Evaluation of the Effect of Cocoa Supplementation on Biochemical and Clinical Profile and Sensory-motor Processing of Peripheral and Autonomic Diabetic Neuropathy: Randomized Clinical Trial
Phase: NA
Conditions: Diabetic Neuropathies
Sponsor: Anahuac University

“EVALUATION OF THE EFFECT OF COCOA SUPPLEMENTATION ON THE 
BIOCHEMICAL AND CLINICAL PROFILE AND SENSORY-MOTOR PROCESSING OF 
PERIPHERAL AND AUTONOMIC DIABETIC NEUROPATHY: RANDOMIZED CLINICAL 
TRIAL” 
 
NCT05247034 
 
March 25 2025, last update 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

SUMMARY 
Background: Type 2 diabetes (T2D) is a highly prevalent disease in Mexico and is associated 
with the development of chronic-degenerative complications such as diabetic neuropathy. The 
latter manifests as a set of disorders that occur as a consequence of a chronic hyperglycemic 
state that can induce oxidative stress and inflammation, resulting in damage to the autonomic 
and peripheral nervous systems. Diabetic neuropathy increases the risk of pain and skin 
lesions, ulcers, or even amputations, affecting the quality of life of those who suffer from it. In 
Mexico, it has been reported that this complication occurs in 29% to 90% of patients with 
diabetes. Cocoa is a food with a high flavonoids content, which are phenolic compounds with 
antioxidant and anti-inflammatory effects. Additionally, its consumption has been associated 
with decreased hyperglycemia and insulin resistance, improved mitochondrial function, and, 
based on the above, a potential effect on diabetic complications has been suggested. This has 
been demonstrated in in vivo and in vitro models but not in human populations. Once symptoms 
of diabetic neuropathy appear, palliative treatments are prescribed, as no pharmacological 
compounds have yet been shown to reverse the consequences of peripheral and autonomic 
diabetic neuropathy. Additionally, clinical trials of compounds with antioxidant properties have 
only conducted subjective evaluations based on questionnaires about perceived improvement 
in diabetic neuropathy and some biochemical markers or nerve conduction tests, with 
inconclusive results. Methodology: A double-blind, randomized, controlled clinical trial is 
proposed to evaluate the effect of cocoa supplementation in patients with type 2 diabetes and 
peripheral and autonomic diabetic neuropathy on a) the biochemical profile, including the 
assessment of glycemic and lipid profiles, b) the clinical profile through standardized 
questionnaires, anthropometric measurements, and blood pressure; and c) somatosensory 
processing through the rate-dependent depression of the H-reflex test. Hypothesis: The 
hypothesis of this study is that cocoa supplementation will have a beneficial effect on the 
biochemical and clinical profile and somatosensory processing of peripheral and autonomic 
diabetic neuropathy. Statistical analysis: For the evaluation of intra-group variables, a 
statistical analysis will be performed using repeated measures ANOVA with Tukey post hoc, or 
if applicable, Friedman with Dunn post hoc, as well as Student's t-test for dependent groups, 
or if applicable, Wilcoxon. The intergroup comparison will be made with Student's t-test for 

independent samples, or if applicable, with U Mann Whitney, considering p <0.05 as statistical 
significance and using GraphPad Prism software version 5. 
2. THEORETICAL FRAMEWORK 
2.1 Type 2 Diabetes 
Type 2 diabetes (T2D) refers to a relative insulin deficiency secondary to a progressive 
dysfunction of its secretion, often with a prior period of insulin resistance (IR). This type of 
diabetes accounts for 90-95% of all cases. (1) According to the International Diabetes 
Federation (IDF), in 2019, the global prevalence of diabetes in adults aged 20 to 79 years was 
9.3%, and specifically for the North America and Caribbean region, it was 13.3%. (2) In Mexico, 
according to the 2018 National Health and Nutrition Survey (ENSANUT), 10.3% of adults over 
20 years old have a diagnosis of T2DM, corresponding to 8.6 million people. (3) This is relevant 
as 2013 data estimated that the total cost attributable to DM in Mexico was $7,736 million, and 
specifically for ISSSTE, $484 million, figures that could become unsustainable in the long term, 
especially with the increase in the number of cases. (4) 
2.2 Pathophysiology 
Diabetes mellitus (DM) is a chronic metabolic disorder characterized by persistent 
hyperglycemia. It occurs due to dysfunction in insulin secretion, resistance, or both (2). IR is 
the inability of the hormone to stimulate peripheral glucose uptake, as well as other signaling 
pathways in different tissues (5, 6), and develops as a result of fat accumulation in muscle and 
organs such as the liver and pancreas. In the pancreas, β-cell dysfunction is generated, as well 
as inflammation of the islets of Langerhans and cellular apoptosis. (7) 
2.3 Diabetic peripheral and autonomic neuropathy 
When metabolic dysregulation related to hyperglycemia persists, macrovascular and 
microvascular complications may develop. The former involve cardiovascular diseases, such 
as those of the large blood vessels, while the latter includes retinopathy, nephropathy and 
diabetic neuropathy (DN) and are caused by pathological changes in capillaries, which 
participate in transporting oxygen and nutrients to the body's systems, including the nervous 
system. (2, 8) Studies in Mexican population have reported that between 29% and 95% of 
patients living with DM develop DN, which can be observed as early as five years after the 

diagnosis of the disease. (9-11) The two most important predictors for the development of DN 
are the duration of DM and glycated hemoglobin (HbA1c) levels. Additionally, other factors 
increase the risk of DN, such as smoking, obesity, hypertension, and dyslipidemias (elevated 
triglycerides and LDL-c, as well as decreased HDL-c), making adequate metabolic control 
vitally important. (12, 13) 
DN is a group of disorders that present signs and/or symptoms of peripheral nerve dysfunction 
with various clinical manifestations in both peripheral and autonomic neuropathy. (8, 14) 
Diabetic peripheral neuropathy (DPN) can be divided into distal symmetric peripheral 
neuropathy and focal and multifocal asymmetric neuropathy, which occur as a consequence of 
the progressive loss of nerve fibers and the resulting instability. (15) Symptoms of DPN depend 
on the type of fibers affected, the most common being numbness and tingling in the case of 
large fibers, and allodynia – pain caused by stimuli that are not usually painful-, hyperalgesia, 
paresthesia and pain, in the case of small fibers (16), symmetric sensory loss or glove or 
stocking sensory loss in the feet, above the ankles and in the hands, is also evident during the 
clinical examination. (12, 15)  
The pathogenesis of DPN is not well defined and the pain often does not respond to first-line 
analgesics or requires doses that may cause adverse side effects. (17) Neuropathic pain 
occurs in approximately 30% of patients living with diabetes (18) and occurs as a consequence 
of sensory abnormalities, changes in the primary afferent nerves and central sensitization. (19) 
On the other hand, decreased pain sensation can result in an increased risk of skin lesions, 
infections and deformities leading to the development of Charcot foot, ulcers or even 
amputations. (14)  
Regarding autonomic neuropathy (ADN), this refers to damage to the nerves that control 
internal organs, causing alterations in various systems such as the cardiovascular, 
gastrointestinal and urogenital systems, and thus, the development of signs and symptoms 
such as arrhythmias, tachycardia at rest, nausea, vomiting, constipation, urinary incontinence, 
erectile dysfunction and decreased sexual libido. (20) 
2.4 Pathophysiology of DN 
Oxidative stress has been described as one of the mechanisms by which DN develops, 
resulting from an imbalance between the production of reactive oxygen species (ROS) and 

