
OLE: 400mg (51.12mg oleuropein 
and 9.67 hydroxytyrosol) in a single 
dose. 
Placebo: matched control. 
 
RCT. 
-Placebo. 
-OLE. 
 
Two evaluation visits 4 
weeks apart (crossover tx). 
 
The hydroxytyrosol derivatives 
reached their peak between 4-8 
hrs and those of oleuropein at 24 
hrs.  
Ex vivo, IL-8 production was 
reduced. 
 
Lockyer, S et al. (2015).  
N=60 (26 men and 34 women). 
55±8.8 years of age. 
OLE: 500mg/day (.62mg luteolin 
and 16% oleuropein). 
Placebo. 
 
RCT. 
-Placebo. 
-OLE. 
 
12 weeks. 
OLE reduced levels of IL-6, 
TNF-α and IL-8. 
 
Javadi, H., et al. (2019).  
N=70 women. 
Age ≥18 years. 
BMI ≥30-≤40 kg/m2 
OLE: 
500mg/day 
(50mg 
of 
oleuropein). 
Placebo: 125mg starch. 
WRD: weight reduction diet. 
 
RCT. 
Groups: 
-Placebo+WRD 
-WRD+OLE. 
 
8 weeks. 
OLE 
significantly 
reduced 
weight, BMI, fat mass, fasting 
glucose, LDL, leptin, adiponectin 
and free fatty acids compared to 
Placebo+WRD. 
Haidari, F., et al. (2021).  

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Table 3. Effects of olive leaf extract (OLE) supplementation in clinical trials in humans. 
Problem statement 
 
According to the National Self-Reported Wellbeing Survey, in Mexico women showed a lower mood balance in 
all age groups compared to men (mainly between 33-40 years old). Regarding anxiety, 19.3% of the population 
revealed severe symptoms; 31.3% manifested symptoms of minimal or some degree of anxiety. For the female 
population the percentages were 23.2% and 32.8%, respectively. In general, this disorder affects the selective 
transport function at the blood-brain barrier (BBB), which favors the translocation of peripheral proinflammatory 
cytokines and the production of reactive oxygen species, which increases oxidative stress (OS). 
OS and inflammation generate a positive feedback loop in which the preservation of one another is favored, and 
thus, perpetuate anxiety-like behavioral alterations and sometimes aggravate them. This increases in OS and 
inflammation are also associated with excessive weight due to the meta-inflammation produced by it. The intake 
of olive leaf extract (OLE) in different subjects or animal models has been shown to reduce the damage 
generated by oxidative stress and the inflammatory state. Since these harmful processes are present in anxiety 
disorder, OLE emerges as a possible modulator of anxious symptomatology. 
Rationale 
 
The prevalence of an anxiety disorder is 60% higher in women than in men. In Jalisco, 5 out of every 20 
individuals have a mental disorder, the most frequent being anxiety, with 16%; In the Guadalajara Metropolitan 
Area (GMA) these alterations have a prevalence of 15%, where the majority of those who suffer from it are adult 
women between ages from 25-50 years. Furthermore, obesity and overweight increases the probability of 
suffering from an anxiety disorder by 30-40%; and the frequency of this disorder is double in obese women. 
According to ENSANUT 2018, the prevalence of excessive weight in the state of Jalisco, in women, is 75.9%, of 
which 36.7% corresponds to overweight and 39.2% to obesity. 
Based on the above, we consider that OLE represents a good option to help with symptoms from anxiety disorder 
by using it as a dietary supplement, due to its known beneficial effects, mainly on oxidative stress and 
inflammation, processes that converge in both anxiety and excessive weight. 
N=64 women. Age 49-69 years.  
OLE: 
250mg/day 
with 
40% 
polyphenols. 
Ca: 1g/day of Calcium. 
 
RCT 
Grupos: 
-Ca. 
-Ca+OLE. 
 
1 year. 
No adverse effects associated 
with tx were reported; OLE 
increased osteocalcin levels by 
32% and reduced TC, LDL and 
TG. 
 
Filip, R., et al. (2015).  
N=148 (126 women and 22 men) 
with stage 1 hypertension. Age 25-
60 years without medication for 
hypertension (HTN). 
OLE: 500mg/day. 
Captopril: 12.5mg/day. 
 
RCT 
Groups: 
-Captopril. 
-OLE. 
 
8 weeks. 
OLE lowered blood pressure 
levels more than captopril; also 
improved the TG, TC and LDL-c 
profile. 
 
Susalit, E., et al. (2011).  
N=79 (51 men and 28 women) with 
diabetes mellitus (DM). 18-79 
years old. 
BMI <40kg/m2, HbA1c <10%. 
OLE: 500mg/day. 
 
RCT 
Groups: 
-Placebo. 
-OLE. 
 
12 weeks. 
Lower levels of HbA1c and 
fasting insulin were observed in 
the group supplemented with 
OLE. 
 
Wainstein, J., et al. (2012).  

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            Research question 
 
What is the effect from supplementation with olive leaf extract (OLE) on the concentration of mediators of meta-
inflammation (TNF-α, IL-6, leptin, cortisol) and anxiety symptomatology in adult women with excessive weight? 
Hipothesis 
 
Dietary supplementation with olive leaf extract (OLE) reduces meta-inflammation and anxiety symptoms in adult 
women with excessive weight. 
General and specific objectives 
General: 
To evaluate the effects from the consumption of olive leaf extract as a dietary supplement on anxiety symptoms, 
meta-inflammation and metabolic comorbidities in women with excessive weight from the Guadalajara 
Metropolitan Area (GMA) in Jalisco, Mexico. 
Specific: 
 
 
1. To evaluate the presence/absence of changes at the 3rd month, with respect to the beginning of the 
study prior to supplementation, in the expression patterns of the lipid profile, glycated hemoglobin 
(HbA1c) and inflammatory mediators (TNF-α, IL- 6, Cortisol and leptins) as indicators of meta-
inflammation in both study groups (supplemented with olive leaf extract or placebo). 
2. Evaluate changes in weight and percentage of fat and muscle monthly. 
3. Analyze the dietary-nutritional component of the participants during the study period to know the possible 
influence it may have on the indicators to be evaluated. 
4. Analyze the effect of supplementation with the extract or placebo in levels of anxiety symptomatology, at 
the end of each of the three months of the study. 
5. Identify if there are significant differences, in the evaluated parameters, between the two study groups 
(supplemented with OLE or placebo). 
Tipe of study 
• 
Longitudinal clinical trial, double-blind randomized placebo. 
Research location 
• 
University Center for Health Sciences (CUCS) of the University of Guadalajara (UdeG) (recruitment of 
participants). 
• 
Resolution Microscopy Laboratory, CUCS, UdeG and Guadalajara Metropolitan Area (GMA) (laboratory 
analysis and subjects’ recruitment, respectively). 
• 
Neuroscience Care Unit, Department of Neuroscience, building L, Door 18, CUCS, UdeG (evaluation 
office). 
Study period 
The scientific literatura review pase began in February 2024 and the study is expected to conclude in January 
2026. 
Study subjects 
Adult women with excessive weight from the GMA, Jalisco, Mexico. 
 