endogenous antioxidant systems (12) and the state of inflammation. (21) Hyperglycemia can 
induce oxidative stress by causing the accumulation of glucose and glycolysis intermediates, 
thereby altering the electron transport chain, reducing H2O and ATP production, and increasing 
the production of superoxide (O2-). (12) 
Additionally, hyperglycemia can stimulate macrophages to secrete proinflammatory cytokines 
such as tumor necrosis factor-alpha (TNFα), and, in these patients, there is an increase in the 
expression of C-reactive protein (CRP) and Interleukin-6 (IL-6). (21) These mechanisms are 
related since the increase in ROS perpetuates the state of inflammation, just as 
proinflammatory cytokines contribute to the increase in ROS. 
Hyperglycemia causes the metabolic pathways to be used to regulate the polyol pathways and 
the hexosamine pathway, which generates an increase in ROS and inflammation, and 
consequently, mitochondrial damage and dysfunction in the nervous system. (12, 22) 
About the polyol pathway, when there is hyperactivity of this pathway, sorbitol production 
increases by activating the aldose reductase enzyme system. This can alter the NADPH/NADP 
ratio and decrease reduced glutathione and oxidized glutathione, causing oxidative stress. (22, 
23) NADPH is derived from the pentose pathway and generates glutathione peroxidase from 
glutathione, and the consumption of this NADPH is what is associated with the development 
of oxidative stress. (12) Sorbitol formed as a result of the polyol pathway is oxidized to fructose 
by the enzyme sorbitol dehydrogenase, which uses NAD+ as a cofactor. Excess fructose 
promotes glycation and NADPH depletion, exacerbating intracellular oxidative stress. 
Furthermore, in preclinical studies in diabetic rats, the accumulation of sorbitol and fructose in 
peripheral nerves has been observed. (12) Sorbitol is not able to cross cell membranes, so it 
accumulates in cells, generating hyperosmolarity and a concomitant flow of taurine, myoinositol 
and adenosine. This causes inhibition of ATP synthesis, decreasing the activity of Na+/K+ 
ATPase and protein kinase C (PKC), an alteration in axonal transport and structural 
degradation of the nerves. (24) 
On the other hand, excess glucose causes glycation of structural and functional proteins and 
produces advanced glycation end products (AGEs), which interact with specific receptors to 
modify gene expression and intracellular signaling pathways that promote the release of 
proinflammatory cytokines and ROS. (25) AGEs also decrease axonal transport in neurons, 

leading to their degeneration. Likewise, they bind to their receptor (RAGE – receptor for 
advanced glycation end products), and, by activating it, trigger inflammatory pathways that 
contribute to oxidative stress. (24) 
Hyperglycemia mainly affects those cells that have a limited capacity to regulate glucose 
uptake, such as vascular cells, Schwann cells and neurons of the peripheral and central 
nervous system, directly damaging the cells or affecting cell function, leading to degeneration 
of peripheral nerves, (12) in particular, it has been described in animal models that when 
Schwann cells cannot provide the factors necessary for the proper functioning of axons, they 
begin to degenerate, in addition, alterations in the soma of dorsal root ganglion neurons could 
also contribute to the degeneration of peripheral nerve fibers. (25) 
Progressive DN involves the retraction and death of peripheral terminal sensory axons, with a 
relative preservation of the soma. It is believed that the entire neuron, both the soma and the 
axon, is affected by DM, however, it is not yet clear whether the first to be damaged are the 
peripheral axons and their associated Schwann cells or the soma of the dorsal root neurons 
that support the axons. Schwann cells are affected by persistent hyperglycemia, which causes 
significant damage to the axon. (25) 
Another comorbidity associated with the development of DN is dyslipidemia since under normal 
conditions, for the degradation of fatty acids and their subsequent use as an energy substrate 
in Schwann cells, they must go through the β-oxidation pathway, which results in a molecule 
of acetyl coenzyme A (CoA), which subsequently enters the Krebs cycle, forming NADH and 
FADH2. (25) 
NADH and FADH2 are used in oxidative phosphorylation to produce ATP and low levels of 
ROS, which can be neutralized by the body's endogenous antioxidant enzymes. (25, 26) 
However, when there is an overload of substrates, as in the case of T2D, the β-oxidation 
transport system to the Krebs cycle becomes saturated and the acetyl CoA molecules are 
converted to acylcarnitines. (25) This accumulation of acylcarnitines appears to be toxic to 
Schwann cells and dorsal root ganglion neurons, so they are released from these cells, 
inducing axonal degeneration, mitochondrial dysfunction, and a maladaptive stress response 
in Schwann cells. (25, 27) 

Moreover, substrate overload also affects oxidative phosphorylation, decreasing ATP 
production and increasing ROS levels that cannot be neutralized, leading to mitochondrial 
dysfunction and metabolic and oxidative damage in these important cells. (25) 
2.5 Diagnosis 
Patients with DM should be evaluated for diabetic neuropathy at diagnosis and for 5 years 
thereafter, and an annual review is recommended through a detailed clinical history, physical 
examination, and clinical tests. (14) The diagnosis of DN is made when other causes that could 
be causing the signs and symptoms are excluded and can be supported by tools such as 
questionnaires and tests. (16) 
Among the causes of neuropathy that must be excluded to make the diagnosis are toxic or 
drug-induced 
neuropathy, 
alcoholic 
neuropathy, 
neuropathy 
associated 
with 
HIV, 
hypothyroidism, and neuropathy due to vitamin B12 deficiency. Especially, those subjects who 
take metformin as a pharmacological treatment for T2D are at high risk of developing vitamin 
B12 deficiency since it decreases the absorption of this vitamin in the terminal ileum and is 
related to alterations in intrinsic factor levels. (28, 29) The signs and symptoms of vitamin B12 
deficiency are very similar to those observed in diabetic neuropathy, and therefore, their 
differentiation can be complex. Thus, the determination of serum vitamin B12 is suggested to 
exclude non-diabetic causes of neuropathy. (29) 
On the other hand, among the tools that can support the diagnosis of DN are biopsy, which is 
an invasive test; nerve conduction tests, which are expensive and are only useful to evaluate 
fast conduction fibers, which are not very practical; and the sensory test with the monofilament. 
(30) 
The Michigan Neuropathy Screening Instrument (MNSI) is a validated tool available in Spanish, 
which evaluates the presence of diabetic peripheral neuropathy. This instrument can be applied 
quickly, includes the history of symptoms and physical examination to detect alterations in the 
foot. It has a sensitivity of 65% and specificity of 83%, and a score of ≥2 has been proposed 
as a cut-off point for the diagnosis of DPN. (31, 32) 
Another tool that allows the severity of DN to be evaluated is the Toronto Clinical Scoring 
System (TCSS), which evaluates the symptoms, reflexes, and sensitivity by anatomical 

regions. According to the score obtained, 3 grades are established: 0-5 points, no DN; 6-8 
points, mild DN, 9-11 points, moderate DN and 12-19 points, severe DN. (33, 34) 
There is no standard for the evaluation of ADN, however, the clinical history is essential, for 
example, for the evaluation of gastrointestinal ADN, tests that identify symptoms such as 
nausea, vomiting, abdominal pain, constipation or diarrhea can be used (35), such as the 
Bristol scale and the BEST questionnaire. 
2.6 RDD of the H-reflex  
The Hoffman reflex (H-reflex hereafter) is produced by an electrical pulse and is recorded in a 
given muscle, and is analogous to the muscle spindle stretch reflex generated by a mechanical 
stimulus (myotatic reflex). The H-reflex represents the excitability of the alpha motor neuron 
(αMN) when presynaptic inhibition and the intrinsic excitability of αMNs remain constant. This 
test allows the evaluation of the response of the nervous system in conditions of damage to 
the nerve fibers and the somatosensory processing in the spinal cord. (36) Through the H-
reflex, it is possible to measure the efficacy of synaptic transmission of afferent fibers (Ia 
sensory) to αMNs. The evoked afferent response starts at the point of electrical stimulation, 
resulting in action potentials that travel along the afferent fibers until they synapse in the αMNs, 
while on the efferent side, the action potentials travel along the fibers until they reach the 
neuromuscular junction and produce a contraction response that can be recorded in the 
electromyogram. (36) 
An additional test based on the H reflex is the rate-dependent depression (RDD) test, which is 
a measure of the change in the amplitude of the spinal H reflex during consecutive paired 
electrical stimulations at different frequencies (<1 Hz). Recently, it has been described that 
RDD is affected after disinhibition of spinal sensory processing, e.g., in animal models of 
experimental diabetic neuropathy (18, 37, 38) and in T1D in humans (18), so the paired-pulse 
H reflex allows the objective and noninvasive detection of alterations in nerves and spinal 
sensory processing. 
2.7 Treatment 
Up to 50% of DN can be asymptomatic, and if not recognized and treated in time, it increases 
the risk of injuries, decreased quality of life, and even mortality, as in the case of cardiovascular 
ADN. Currently, the management of DN consists of lifestyle modifications to control glycemia 

and pharmacological treatment, especially in the case of painful neuropathy. These drugs 
include tricyclic antidepressants, antiepileptics, opioid agonists, morphine derivatives, selective 
serotonin reuptake inhibitors, and nonsteroidal anti-inflammatory drugs, which are only 
palliative and may have adverse side effects and/or limited long-term efficacy. (13, 16)  
Therefore, strategies have been sought to mitigate the symptoms associated with DN. For 
example, the consumption of flavonoids could have a beneficial effect on patients living with 
DM by controlling the glycemic profile (glycemia and HbA1c), lipid profile (triglycerides, LDL-c, 
and 
HDL-c), 
and 
possibly 
reducing 
blood 
pressure 
and 
inflammation 
markers 
(neutrophil/lymphocyte ratio) without developing adverse effects. (39-41) 
2.8 Flavonoids 
Flavonoids are phenolic compounds present in plant-based products such as fruits and 
vegetables, especially those with intense colors such as berries and green leaves, red wine, 
green tea, and cocoa, and are recognized for their antioxidant, analgesic, and anti-
inflammatory effects. Some molecular mechanisms underlying these effects include the 
inhibition of the nuclear factor κB (NF-κB), thereby decreasing the transcription and secretion 
of inflammatory cytokines. (39, 42) 
Cocoa (Theobroma cacao) has a high polyphenol content: 12 to 18% of the seed's dry weight. 
Flavonols are the most important polyphenols in cocoa, especially characterized by the 
presence of monomers and isomers of catechin and epicatechin, such as (-)epicatechin and 
(+)catechin. (42, 43) These polyphenols have shown beneficial effects in overweight subjects 
on parameters such as body weight, fat mass, and lipid profile by modulating fatty acid 
synthesis, which are closely related to glucose metabolism. (44) Additionally, polyphenols have 
been associated with increased nitric oxide synthase activity, which increases nitric oxide levels 
and reduces blood pressure. (43) 
At the neurological level, it has been observed that the consumption of cocoa and its flavonoids 
can reduce the risk of neurodegenerative diseases, especially those derived from oxidative 
stress such as Alzheimer's and Parkinson's disease; reduce depression due to its tryptophan 
content and its consequent conversion to serotonin; provide protective effects against 
neurotoxicity induced by β amyloid protein and against cognitive decline associated with aging 
and protect nerves from injury and inflammation. (45, 46) 