 
 

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Inclusion criteria: 
• 
Females. 
• 
Residing in GMA.  
• 
Initial age ≥18-≤ 40 años 
• 
Initial Body Mass Index (BMI) ≥25-≤40 kg/m2 
• 
Blood pressure: ≤129mmHg systolic and ≤80 diastolic.   
• 
Anxious symptomatology score 6 to 14 (mild) through the Hamilton anxiety scale (HAS). 
Non-inclusion criteria: 
• 
Use of psychoactive drugs, suffering from major psychiatric disorders (depression, attention deficit 
hyperactivity disorder, bipolar disorder, eating disorders), pregnancy, breastfeeding, hypotension, alcohol 
use disorder (AUD). 
• 
Suffer from liver, kidney or thyroid disease or cancer. 
• 
Olive allergy. 
• 
Consume any medication that influences weight 
• 
Consume some other type of food supplement from organic origin. 
Exclusion criteria: 
• 
Less than 70% adherence frequency to the consumption of the extract/placebo. 
• 
Getting pregnant during the study. 
Sample size. 
Sample size was chosen based on similar studies in the literature that identified statistically significant 
differences. Studies that evaluated inflammatory markers and the effect of olive leaf polyphenol supplementation 
on proinflammatory cytokines generally used samples between 20-35 subjects per group (intervention group and 
control group) (Javadi, 2019; Lockyer, 2017). The calculation of the comparison of means taking as reference 
the study by Javadi et. to the. 2019 (Control 78.4±13.2ng/L vs Experimental 69.7±9.8 ng/L IL-6; post-intervention 
with polyphenols) through the OpenEpi statistical calculator suggests recruiting 29 participants in total to 
demonstrate a moderate effect (>0.60) for the groups comparison to intervene with statistical significance and 
power of 80% (α= 0.05). We decided to use 35 participants (n=70) per group due to possible eliminations. 
Sample aleatorization 
Block randomization was performed using Random Allocation Software. Subsequently, participants will be 
stratified according to their Body Mass Index (BMI). From this stratification, 7 blocks of 10 individuals will be 
produced. According to their correspondence, the codes in Figure 1 will be distributed into two equal containers 
separated by denomination (case or placebo) that the participants in each block will take. The correspondence 
of an individual to a certain group will be blinded for the researcher and the participants. 
 
 

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Figure 1. Block randomization, of 10, delivered by the Random Allocation Software. 
Group Division 
Participation in the study will be promoted inside CUCS from UdeG through information flyers and official digital 
media, with prior authorization. 70 women from the GMA who meet the inclusion criteria will be recruited. They 
will be given an informed consent document and, if they accept, they will be included in the study. Each of the 
participants will be assigned in a double-blind and randomized manner to one of two groups: supplemented with 
olive leaf extract (EHO) (n=35) or placebo group (cellulose; n=35). They will be treated as indicated in Figure 2. 
Most measurements will be carried out at the beginning of the study and three months after it, when the clinical 
trial concludes. Anxious symptomatology will be evaluated using alternately the Hamilton Anxiety Scale (HAS) 
and the Beck Anxiety Inventory (BAI) (at the beginning and subsequently every 30 days, for 3 months); Patients 
who present scores that refer to moderate/severe anxiety will be referred to the relevant medical care service 
free of charge by direct contact (Department of Neurology of the Fray Antonio Alcalde Civil Hospital and 
Neuroscience Care Unit, Dept. of Neurosciences building L, CUCS , UdeG). Adherence to the protocol will be 
verified every 15 days and a 24-hour dietary recall and food frequency will be applied to keep a record of eating 
patterns throughout the clinical trial. Weight and fat and muscle percentages will be recorded monthly using an 
Omron® HBF-514C scale. ELISA assays will be used to measure levels of TNF-α, IL-6, Cortisol and leptins; 
height will be measured through a stadiometer and, finally, lipid profile and glycosylated hemoglobin will be 
measured through blood chemistry; All these parameters will be taken at the beginning and at the 3rd month of 
the trial (height will only be taken at the beginning). 
 
Figure 2. Division, treatment and evaluation for each participant group.  

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Dependent and independt variables 
 
Independent variable:  
olive leaf extract (OLE), placebo and anxiogenic state. 
 
Intervener variable:  
Age and dietary-nutritional component. 
 
Dependent variables:  
TNF-α, IL-6, cortisol, leptines, weight, BMI, muscle and body fat percentage, lipid profile, glycosylated 
hemoglobin and anxious symptomatology. 
Biosafety considerations 
 
The procedures to be followed are specified in accordance with NOM-087-ECOL-SSA1-2002, NOM-054-
SEMARNAT-1993 and NOM-052-SEMARNAT-2005; the competence in biosafety of the researchers 
responsible for the High-Resolution Microscopy Laboratory, University Center for Health Sciences (CUCS), 
UdeG, and the equipment available to carry out the experiments, in ANNEX 4. 
For the management of chemical waste, NOM-118-STPS-2000 and the manual for the management of 
hazardous chemical waste (CRETI) will be considered, 
both attached in the following link: 
https://drive.google.com/drive/folders/16mo95bffasZs7ug0Gweyanl8I8x3F_xg 
Ethical considerations 
 
This project adheres to the requirements by the mexican Regulation of the General Health Law on Health 
Research (mainly articles 13, 14, 15, 16, 17, 20 to 27) and the CIOMS guidelines (1st to 6th, 8th to 15th and 
18th). To safeguard the rights and autonomy of the participants, they will be made aware of the objectives, risks 
and benefits expected in this study through an informed consent (ANNEX 1); right there they will be informed of 
the right to withdraw without any opposition. The purpose of this research is supported by previous scientific 
studies. Regarding its social value, if successful, it could improve the quality of life of women who suffer from 
meta-inflammation and anxiety; Its cost is not much higher than the drug buspirone, one of the most used in the 
treatment of anxious behavior disorders, however, the field of its beneficial effects is broader. Likewise, olive 
leaves, which can represent up to 5% of the mass of olives in olive mills, could be revalued as raw materials 
(Garro, 2023). Also, participants will be given personalized nutritional advice for 3 months after their participation 
in the study, in the form of compensation and gratitude for their participation. On the other hand, if at any time 
during the study they present moderate/severe levels of anxiety and/or physical damage resulting from this 
research, they will be referred free of charge to the corresponding medical service by direct telephone contact 
(Dr. José Luis Ruíz Sandoval, head of the service of neurology, Hospital Civil Fray Antonio Alcalde; with free 
access to the clinical care unit of the Dept. of Neurosciences, CUCS).  
The study presents equity in the distribution of research burdens, the risks reported by the consumption of olive 
leaf extract are not significant and are outweighed by the known benefits. The use of cellulose as a placebo is 
because it will allow us to discern whether the extract has a significant therapeutic effect when comparing both 
groups. Likewise, current treatments for anxiety disorders are mainly through neuromodulators derived from 

13 
 
benzodiazepines that have a delayed action and may have unwanted side effects, plus, their effect on meta-
inflammation is unknown. 
For its part, although some studies in animal models report a neuromodulatory effect of olive leaf extract, its 
main action is through an antioxidant and anti-inflammatory effect; It is through these mechanisms that an 
anxiolytic action is attributed to it in these same studies; Through its anti-inflammatory action, we will evaluate 
its possible anxiolytic effect, as well as its effect on meta-inflammation caused by excessive weight (processes 
associated with each other). 
This research also adheres to the WMA Declaration of Helsinki-Ethical Principles for Medical Research on 
Human Beings 64th Assembly, Fortaleza, Brazil, October 2013. In accordance with article 17 of the Regulations 
of the General Health Law on Matters of Health Research, the risk of the research is minimal since it 
contemplates blood extraction, application of psychological tests and administration of a nutritional supplement 
(olive leaf extract) whose dose to be used does not exceed the manufacturer's recommendation and does not 
report adverse effects in the literature. The informed consent, the data collection format (contained in the 
nutritional clinical history) and the conflict-of-interest report are attached at the end of this document (ANNEX 1, 
2 and 3, respectively). The confidentiality of the data will be given by identifying each participant by means of a 
code (randomization number) that only the main researcher responsible for the study will have access to decipher 
(name-code agreement). 
Methodology 
Cytokine quantification to asses meta-inflammation 
Enzyme-linked immunosorbent assay (ELISA) kits will be used according to the manufacturer's instructions 
(Table 4) and will be interpreted based on reference values for each molecule. The procedure for blood collection 
is mentioned in ANNEX 4. 
Table 4. ELISA assay specifications 
Kit 
Detection range 
Sensitivity 
LEGEND MAXTM Human IL-6 
ELISA kit 
7.8 pg/mL – 500 pg/mL 
1.6 pg/mL 
LEGEND MAXTM Human TNF-
α ELISA kit 
15.6 pg/mL – 1000 pg/mL 
3.5 pg/mL 
InvitrogenTM 
Kit 
de 
ELISA 
human cortisol 
100-3,200 pg/mL 
17.3 pg/mL 
InvitrogenTM 
Leptin 
Human 
ELISA Kit 
15.6-1000pg/mL 
<3.5 pg/mL 
 
Asses of nutritional parameters 
A nutritional clinical history will be prepared that contains information on eating habits (24-h recall and food 
frequency), clinical history and family history. Anthropometric parameters such as weight, height, age, BMI will 
be recorded; percentage of fat and muscle through bioimpedance (ANNEX 2). The lipid profile and glycated 
hemoglobin will be evaluated by biochemical tests. The procedure for blood collection is mentioned in ANNEX 
4. 
Olive leaf and placebo treatment 
Nutricost ® 20% oleuropein olive leaf extract will be administered for 3 months, 1 capsule per day, at a dose of 
750mg daily. The placebo group will receive 100 mg of microcrystallized cellulose during the same period. 
Anxious symptomatology 
It will be measured through the Hamilton Anxiety Scale (HAS) and the Beck Anxiety Inventory (BAI) adapted to 
the Mexican population (ANNEX 6; “Clinical Practice Guide GPC, IMSS 392-10”. Complete at: 
https://drive.google.com/drive/folders/16mo95bffasZs7ug0Gweyanl8I8x3F_xg). HAS will be applied at the 
beginning and individuals with anxiety scores between 6-14 will be included; BAI will be applied to support the 
results obtained through the EAH, every 30 days alternately, for three months. This evaluation period is based 

14 
 
on the cumulative effect of the consumption of the extract to produce a possible metabolic readjustment that 
improves the state of meta-inflammation and consequently the anxiety symptomatology. The alternation of the 
tests (HAS and BAI) is done to avoid bias in patients that allows them to recognize answers to obtain better 
scores. 
If any participant presents moderate/severe anxiety scores, they will be referred to relevant medical care (Dr. 
José Luis Ruíz Sandoval, head of the neurology service, Hospital Civil Fray Antonio Alcalde; free access to the 
clinical care unit of the Department of Neurosciences, CUCS). 
Adherence 
It will be carried out biweekly from the return of the container where the respective treatment was delivered; The 
adverse effects questionnaire, the 24-hour food recall and food frequency will also be applied here (ANNEX 5). 
Statistical analysis 
It will be carried out through a descriptive and inferential analysis, taking p<0.05 as the significance level. The 
Shapiro-Wilk test will be used to observe the distribution of the data. 
• 
Descriptive analysis: measures of central tendency (arithmetic mean or median) and measures of 
dispersion (deviation or standard error of the mean). 
• 
Inferential analysis: Parametric tests (“Student's t”, ANOVA). If the data do not follow a normal distribution, 
non-parametric tests will be reported (X”, Kruskal-Wallis, Mann-Whitney U). 
• 
Post hoc: If parametric tests are used, the Sidak analysis will be performed. If non-parametric tests are 
used, the Tukey test will be carried out after the ANOVA test. 
Project viability 
Funding 
The responsible researchers have the adequate budget to start part of the proposed techniques and 
supplementation (OLE/placebo); likewise, the project will be submitted to the PIN 2023-IV financing call. 
Infrastructure 
The project will be carried out in the High-Resolution Microscopy Laboratory, the Dept. of Neurosciences, CUCS, 
UdeG and GMA. The equipment the laboratory has is specified in ANNEX 4. 
 