On the other hand, the consumption of catechins has been associated with a decrease in 
hyperglycemia and insulin resistance through the modulation of proinflammatory cytokines 
such as IL-1β, IL-6 and TNFα, and the activation of signaling pathways that allow maintaining 
an adequate function of the mitochondrial respiratory chain, and thus, protecting the cell islets 
and improving insulin resistance. (43) In preclinical studies, the possible decrease of 
neuropathic pain through different mechanisms has been reported, one of the most important 
being the modulation of the inflammatory response. (47) 
In a study in rats with streptozotocin-induced diabetes, doses of 25 and 50 mg/kg of catechins 
were administered intraperitoneally for 28 days, observing attenuation of diabetic autonomic 
neuropathy through the improvement of antioxidant enzymes in the vagus nerve, in addition to 
the decrease of malondialdehyde (MDA), a marker of oxidative stress. (39, 48) It is therefore 
relevant to objectively and quantitatively evaluate the effect of cocoa supplementation on the 
biochemical and clinical profile and sensorimotor processing of diabetic peripheral and 
autonomic neuropathy in adults with T2D. 
3. JUSTIFICATION 
T2D increases the risk of developing macrovascular and microvascular complications, which 
lead to a significant portion of the national budget being allocated to the treatment of these 
chronic-degenerative conditions. In 2013, it was estimated that the total cost attributable to 
diabetes in Mexico was USD 7.736 billion, with USD 484 million specifically for the ISSSTE 
(Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado), figures that could 
become unsustainable in the long term. (4) 
DN is a common complication of T2D that affects the quality of life of those who suffer from it. 
Currently, the management of diabetic neuropathy includes lifestyle modifications, glycemic 
control, and pharmacological treatment, which focuses on reducing pain and symptoms (13); 
however, the medications used are palliative and have adverse or undesirable side effects.  
The consumption of foods rich in flavonoids has been linked to the regulation of processes 
such as oxidative stress and metabolic and inflammatory control. Therefore, supplementation 
with cocoa, which is rich in flavonoids, could reduce neuropathic damage and improve the 
quality of life of those affected, thereby reducing the costs associated with the treatment of 
diabetes and its complications. (39, 43) For these reasons, the objective and quantitative 

evaluation of DN is crucial to establish potential beneficial effects in patients. This study 
proposes implementing the RDD of the H-reflex test as an indicator of alterations in sensory 
processing at the spinal cord level and nerve fibers, according to previous results in animal 
models of experimental diabetic neuropathy and in humans with T1D. (17, 18) Additionally, this 
research will be complemented by the application of quality-of-life instruments, and clinical 
evaluation. Therefore, the research question for this study is: What is the effect of cocoa 
supplementation on the biochemical and clinical profile and the somatosensory processing of 
peripheral and autonomic diabetic neuropathy? 
3.1 Relevance 
DM has a prevalence of 10.3% in Mexico, and of this percentage, between 29 and 95% develop 
diabetic neuropathy (9), which causes pain, sensory and autonomic abnormalities and 
therefore decreases the quality of life of those who suffer from it. Studies have demonstrated 
the beneficial effect of polyphenols on neuropathic damage; however, to date their effect has 
not been studied objectively and quantitatively. This study considers the evaluation of three 
important aspects in the evaluation of the effect of cocoa consumption in patients with DN: the 
biochemical and clinical profile and somatosensory processing. In this sense, this project is 
comprehensive and innovative, and the results obtained could serve as indicators of the 
possible mechanisms by which flavonoids contribute to the improvement of metabolic control 
and therefore to the reduction of symptoms of peripheral and autonomic DN. 
3.2 Feasibility 
In order to carry out this research project, work will be carried out at the facilities of the Anáhuac 
México Norte University, at the Kidney Specialty Center (State of Mexico) and at the Nueva 
Oxtotitlán clinic (Toluca), where the tests will be carried out with the patients. The equipment 
to carry out the electrophysiology tests (electrical recording and stimulation) is already 
available, as well as the cocoa capsule supplement, so the study can be carried out without 
setbacks. Cocoa supplementation does not affect the patients' dietary regimen or compromise 
their regular medication. In addition, the RDD of the H reflex test does not produce adverse 
effects, is painless and noninvasive. 
4. STATEMENT OF THE PROBLEM 

T2D is associated with the development of chronic-degenerative complications such as 
diabetic peripheral and autonomic neuropathy. This manifests itself as a set of disorders that 
occur as a consequence of a chronic state of hyperglycemia that favors a state of inflammation 
and oxidative stress, resulting in damage to the peripheral and autonomic nervous system. 
This hyperglycemia contributes to the increase in the inflammatory state and the production of 
reactive oxygen species, as well as to the increase in oxidative phosphorylation and overload 
of the electron transport chain. Among the cells that are most affected are the Schwann cells, 
which, when damaged, cause alterations in the functioning of the axons and nerve fibers. 
Flavonoids are phenolic compounds present in foods such as cocoa, and whose effects are 
recognized as antioxidant and anti-inflammatory mechanisms. Additionally, in preclinical 
studies, their consumption has been associated with a decrease in hyperglycemia and insulin 
resistance, improvement in mitochondrial function and attenuation of autonomic diabetic 
neuropathy, so they could be very useful in these patients. 
The Hoffman reflex objectively and noninvasively assesses the response of the somatosensory 
system. A study in subjects with T1D reported that the H reflex evoked by paired pulses in the 
lower extremities may be an indicator of dysfunction in somatosensory processing at the spinal 
level. To date, few studies have evaluated the dysfunction of sensory and autonomic fibers 
simultaneously in DN, so this study would provide valuable data since it additionally includes 
the evaluation of markers related to oxidative stress and inflammation, in addition to 
considering the evaluation of symptoms reported by subjects with peripheral and autonomic 
DN during 3 months of cocoa supplementation through a double-blind, randomized controlled 
clinical trial. 
5. RESEARCH QUESTION 
What is the effect of cocoa supplementation on the biochemical and clinical profile and 
somatosensory processing of diabetic peripheral and autonomic neuropathy? 
6. HYPOTHESIS 
Cocoa supplementation has a beneficial impact on the biochemical and clinical profile and 
somatosensory processing of diabetic peripheral and autonomic neuropathy. 
7. GENERAL OBJECTIVE 

To evaluate the effect of cocoa supplementation on the biochemical and clinical profile, and 
somatosensory processing of diabetic peripheral and autonomic neuropathy during 3 months 
of treatment. 
7.1 SPECIFIC OBJECTIVES 
Biochemical profile: 
- To evaluate the effect of cocoa supplementation on the glycemic profile (HbA1c and fasting 
blood glucose). 
- To evaluate the effect of cocoa supplementation on lipid profile (triglycerides, HDL-c, LDL-c 
and TG/HDL). 
- To evaluate the effect of cocoa supplementation on markers of inflammation 
(neutrophil/lymphocyte ratio). 
Clinical profile: 
- To evaluate the effect of cocoa supplementation on the “Toronto Clinical Scoring System” 
score. 
- To evaluate the effect of cocoa supplementation on anthropometry (weight, waist and 
abdominal circumference, waist to height ratio). 
- To evaluate the effect of cocoa supplementation on blood pressure. 
- To evaluate the effect of cocoa supplementation on quality of life. 
- To evaluate the effect of cocoa supplementation on gastrointestinal symptoms. 
Somatosensory processing: 
- To evaluate the effect of cocoa supplementation on sensorimotor processing in the spinal 
cord using the RDD of the H reflex test. 
8. METHODOLOGY 
8.1 Study Type: Explanatory 
8.2 Design: Double-blind, randomized controlled clinical trial. 