 
 

15 
 
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e25. 
 

18 
 
ANNEX 1                                              Informed consent  
“Olive leaf extract effects on meta-inflammation and anxiety symptoms in women with excess weight” 
Research objective 
This study seeks to see if olive leaf extract (OLE) has any effect that could help lowering the number of molecules 
in your blood that increases due to excessive weight and promotes the symptoms of mild anxiety. 
Why were you included?  
Because you’re a woman who lives in the metropolitan area of Guadalajara, between 18 and 40 years old, with 
excessive weight and normal or slightly elevated blood pressure. 
Study diagram 
You will be chosen at random, by drawing, to take for 90 days (3 months) one of two 
supplements: olive leaf extract or cellulose (placebo), a fiber very different from olive leaf 
extract. 
Neither you nor the other researchers, except Dr. Ma. Sonia Luquín, will know which 
supplement you received. The extract is what will help you, in theory, reduce the effects 
of the molecules that increases with excessive weight and provokes anxiety symptoms.  
Cellulose can't help you with that kind of thing, but it can improve your digestion because 
it helps your intestine movility. 
Neither of these two compounds have reported serious ill effects.      
How can you be excluded from the study?  
1. If you add 13 days of not taking the supplement during the 3 months. 
2. If you get pregnant. 
3. If you need to take some type of medication for depression, other behavioral disorders or a more serious 
chronic illness (diabetes, cancer, liver and kidney diseases, etc.). 
4. If you present any negative effect from the supplement or if you start taking another type of organic herbal 
supplement in conjunction with participating in the study. 
Research justification 
Olive leaves are used in many food preparations. They have compounds that reduces the increased negative 
molecules in your blood that happens when you have excessive weight and anxiety symptoms. Anxiety is closely 
related to stress. Cortisol levels (a molecule in your blood) are used to evaluate the presence of stress; Cytokines 
(other molecules in your blood) are used as an object to understand inflammation. Excessive inflammation is 
bad; It happens when you have excessive weight or anxiety. More cytokines equals more inflammation. Finally, 
leptin levels (another molecule in your blood) tell us about how fat reserve in your body behaves. From these 
measurements plus the profile of other fats (LDL, HDL cholesterol levels, triglycerides and total cholesterol), 
glycated hemoglobin (to know if you have diabetes), body mass index (to know if you have excessive weight), 
weight, height and the percentages of fat and muscle in your body, a complete evaluation will be made on the 
possible benefits that the extract may generate in you, on excessive weight and anxiety symptoms. 
 
 
 

19 
 
Researcher objectives 
1. Know the anxious symptoms by asking questionnaires called: Hamilton Anxiety Scale (HAS) and Beck 
Anxiety Inventory (BAI) 
2. Evaluate inflammation, fat reserve and stress due to excessive weight and anxiety symptoms by 
measuring proinflammatory cytokines (TNF-α and IL-6), leptin and cortisol in your blood. 
3. Know the profile of other fats and glycated hemoglobin (blood sugar levels during the last 3 months). 
4. Measure Weight, Height, BMI, body muscle and fat percentage. 
5. Supplement for 3 months with olive leaf extract (OLE) or cellulose according to the corresponding 
selection. 
6. Know every 15 days your adherence to the treatment, the presence of bad effects from taking it and if 
you wish to continue in the study; apply the 24-hour food reminder and food frequency. 
Procedures 
When participating in the trial, you may be given to take every day, for 3 months, a capsule with olive leaf extract 
(750 mg/day) or a capsule with cellulose (100 mg; a substance without pharmacological effects but that helps 
improve bowel movement). This will depend on a random selection, that is, by a draw without any preference 
per person. 
1.- Objective 1: To evaluate anxious symptoms, two questionnaires will be applied at different times of the study. 
The first, the Hamilton Anxiety Scale, consists of 14 questions that address your current mood and which you 
will answer with “absent, mild, moderate, severe and very severe”; The second questionnaire, the Beck Anxiety 
Inventory, consists of 21 questions that refer to situations from the last two weeks of your life to which you will 
answer with “no; mild; moderate; severe” according to how you identify yourself with respect to the question. 
Applying the two questionnaires lasts approximately 25 minutes in total. They will be applied every 30 days 
alternately for 3 months. 
Risks: When answering these questionnaires the risks are very low. It may present stress, confusion, sadness, 
among other negative emotions. 
2.-Objective 2 and 3: To evaluate the levels of cytokines, cortisol, leptins, profile of other fats and glycated 
hemoglobin, a small puncture will be made in the middle of the arm and approximately 20 ml of blood will be 
extracted in total. 20 ml will be taken before starting the supplementation and after 3 months, when you finish 
supplementing, another 20 ml will be taken. This means that you will receive two injections (with 20ml of blood 
each) throughout the study. 
The person drawing your blood should use a new syringe or needle, gloves, a gown, and a face mask; he will 
need to clean the punction site with an alcohol swab before and after drawing blood. If the person does not has 
and do all of these, you can refuse to have blood drawn. 
Risks: There are few associated risks, but there may be excessive bleeding, fainting or a feeling of dizziness, 
accumulation of blood under the skin (bruises), and occasionally more than one sting if the vein is not found; In 
very unlikely cases the punction can become infected. 
3.-Objective 4: Some body parameters will be evaluated. You will be required to provide us with your age. Your 
weight, height, percentage of fat and muscle will be recorded with a special scale in which you only need to stand 
on and hold a bar integrated into the scale with both hands. To know your body mass index, you only need to 
know your weight and height. Measurement of weight and percentage of fat and muscle will be carried out 
monthly. 
This processes will be carried out by nutricionist Mario Hernández Garibay. 

20 
 
Risks: There are no significant risks associated with obtaining this information. 
4.- Objective 5: By participating in this research you agree to ingest daily (every day) for 3 months a capsule of 
olive leaf extract or cellulose, a non-digestible fiber, according to the selection made at random, by lottery. The 
capsule is taken with a little water; This may be a little uncomfortable, until you get used to it. 
Risks: The associated risks are few, if you do not tolerate the ingestion of the olive leaf extract or cellulose you 
could experience headache, muscle pain, low fever (low-grade fever), sweating, nausea, sore throat, runny nose 
and dizziness, in which case the ingestion of the capsule is suspended and if it is considered necessary, you will 
be referred to the corresponding medical attention (Dr. José Luis Ruíz Sandoval, 3331973293). 
5.-Objective 6: To find out your adherence to the treatment, you will be asked to return the container where the 
corresponding compound was last given and we will see how many capsules you took; at that time you will be 
given more capsules. You will be given a questionnaire to find out if you have had adverse (bad) effects that you 
attribute to the treatment and, as mentioned, if you need medical attention, you will be referred to it (Dr. José 
Luis Ruíz Sandoval, 3331973293). You will be given a 24-h food reminder, and you will be given the option to 
continue or abandon the study. This will be done in the Department of Neurosciences at the University Center 
for Health Sciences (CUCS) of the University of Guadalajara, biweekly, for 3 months. In the event that it is difficult 
or uncomfortable for you to travel to CUCS, it can be done at home if you do not have any inconvenience. If you 
have any inconvenience, the evaluation can be rescheduled in nearby days. 
This processes will be carried out by nutricionist Mario Hernández Garibay (3316052068). 
Risks: There are no significant risks associated with obtaining this information.  
Expected benefits 
Apart from the aforementioned benefits on inflammation and anxiety symptoms, olive leaf extract reduces blood 
sugar levels and levels of total cholesterol, LDL and triglycerides (this is associated with a lower risk of suffering 
from diabetes and chronic diseases); It also reduces blood pressure and is capable of improving the function of 
the immune system, which is the system responsible for defending the human body against invading 
microorganisms.  
Finally, the main objective of consuming this compound is to help reduce the anxiety symptoms that often 
motivate the greater consumption of foods with a lot of fat or sugar. 
As a thank you for your participation, at the end of the trial you will be given personalized nutritional 
assistance for three months free of charge (Nutritionist Mario Hernández Garibay 3316052068).  
Once the months of participation in the study have ended, you will NOT be given more olive leaf extract. 
Information privacy 
Your name will only be used to sign this consent. For privacy purposes, a numerical identification method will be 
used according to the draw for your group assignment (extract or placebo) in the study and not your name; Your 
signature (without your name) will also be used when answering the treatment adherence questionnaire. 
Regarding personal information, you will be asked to provide your age, marital status, your cooperation in 
recording the parameters mentioned above (BMI, height, weight, % fat and muscle; as well as the 
aforementioned analyzes for which we will be taking your blood) and information to know nutritional aspects; 
Next, you will be shown and explained the nutritional questionnaire that will be applied to you. The information 
you provide us will be used solely for the purposes of this research; You will be informed of all your results once 
obtained. 
If we would like to use your information for another research, other than this one, your permission will 
be requested again; Just as if your data needs to be transferred to another researcher, you will be 
required to agree. You are free to reject if you wish. 

21 
 
Knowledge of your name and data will be exclusive to the main researcher, Dr. Ma. Sonia Luquín de Anda and 
no one else. The responsibility and privacy of the data provided will fall on her. Your data will NOT be shared 
with any type of company or individual unrelated to the research. 
The other researchers will know the results of the evaluations carried out, but they will not be able to identify 
them by name, they will only identify them by means of the numerical code assigned to them. 
Doubts and clarifications 
You will be in direct communication with the responsible researchers at the times when the aforementioned data 
and blood extraction is required; as well as three to four times a week depending on availability and need, 
throughout the study. 
Dr. Ma. Sonia Luquín de Anda 3334408913. 
Dr. Joaquín García Estrada 3332016340. 
Nutricionist Mario Hernández Garibay 3316052068. 
You will have the possibility to clarify any type of doubt that may arise associated with this research, as well as 
receive the medical attention of Dr. José Luis Ruiz Sandoval (3331973293), necessary in case you present any 
type of negative health response associated with participation in this research, such as those discussed above. 
To schedule an appointment with the Doctor, you will contact him, and he will give you the date and place of the 
medical consultation. Free medical care will not be given for illnesses that you suffer from before starting the 
study. 
Any new information on the extract that indicates the need to immediately stop the study or specifically exclude 
you will be informed to you and will be carried out promptly under the responsibility of the principal investigators.  
If you have any bad results in any of the blood tests that need to be told to you soon, you will be informed as 
soon as possible. 
You can leave the investigation at any time you wish. Participation or non-participation, as well as 
abandoning the research, will not represent any type of negative repercussion.   
Participating in the study will not result in any additional financial expense; In the event that one day 
you cannot attend, or it is inconvenient for you to go to the CUCS for the respective evaluation, this 
can be done at home if you consider it convenient or, failing that, it can be rescheduled according to 
your availability, on nearby days.  The negative side effects known from consuming olive leaf extract 
or cellulose are generally not serious. As mentioned, if you experience any type of adverse effect, you 
will be referred for medical attention to Dr. José Luis Ruíz Sandoval, for whom you have contact 
information. 
 
 
 
 

22 
 
Freedom of consent 
By signing this document, you agree to be part of this research. It is important to remind you that you have the 
freedom, at any time if you wish, to stop participating.  
I repeat that your participation in this research will not generate any cost to you; If adverse effects occur due to 
participation in this study, medical attention will be provided (Dr. José Luis Ruíz); It is reaffirmed that you are 
free to accept as well as free to stop participating at any time.  
Finally, I remind you that, as a thank you for your participation, if you wish, you will be given individualized 
nutritional attention at no cost at the end of the study, biweekly, for a period of 3 months by Nutricionist Mario 
Hernández Garibay.   
By signing this document, you are agreeing to participate in this study and be part of everything described above: 
 
_____________________________                     
Date, Name and sign from the participant 
 
_________________________                                                                       ___________________________ 
Witness 1. Date, name and sign.                                                                    Witness 2. Date, name and sign.   
 