8.3 Study population: Adult subjects aged 40-60 years diagnosed with type 2 diabetes 
mellitus and diabetic neuropathy who attend, by open call, to Anáhuac México Norte 
University, the Kidney Specialty Center in the State of Mexico, and the Nueva Oxtotitlán clinic 
(Toluca). 
8.4 Sample Size Calculation: 
 
 
A total of 18 subjects per group were obtained, considering the following data: 
s= Baseline standard deviation: 0.84 (obtained from a study in which probucol and 
methylcobalamin are used in subjects with DN and the TCSS is used to assess the effect of 
the intervention.) (49) 
d= Proposed difference: 0.776 (refers to the expected difference → 7.5% decrease with 
respect to the control group) 
Zα= 1.96 (value in tables, corresponding to a significance level of 95% for two tails) 
Zβ= 0.84 (value in tables, corresponding to a power of 80%) 
8.5 Selection criteria 
Inclusion Criteria 
Non-inclusion Criteria 
- Adults aged 40-60 years with a diagnosis 
of type 2 diabetes mellitus (T2D) and 
diabetic neuropathy   
- Minimum duration of T2DM diagnosis: 5 
years.  
- MNSI (Michigan Neuropathy Screening 
Instrument) score ≥ 2.  
- Both genders.  
- Must sign the informed consent form 
- Subjects with known sensitivity or 
hyperreactivity to cocoa or any of its 
components.  
- Pregnant women.  
- Subjects with neurological diseases, 
such as multiple sclerosis, epilepsy, or 
previous cerebrovascular events.  
- Subjects 
with 
implanted 
electronic 
devices, e.g., pacemakers.  
- Subjects with peripheral nerve damage in 
the lower extremities caused by trauma.  
𝑵=
𝟐(𝒁𝜶+𝒁𝜷𝟐)𝒔𝟐
𝒅𝟐
 = 𝑵=
𝟐(𝟏.𝟗𝟔+𝟎.𝟖𝟒𝟐)(𝟎.𝟖𝟒)𝟐
(𝟎.𝟕𝟕𝟔)𝟐
 = 𝑵=
(𝟏𝟓.𝟔𝟖)(𝟎.𝟕𝟎𝟓𝟔)
(𝟎.𝟔𝟎𝟐𝟏)
 

- Subjects with a diagnosis of disc 
herniation below T10.  
- History/diagnosis of herpes zoster.  
- Serological diagnosis of rheumatoid 
arthritis 
or 
systemic 
lupus 
erythematosus. HIV/AIDS.  
- Venous 
insufficiency 
(C2-C6 
class, 
asymptomatic/symptomatic according to 
Nicolaides CEAP classification) in lower  
limbs.  
- Clinical hypothyroidism.  
- Use of orthopedic equipment (e.g., 
prosthetics, 
splints, 
wheelchair, 
crutches).  
- Cancer diagnosis, use of oncological 
drugs and/or radiotherapy, or history of 
cancer or tumors treated with oncological 
drugs or radiotherapy.  
- Smoking, considered positive in those 
with a smoking index greater than 10.  
- History of diabetic foot (Wagner Scale 3-
5) or amputation. 
- Presence of ulcers and open and/or 
extensive wounds (>2 cm)  
- Allodynia  
- Sciatic nerve root compression  
- Gout/Hyperuricemia 
Elimination Criteria 
- Subjects who modify their pharmacological treatment during the study.  
- 
Subjects who do not attend one of the intermediate consultations  
- 
Subjects who withdraw informed consent or decide not to continue participating in the 

study  
- Subjects with less than 80% adherence to cocoa supplementation  
- Subjects who miss more than one consultation or do not attend the final consultation. 
 
8.6 Study Variables 
Type  
Variable 
Conceptual 
Definition 
Operational Definition 
Indicator 
(unity) 
Scale 
Independent 
Cocoa 
supplementation 
Cocoa powder 
capsules 
4 cocoa powder capsules providing 
50 mg of flavonoids (12.5 mg per 
capsule), 
administering 
two 
capsules in the morning and two 
capsules at night, every day, for 
three months. (Appendix 2) 
Present 
(supplement) 
/ Absent 
(placebo) 
Qualitative 
nominal 
dichotomous 
Dependent 
Peripheral 
diabetic 
neuropathy 
 
 
 
 
 
 
Presence of signs 
and/or symptoms 
of peripheral 
nerve dysfunction 
in people with 
diabetes, after 
excluding other 
causes. (50) 
It is obtained after applying the MNSI 
questionnaire regarding symptoms 
(filled out by the patient and with a 
maximum score of 13 points) and the 
physical examination (evaluated by 
the researcher) with a maximum 
score of 13 points. (Appendix 3) (51) 
 
Toronto Clinical Scoring System: is a 
clinical evaluation system carried out 
by the researcher, assigning a score 
to the symptoms, reflexes and 
sensory tests. (33, 34) (Appendix 4) 
 
 
 
 
 
 
 
 
H reflex test: This is performed by 
electrical stimulation, placing the 
active electrode on the Achilles 
tendon, the positive electrode above 
the gastrocnemius and the reference 
electrode at the height of the 
gastrocnemius heads. Stimulation is 
carried 
out 
behind 
the 
knee 
(anatomical site of the tibial nerve). 
The electrical stimulus consists of 
the application of 10 pulses (1 every 
10 seconds) and the maximum 
intensity applied is according to the 
sensitivity and tolerance of each 
individual. (18, 36) 
Total sum of 
points obtained 
from the 
application of 
the MNSI 
questionnaire 
No unit 
 