________________________________ 
Responsable researcher: 
Dr. Ma. Sonia Luquín de Anda 
sonia.luquin@academicos.udg.mx 
3334408913 
 
___________________________                                        _________________________       
Dr.  Joaquín García Estrada                                                   Nutricionist Mario Hernández Garibay 
jgarciaestrada@gmail.com                                                     marioahg93@gmail.com 
3332016340                                                                            3316052068   
 
 
 
 
 
 
 

23 
 
ANNEX 2 
Nutriotional History 
Date: _____________________ 
Expedient: _________________ 
Age: __________       Sex: _________                         
Birthdate: ________________  
Scolarship:________________                   Occupation: _______________ 
CLINICAL INDICATORS 
Diarrhea: _____   Constipation: _____ Gastritis: ____  Nausea: ______ Pyrosis: ______ Threw up: ______  Colitis: _____        
Teeth: ______         Other: _____             
Observations (signs and symptoms): 
 
Has a diagnosed illness: _____________________ 
Has suffered a major illness: ____________________ 
Take some medication:        Wich:                          Dose:           Since:             
Family background: 
Obesity:        DBM:        AHT:       Cancer:       Hypercolesterolemia:       Hypertrigliceridemia:      Thyroid diseases:     
Other:      
Physical Activity 
Tipe:                                  Intensity:                     Frecuency:               Timing:                
Reasons: 
 
Arterial tension (left arm) 
Time:                  Score:                       
Waist: __________ BMI:_________  Weight: __________ Height: __________   
% Body fat: _________ % Muscle: ___________          
Hip: __________ 
Waist hip index: ___________         
Men 
Women 
Health risk 
Less than 0.95 
Less than 0.80 
Very low 
0.96 to 0.99 
0.81 to 0.84 
Low 
≥ 1 
≥ 0.85 
High 
 

24 
 
24 hr-food recall. 
 
Time 
Place 
Food 
Portions 
Preparation method 
Breakfast 
 
 
 
 
 
Collation 
 
 
 
 
 
Meal 
 
 
 
 
 
Collation 
 
 
 
 
 
Dinner 
 
 
 
 
 
 
 

25 
 
 
 
Food frequency 
 
0 
1 
2 a 3 
4 a 5 
6 o más 
Vegetables 
Chard, cabbage, spinach, lettuce, alfalfa germ. 
 
 
 
 
 
Broccoli, beet, cauliflower. 
 
 
 
 
 
Squash, carrot, onion, tomato, mushroom, pumpkin 
flower, cactus. 
 
 
 
 
 
Fruits 
Strawberries, pineapple, guava, watermelon, 
blueberries, blackberries, peaches, tangerines, 
lemon, lime, apple, grapefruit, mango, banana, 
orange. 
 
 
 
 
 
Fat-free cereals 
Amaranth, rice, oatmeal, bolillo, sweet potato, 
commercial cereal, corn, pasta, Maria cookies, 
granola, macaroni, midnight, popcorn, potato, corn 
tortilla. 
 
 
 
 
 
Cereals with fat 
Baked corn or nopal toast, telera, commercial cereal 
bars, croissants, fried foods (commercial potatoes), 
commercial cookies, sweet bread, tamale, cake, flour 
tortilla. 
 
 
 
 
 
Legumes 
Chickpea, bean, kidney bean, broad bean, soybean. 
 
 
 
 
 
Protein with very low fat content 
Clam, fresh or water-drained tuna, beef steak, 
shrimp, jerky, smoked chop, egg whites, beef brisket, 
fish fillet, machaca, chicken breast, fish, commercial 
low-fat cheeses. 
 
 
 
 
 
Protein with low fat content 
Skirt steak, tuna in oil, pork chop, pork fillet, pork 
loin, chicken or beef liver, ground beef, white fish, 
salmon, light commercial cheeses. 
 
 
 
 
 
Protein with moderate fat content 
Pork crackling, egg, chicken with skin, turkey 
sausage, turkey salami, commercial reduced-fat 
cheeses. 
 
 
 
 
 
Protein with high fat content 
Beef jerky, backbone, ham, pork or beef tongue, 
moronga, chicken nugget, pigeon, crispy chicken, 
yellow cheese, Vienna sausage and cheeses in 
general. 
 
 
 
 
 
Dairy 
Milk (specify presentation), yogurt (specify 
presentation) and ice cream. 
 
 
 
 
 
Oils and fats 
Cooking oil, olives, salad dressing, coconut, cream, 
shortening, margarine, mayonnaise, butter, avocado, 
bacon, cream cheese. 
 
 
 
 
 
Oils and fats with protein 
Almond, hazelnut, peanut, chilorio, chistorra, 
chorizo, peanut butter, serrano ham, walnut, 
sesame, pistachio, pepitas, pâté, sunflower seed. 
 
 
 
 
 

26 
 
ANNEX 3 
DECLARATION OF CONFLICT OF INTEREST 
 
 
Through this document I state that the intervention called “Olive leaf extract effects on meta-inflammation 
and anxiety symptoms in women with excessive weight” has NOT been financed in whole or in part by any 
company with economic interests associated with the products, equipment or similar referred to here.  
 
Likewise, none of the responsible researchers have relationships with any associated company that could be 
perceived as a potential conflict of interest. 
                                                                     
 
 
________________________________ 
L.N. Mario Hernández Garibay 
marioahg93@gmail.com 
3316052068 
 
___________________________                                        _____________________________      
Dr. en C. Joaquín García Estrada                                          Dra. en C. María Sonia Luquín de Anda 
jgarciaestrada@gmail.com                                                     sonia.luquin@academicos.udg.mx 
3332016340                                                                            3334408913 
 
 
 
 

27 
 
ANNEX 4                                  
Waste type 
 
Physical 
state 
Management 
Packing 
 
Color 
to 
discard 
Storage 
Blood (labeled “Liquid 
Biological Hazardous 
Waste”) 
 
Liquid 
Lab coat, gloves, 
safety 
glasses, 
closed shoes and 
face mask. 
 
Hermetic (yellow or red lid). 
To discard, it must be 
neutralized with chlorine 
previously. 
 
Red 
-70°C 
Sharps 
(labeled 
“Biological-infectious 
sharps 
hazardous 
waste”) 
 
Solid 
Lab coat, gloves 
and face mask. 
 
Dispose 
of 
in 
rigid 
polypropylene containers 
 
Red 
------ 
Globes 
Solid 
They should not 
have any holes or 
damage 
when 
used. 
Discard in red bag. 
 
Red 
Room temperature. 
ELISA plate 96 wells 
 
Solid 
Adequate 
ventilation, gloves, 
safety 
glasses, 
face 
mask 
and 
gown. 
Rinse with plenty of water 
before discarding or in case 
of 
accidental 
spillage, 
collect 
with 
mechanical 
methods and then dispose 
of in a red bag. 
 
Red 
Cool, 
dry 
and 
well-
ventilated place. Store 
in 
properly 
labeled 
containers. 
 
 
In accordance with NOM-087-ECOL-SSA1-2002 
• 
Blood sampling will be carried out in the Department of Neurosciences, of the University Center for Health 
Sciences (CUCS) in an area equipped for this process, where a puncture will be performed in the middle 
of the arm and 20 ml of blood will be extracted. A new rigid polypropylene sharps waste container with 
needle separator and storage opening will be brought. Subsequently, the container will be transported in 
a polyethylene bag, to ensure its resistance, to the High-Resolution Microscopy Laboratory, University 
Center for Health Sciences, University of Guadalajara. In the laboratory, the sharps container will be 
treated accordingly. 
• 
The blood collected will be labeled in the manner referred to and with the participant's respective 
identification code. It will be transported in a disinfected hermetic container that allows it to be kept at 4°C 
with a splint that prevents compaction of the tubes where it was packaged. 
• 
In the laboratory, the collected blood will be stored in an isolated area that maintains it at -70°C with its 
respective labeling and for no more than 30 days. 
• 
The laboratory will be classified as Level I in the generation of RPBI since it will handle samples between 
1 and 50 per day. 
Nitrile or latex gloves, a gown and wrap-around lenses will be mandatory for handling and processing the 
samples. 
 
During the processing of the samples through the ELISA assay, if any contingency associated with its reagents 
arises, the procedure will be followed in accordance with the manufacturer's recommendations, which are 
mentioned 
in 
the 
attached 
safety 
sheets 
(https://drive.google.com/drive/folders/16mo95bffasZs7ug0Gweyanl8I8x3F_xg). 
In accordance with NOM-052-SEMARNAT-2005 and NOM-054-SEMARNAT-1993, blood and some of the 
components used in the compounds of the ELISA tests refer to incompatibility with other residues, a hazard code 
(CPR) and specific handling. shown in the following table: 
 
 

28 
 
Component 
CPR 
Group 
and 
incompatibility 
Storage by compatibility 
Management 
Minimization 
Blood 
B; 
infectious 
biological. 
 
--- 
-70°C 
Nitrile 
gloves, 
gown, 
face 
mask, 
safety 
glasses 
and 
closed shoes. 
 
Inactivation 
with 
chlorine 
and 
subsequent 
disposal 
to 
the 
drain. 
 
Hydrogen chloride. 
 
C; corrosive. 
 
Group 
1. 
Incompatible with 
sulfuric acid and 
methanol. 
 
Cool, dry, well-ventilated, 
corrosion-resistant 
area. 
Away from flammable or 
oxidizing 
substances, 
organic substances, alkalis 
or near metals. 
 
Ventilated area, 
nitrile 
gloves, 
gown, 
face 
mask, 
safety 
glasses 
and 
closed shoes. 
 
Neutralize 
with 
sodium hydroxide 
or 
calcium 
carbonate and pour 
into the drain. 
 
Tritón x-100. 
Tea; 
Environmental 
toxicity. 
 
--- 
Cool, dry, well-ventilated 
area. Away from oxidizers, 
oxidants and strong acids. 
 
Nitrile 
gloves, 
gown, 
face 
mask, 
safety 
glasses 
and 
closed shoes. 
Disposal 
of 
the 
container 
at 
a 
hazardous 
waste 
collection point. Do 
not pour into drain. 
3,3’, 5, 5’ Tetramethylbenzidine 
dihydrochloride. 
 
Tt; 
Chronic 
toxicity. 
 
--- 
Cool, 
dry 
and 
well-
ventilated place. Far from 
oxidizers. 
 
Nitrile 
gloves, 
gown, 
face 
mask, 
safety 
glasses 
and 
closed shoes. 
 
Disposal 
of 
the 
container 
at 
a 
hazardous 
waste 
collection point. Do 
not pour into drain. 
 
Sulfuric acid. 
 
Tt 
and 
C; 
Chronic 
and 
corrosive 
toxicity. 
 
Group 
2. 
Incompatible with 
hydrogen chloride 
(group 
1). 
Generates 
heat 
and 
solubilizes 
metals. 
Keep tightly closed in a dry 
place. Away from metals, 
acids, 
water, 
hydrogen 
peroxide and halogenated 
hydrocarbons. 
 
Nitrile 
gloves, 
gown, 
face 
mask, 
safety 
glasses 
and 
closed shoes. 
 
Disposal 
of 
the 
container 
at 
a 
hazardous 
waste 
collection point. Do 
not pour into drain. 
 
Metanol. 
I 
and 
Tt; 
Flammable 
and 
chronic 
toxicity. 
 
Group 
4. 
Incompatible with 
hydrogen chloride 
and sulfuric acid 
(group 1 and 2). 
Generates 
heat, 
flammable, violent 
polymerization and 
toxic gases. 
 
Well-ventilated 
place 
protected 
from 
sunlight, 
away from oxides, acids, 
halogenated hydrocarbons, 
flames and hot surfaces. 
 
Ventilated area, 
nitrile 
gloves, 
gown, 
face 
mask, 
safety 
glasses 
and 
closed shoes. 
 