6-8 points: mild DN 
9-11 points: 
moderate DN 
12-19 points: 
severe DN  
 
Total sum of 
points obtained 
from the 
application of 
the TCSS 
No unit 
 
 
Quotient 
R2/R1 
µV 
Discrete 
quantitative 
Qualitative ordinal 
Discrete 
quantitative 
Discrete 
quantitative 

Dependent 
Autonomic 
diabetic 
neuropathy 
Damage to the 
nerves that control 
internal organs, 
causing 
alterations in 
various systems 
such as the 
cardiovascular, 
gastrointestinal 
and urogenital 
systems. (20) 
- BEST questionnaire: Includes: 1) 
severity of intestinal symptoms; 2) 
impact of symptoms on the ability to 
enjoy everyday things; 
3) patient perception of the condition; 
and 4) impact of symptoms on mood 
and humor. Scale from 0 to 100 
according 
to 
the 
subject's 
responses. (Appendix 5) (52) 
 
-Bristol scale: It is made up of 6 
categories that include an image and 
an explanation, ranging from 1 to 7, 
with 1 being hard, separated pieces 
that pass with difficulty and 7 being 
watery stools. (Appendix 6) (53) 
No unit 
 
 
 
 
 
 
 
 
 
No unit 
Continuous 
quantitative 
Discrete 
quantitative 
Dependent 
Weight 
Total body mass 
of an individual 
(54) 
Weight of an individual in kg 
determined 
by the scale. 
The 
measurement is carried out without 
shoes 
and 
with 
the 
minimum 
possible clothing. The subject must 
be placed in the center and remain 
motionless during the measurement. 
(54) 
kg 
Continuous 
quantitative 
Dependent 
Waist 
circumference 
Body 
measurement 
used as an 
indicator of central 
adiposity, as well 
as cardiovascular 
risk (55) 
The tape is placed in a horizontal 
plane around the waist, taking the 
midaxillary line as a reference, 
locating the midpoint between the 
lower costal margin and the highest 
lateral edge of the iliac crest. (55) 
cm 
Continuous 
quantitative 
Dependent 
Waist-to-height 
ratio (WtH)  
Index resulting 
from dividing the 
waist 
circumference by 
the height. (74) 
It is obtained by dividing the waist 
circumference in cm by the height in 
cm, with a result of ≥ 0.5 indicating 
an increased risk for cardiometabolic 
disease. (74) 
No unit 
Continuous 
quantitative 
Dependent  
Abdominal 
circumference  
Body 
measurement that 
directly correlates 
with visceral fat. 
(54) 
The top of the hip bone and the top 
of the right iliac crest are located and 
the 
tape 
measure 
is 
placed 
horizontally around the abdomen, at 
the level of the iliac crest, at the end 
of a normal expiration. (55) 
cm 
Continuous 
quantitative 

Dependent 
Arterial Pressure 
Hydrostatic force 
of blood on 
arterial walls 
resulting from the 
pumping function 
of the heart, blood 
volume, 
resistance of the 
arteries to flow, 
and diameter of 
the arterial bed. 
(56) 
A sphygmomanometer is used to 
obtain blood pressure using the 
technique specified in the Clinical 
Practice Guidelines for the diagnosis 
and 
treatment 
of 
arterial 
hypertension in the primary care 
level. (57) 
mmHg 
Continuous 
quantitative 
Dependent 
Neutrophil/ 
lymphocyte ratio 
Index resulting 
from the division 
of the serum 
concentration of 
neutrophils by the 
serum 
concentration of 
lymphocytes. (75) 
It is obtained by dividing the serum 
concentration 
of 
the 
absolute 
number of neutrophils by the serum 
concentration 
of 
the 
absolute 
number of lymphocytes. (75) 
No unit 
Continuous 
quantitative 
Dependent 
Glycemia 
Blood glucose 
concentration (58) 
Obtained from laboratory study by 
spectrophotometry of blood sample, 
with a fasting period of 8 hours. (59) 
mg/dL 
Continuous 
quantitative 
Dependent 
Glycated 
hemoglobin 
(HbA1c) 
Value of the 
fraction of 
hemoglobin that 
has glucose 
attached (58) 
Analyzed by high-performance liquid 
chromatography (HPLC), without a 
specific fasting period for this test. 
(62) 
% 
Continuous 
quantitative 
Dependent 
Triglyceridemia 
Blood triglyceride 
concentration  
(58) 
Obtained from a laboratory study by 
spectrophotometry of the lipid profile 
of a blood sample, with a 12-hour 
fasting period. (59) 
mg/dL 
Continuous 
quantitative 
Dependent 
High density 
cholesterol (HDL-
c) 
HDL-c 
concentration in 
blood. 
Obtained from a laboratory study by 
spectrophotometry of the lipid profile 
of a blood sample, with a 12-hour 
fasting period. (59) 
mg/dL 
Continuous 
quantitative 
Dependent 
Low density 
cholesterol (LDL-
c) 
LDL-c 
concentration in 
blood. 
Obtained from a laboratory study 
using spectrophotometry of the lipid 
profile of a blood sample, with a 
fasting period of 12 hours. (59) 
mg/dL 
Continuous 
quantitative 
Dependent 
Triglycerides/ 
HDL-c ratio 
(TG/HDL) 
Index resulting 
from the division 
of the serum 
concentration of 
triglycerides by 
the serum 
concentration of 
HDL (63) 
It is performed after dividing the 
serum triglyceride concentration in 
mg/dL 
by 
the 
serum 
HDL 
concentration in mg/dL.(63) 
No unit 
Continuous 
quantitative 
Dependent 
Diabetes 39 
questionnaire 
Questionnaire 
designed to 
measure quality of 
life in patients with 
type 1 and type 2 
diabetes. (64) 
Questionnaire containing 39 closed 
items grouped into 5 sections: 
energy 
and 
mobility, 
diabetes 
control, anxiety-worry, social burden 
and sexual functioning. Subjects rate 
their perception of their quality of life 
No unit 
Discrete 
quantitative 

in a global manner, with a range from 
1 (minimal) to 7 (extremely severe), 
and 
the 
scores 
obtained 
are 
transformed into a scale of 0 to 100. 
(Appendix 7) (64) 
Confounding 
Hypoglycemic 
drugs 
Drugs that lower 
or regulate 
glucose. (65) 
The use of hypoglycemic drugs will 
be considered when the patient is 
taking any drug from the following 
groups: 
Sulfonylureas 
(glibenclamide), 
biguanides 
(metformin), 
glinides, 
thiazolidinediones 
or 
glitazones, 
GLP-1 analogues, DPP4 inhibitors 
(sitagliptin) and alpha glucosidase 
inhibitors (acarbose). (66) 
Present/ Absent 
Qualitative 
nominal 
dichotomous 