It can be filtered 
and distilled. Small 
quantities can be 
allowed 
to 
evaporate 
or 
diluted with plenty 
of water down the 
drain. Disposal at 
hazardous 
waste 
collection point. 
Dimethyl sulfoxide 
 
Tt; 
chronic 
toxicity. 
 
--- 
Inert, dry and cool place. 
Away 
from 
water, 
hot 
places, flames, oxidizers, 
acid chlorides, strong acids, 
phosphorus 
oxides 
and 
inorganic components. 
Nitrile 
gloves, 
gown, 
face 
mask, 
safety 
glasses 
and 
closed shoes. 
Disposal 
of 
the 
container 
at 
a 
hazardous 
waste 
collection point. Do 
not pour into drain. 
 
 
 

29 
 
Among the equipment available at the High Resolution Microscopy Laboratory, University Center for Health 
Sciences, University of Guadalajara are: 
• 
Centrifuge for 10 ml Vacutainer tube. 
• 
Refrigerated centrifuge for microtube. 
• 
2 analytical balances 
• 
Incubator for cell culture. 
• 
Ultramicrotome. 
• 
Stirring and heating grill. 
• 
Digital potentiometer. 
• 
2 horizontal freezers. 
• 
Cold camera 
• 
Motic 3-channel fluorescence microscope. 
• 
Simple light microscope. 
• 
3-laser confocal microscope. 
• 
Inverted Leica fluorescence microscope. 
• 
Electrophoretic chambers with power source. 
• 
Leica brand microtome and vibratome. 
• 
Ultra freezer. 
Among those responsible for the laboratory who have taken biosafety training are Dr. Rocío Elizabeth González 
Castañeda and Dr. Sonia Luquín de Anda. They will train the personnel who will participate in the laboratory 
activities in terms of risks, handling, disposal and safety codes of the different chemical substances. The 
laboratory has space for storage capacity for reagent compatibility. 
The safety protocol to follow in case of emergency within the laboratory is in accordance with what is suggested 
in the “Safety protocol in the CENAPRED environmental samples laboratory”, attached in the following link: 
https://drive.google.com/drive/folders/16mo95bffasZs7ug0Gweyanl8I8x3F_xg.  
There is access to the emergency contact number of the Toxicological Information Center, Cruz Verde de 
Guadalajara, (33) 36-50-30-60. 
 
 

30 
 
ANNEX 5 
Adherence 
Participant number: ____________________                                     Date: _____________________ 
 
Consumed capsules: ________________________ 
Non consumed capsules: ______________________ 
Reason(s): 
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________ 
 
After taking the capsule, have you experienced: 
Headache: _____ Muscle pain: _____ Sore throat: _____ Low fever: _____ Sweating: _____ Nausea: _____ 
Runny nose: _____ Dizziness: _____ Reflux: _____ Heartburn: _____ Constipation: _____ Colitis: _____ 
Diarrhea: _____ 
Others:__________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________ 
 
Do you wish to continue in this research?    Yes_____ No_____ 
Reason (s): 
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________
________________________________________________________________________________________  
 
 
 
 
 
 
 
 

31 
 
 
Time 
Place 
Food 
Portions 
Preparation method 
Breakfast 
 
 
 
 
 
Collation 
 
 
 
 
 
Meal 
 
 
 
 
 
Collation 
 
 
 
 
 
Dinner 
 
 
 
 
 
     
 
 
 
 
 
 
 
 

32 
 
                                                                                                        
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Participant sign: _________________. Researcher name and sign: ___________________. 
 
 
 
Food frequency 
 
0 
1 
2 a 3 
4 a 5 
6 o más 
Vegetables 
Chard, cabbage, spinach, lettuce, alfalfa germ. 
 
 
 
 
 
Broccoli, beet, cauliflower. 
 
 
 
 
 
Squash, carrot, onion, tomato, mushroom, pumpkin 
flower, cactus. 
 
 
 
 
 
Fruits 
Strawberries, pineapple, guava, watermelon, 
blueberries, blackberries, peaches, tangerines, lemon, 
lime, apple, grapefruit, mango, banana, orange. 
 
 
 
 
 
Fat-free cereals 
Amaranth, rice, oatmeal, bolillo, sweet potato, 
commercial cereal, corn, pasta, Maria cookies, 
granola, macaroni, midnight, popcorn, potato, corn 
tortilla. 
 
 
 
 
 
Cereals with fat 
Baked corn or nopal toast, telera, commercial cereal 
bars, croissants, fried foods (commercial potatoes), 
commercial cookies, sweet bread, tamale, cake, flour 
tortilla. 
 
 
 
 
 
Legumes 
Chickpea, bean, kidney bean, broad bean, soybean. 
 
 
 
 
 
Protein with very low fat content 
Clam, fresh or water-drained tuna, beef steak, shrimp, 
jerky, smoked chop, egg whites, beef brisket, fish fillet, 
machaca, chicken breast, fish, commercial low-fat 
cheeses. 
 
 
 
 
 
Protein with low fat content 
Skirt steak, tuna in oil, pork chop, pork fillet, pork loin, 
chicken or beef liver, ground beef, white fish, salmon, 
light commercial cheeses. 
 
 
 
 
 
Protein with moderate fat content 
Pork crackling, egg, chicken with skin, turkey sausage, 
turkey salami, commercial reduced-fat cheeses. 
 
 
 
 
 
Protein with high fat content 
Beef jerky, backbone, ham, pork or beef tongue, 
moronga, chicken nugget, pigeon, crispy chicken, 
yellow cheese, Vienna sausage and cheeses in 
general. 
 
 
 
 
 
Dairy 
Milk (specify presentation), yogurt (specify 
presentation) and ice cream. 
 
 
 
 
 
Oils and fats 
Cooking oil, olives, salad dressing, coconut, cream, 
shortening, margarine, mayonnaise, butter, avocado, 
bacon, cream cheese. 
 
 
 
 
 
Oils and fats with protein 
Almond, hazelnut, peanut, chilorio, chistorra, chorizo, 
peanut butter, serrano ham, walnut, sesame, 
pistachio, pepitas, pâté, sunflower seed. 
 
 
 
 
 

33 
 
ANNEX 6 
Hamilton Anxiety Scale (HAS) adapted for the Mexican population 
 

34 
 
 
 
 

35 
 
Beck Anxiety Inventory (BAI) adapted for the Mexican population