8.7 STRATEGIC PLAN 
Through an open invitation, subjects living with T2D who meet the inclusion criteria will be 
invited to be assessed and tested at Anáhuac México Norte University, the Kidney Specialty 
Center, and the Nueva Oxtotitlán Clinic (Toluca), as appropriate. The invitation will be 
extended, explaining the procedures, duration, and ethical issues; if the subject decides to 
participate, he or she will be asked to sign the informed consent letter (Annex 1). Once the 
subject enters the protocol, he or she will be randomized (using a random number table 
previously generated in a spreadsheet) to the experimental group (diet + capsules with 
cocoa) or the control group (diet + capsules with methylcellulose). 
The study will last three months and patients will be seen on four occasions: at the beginning 
(baseline measurement), at the first and second month (review appointments) and at the 
third month (final measurement). 
Patients will be asked to attend the appointments on an empty stomach, after having 
urinated and defecated. Weight will be determined according to the Lohman technique, that 
is, without shoes and with as few clothes as possible, the individual will be placed in the 
center of the scale and must remain still during the measurement. (54) Waist circumference 
will be measured according to the proposal of the Ministry of Health of Mexico: placing the 
tape in a horizontal plane around the waist, taking as reference the mid-axillary line, locating 
the midpoint between the lower costal margin and the highest lateral edge of the iliac crest 
(55) and the abdominal circumference: the upper part of the hip bone and the upper part of 
the right iliac crest are located and the measuring tape is placed in a horizontal plane around 
the abdomen, at the level of the iliac crest, at the end of a normal expiration. (55) Likewise, 
blood pressure will be measured using a sphygmomanometer (Nebucor brand, model HL-
888JA), according to the Clinical Practice Guide for the Diagnosis and Treatment of Arterial 
Hypertension in the first level of care (57). These evaluations will be carried out every month, 
until completing the three months of the duration of the study. In addition, at the beginning 
of the study the MNSI (Annex 3) will be applied as part of the diagnosis of DN, and both at 
the beginning and at the end, the TCSS (Annex 4), the questionnaire as the physical 
examination (51), the BEST questionnaire (Annex 5), the Bristol scale (Annex 6) and the 
Diabetes 39 quality of life questionnaire (Annex 7) will be applied and laboratory studies will 
be taken to know the biochemical parameters (triglyceridemia, HDL-c, LDL-c, glycemia, 

HbA1c, TG / HDL index and neutrophil/lymphocyte ratio will be determined, as described in 
the table of variables. 
Participants in both groups, being subjects with a cardiometabolic disease, will be 
nutritionally assisted, that is, all patients will be given a meal plan according to the 
characteristics of each one of them, calculated by the Mifflin-St. Jeor energy expenditure 
prediction formula (67) for subjects with obesity, while, according to the Joslin Diabetes 
Center, a distribution of 40-45% carbohydrates, 30-40% lipids, and 20-30% protein is 
suggested. (68) Two capsules will be prescribed – cocoa for the experimental group or 
methylcellulose for the control group – in the morning and two capsules at night. 
8.7.1 H-reflex test  
The H-reflex test will be performed by electrical stimulation through disposable surface 
electrodes (3M) connected to a bipolar constant current electrical stimulator (Digitimer 
DS8R). Electrophysiological signals will be recorded using surface electrodes (3M) 
connected to the signal acquisition and amplification system (LabChart and PowerLab 8/35, 
ADInstruments). The signals obtained will be sampled at 10 KHz with a band-pass filter of 
0.5-500 Hz. The signals will be stored in a computer for later analysis. Once the recording 
and stimulation sites have been identified (see below), the subject's skin will be cleaned with 
an alcohol swab. Micropore® adhesive tape will be used to improve contact between the 
electrodes and the subject's skin. The electrodes for stimulation will be placed as follows: 
the active electrode (anode) at the level of the Achilles tendon, the positive electrode 
(cathode) above the inverted "V" between the calf muscles (gastrocnemius). The reference 
electrode will then be placed at the height of the heads of the gastrocnemius. The subject 
will be stimulated behind the knee, where the tibial nerve runs through its anatomical course. 
To begin the tests, the subject will be asked to indicate with his hand to the researcher when 
he perceives a tolerable physical sensation due to the applied current. The test will start with 
an intensity of 0 mA and then pulses will be given every 0.5 mV until the evoked potential 
(H reflex) is observed in a consistent and clearly identifiable manner based on the latency 
(35-45 ms). The electrical stimulus consists of the application of 1 square pulse (1 ms 
duration each pulse) every 10 seconds (10 pulses in total). The maximum intensity of the 
applied current will be in accordance with the sensitivity and tolerance of the individual in 
both lower limbs during the tests on the sensory and motor nerves. The applied electrical 

pulse should not cause a painful sensation, but it may cause a tingling sensation. The test 
will be suspended if the individual reports pain or does not wish to continue with the research 
protocol. For the recording of the H reflex, the active electrode will be placed between the 
lateral malleolus and the Achilles tendon. Next, the reference electrode will be marked below 
it and 14 cm will be measured from the active electrode towards the middle of the calf. The 
“H” reflex test will be performed in two parts. The first part of the protocol consists of 
determining the curve of stimulus intensity vs. amplitude of the motor responses from the 
appearance of the “M” wave and the “H” wave, for which the electric current will be increased 
in steps of 0.5 µA until the appearance of the waves. For this part, only one electric pulse (1 
ms duration) will be given every 10 seconds. The intensity of the electric current that will be 
used for the second part of the protocol will be that whose value on the curve of amplitude 
of the H wave-electric current intensity reaches 60% of the maximum amplitude. This 
stimulation value guarantees the reproducibility and minimum variability of this wave, which 
also avoids muscle contraction that contaminates the electrical recording. The second part 
is the paired electrical stimulation test in which two electrical pulses (1 ms duration) will be 
produced at different frequencies between the pulses: 0.1, 1, 5 and 10 Hz. The interval 
between the paired pulses will be 10 s, until completing 10 series. 
Electrophysiological recordings will be analyzed with Clampfit 10.0 software. The latency 
and amplitude of the H1 and H2 evoked potentials will be determined for each electrical 
pulse and at all stimulation frequencies, taking the stimulus artifact as a reference. The ratio 
of the amplitude of the paired H2/H1 pulses will then be determined to establish the 
modulation of spinal excitability. A ratio ≥ 0.6 for any stimulation frequency will be considered 
as an indicator of dysfunction in somatosensory processing according to Marshall et al. (18) 
8.8 STATISTICAL ANALYSIS 
A descriptive analysis of all dependent variables, as well as demographic variables, will be 
performed, expressing them as mean ± standard deviation or, where appropriate, as median 
and range (min-max), according to the normality of the data; nominal variables will be 
expressed as frequencies. Subsequently, an intragroup inferential analysis will be carried 
out for weight, waist circumference, abdominal circumference and blood pressure with 
ANOVA for repeated samples with Tukey post hoc, and in case the data distribution does 
not meet the normality criteria, the corresponding nonparametric test will be performed (e.g., 

Friedman, followed by Dunn's post hoc). For biochemical analysis (glycemic and lipid profile 
and markers of inflammation), as well as the evaluations corresponding to diabetic 
peripheral and autonomic neuropathy, and quality of life, a Student's T test will be performed 
for dependent groups, or where appropriate, with Willcoxon. 
The intergroup comparison will be made with Student's T test for independent samples, or 
if applicable, with Mann-Whitney U at each measurement time. A p <0.05 will be considered 
as statistical significance. All of the above will be carried out using version 5 of the GraphPad 
Prism software. 
9. ETHICAL CONSIDERATIONS 
This study complies with the guidelines stipulated in the International Ethical Guidelines for 
Biomedical Research Involving Human Subjects, in the Declaration of Helsinki (69) and the 
Nuremberg Code (70), as well as the NOM-012-SSA3-2012. (71) In terms of national 
regulation, it adheres to the Regulations of the General Health Law on Research, within 
whose article 17, this protocol is considered research with greater than minimum risk due to 
the random method of assignment to the intervention scheme. (72) Considering the level of 
risk, and in accordance with the provisions of article 14, informed consent must be in writing, 
so each subject who participates in the study, as detailed in the Methodology section, will 
be informed and explained the procedures to be carried out in order to subsequently record 
in writing their decision to participate in an informed consent letter, in accordance with 
Chapter I of the General Health Law regarding the ethical aspects of research in human 
beings (Annex 1). 
Likewise, the protocol will be submitted to the research and Bioethics committees of the 
Faculty of Health Sciences of the Universidad Anáhuac México Norte. 
A nutrition plan will be given for the prevention or correction of obesity and to contribute to 
metabolic control, as indicated by the Mexican Official Standard 015-SSA2-2010 for the 
Prevention, Treatment and Control of Diabetes Mellitus. (76) 
10. SCHEDULE OF ACTIVITIES 

ACTIVITY 
August-
December 
2020 
January – 
December 
2021 
January 
– June 
2022 
July – 
December 
2022 
January – 
June 2023 
Submission, evaluation, 
correction 
Health Science Faculty 
Committee protocol 
X 
X 
Patient recruitment and 
experimental development 
X 
Information coding and 
emptying in the database 
X 
X 
Statistical analysis 
X 
Preparation of final report 
X 
Publication of Results and 
obtaining a degree 
X 
 
11. BUDGET 
Institutional financing will be requested (from the CICSA of the UAMN), subject to approval 
of this document, according to the following breakdown: 
Stationery 
$4,000 
Cocoa capsules 
$104,000 * 
Placebo capsules 
$5300* 
ELISA kits (TNFa, IL-6, IL-10, IL-1b) 
$128,000 
Kits (MDA, carbonils, TAS) 
$55,000 
Laboratory studies (glycemic and lipidic profile and CRP) 
$64,430** 
Statistical software (annuity) 
$5,600 
Disposable electrodes for surface recording 
$15,000 
Computer, measuring tape 
$13,500* 
Electrophysiology equipment 
$850,000* 
Electromyography/nerve conduction velocity studies 
$5,000 – 10,000* 
TOTAL  
$1’254,830.00 
TOTAL TO REQUEST 
$207,600.00 
* They are already counted on 
** They are already included as part of the institutions 
 
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Ellinger S, Stehle P. Impact of cocoa consumption on inflammation processes – A critical 
review of randomized controlled trials. Nutrients [Internet]. 2016;8:321. doi:10.3390/nu8060321. 
43. 
Bai L, Li X, He L, et al. Antidiabetic potential of flavonoids from traditional chinese medicine: 
a review. Am J Chin Med [Internet]. 2019;47:933-57. doi: 10.1142/S0192415X19500496. 
44. 
Munguía L, Gutiérrez-Salmeán G, Hernández M, et al. Beneficial effects of a flavonol-
enriched cacao beverage on anthropometric and cardiometabolic risk profile in overweight subjects. 
Rev 
Mex 
Cardiol 
[Internet]. 
2015;26(2):78-86. 
URL: 
https://www.medigraphic.com/cgi-
bin/new/resumenI.cgi?IDARTICULO=58908 
45. 
Andújar I, Recio M, Giner R, Ríos J. Cocoa polyphenols and their potential benefits for 
human health. Oxid Med Cell Longev [Internet]. 2012;2012:23. doi: 10.1155/2012/906252. 
46. 
De Araujo Q, Gattward J, Almoosawi S, et al. Cacao and Human Health: from Head to Foot 
— A Review. Crit Rev Food Sci Nutr [Internet]. 2013. doi: 10.1080/10408398.2012.657921. 
47. 
De Feo M, Paladini A, Ferri C, et al. Anti-inflammatory and anti-nociceptive effects of cocoa: 
a review on future perspectives in treatment of pain. Pain Ther [Internet]. 2020;9:231-40. doi: 
/10.1007/s40122-020-00165-5. 
48. 
Addepalli V, Suryavanshi SV. Catechin attenuates diabetic autonomic neuropathy in 
streptozotocin induced diabetic rats. Biomed Pharmacother [Internet]. 2018;108:1517-23. doi: 
10.1016/j.biopha.2018.09.179. 
49. 
Peng H, Gong Y. Analysis of the effect of probucol-mecobalamin tablets combination on 
oxidative stress in patients with diabetic peripheral neuropathy. Neurosci Lett [Internet]. 
2021;741:135484. doi: 10.1016/j.neulet.2020.135484.  
50. 
Samper Bernal D, Monerris Tabasco MM, Homs Riera M, Soler Pedrola M. Etiología y 
manejo de la neuropatía diabética dolorosa. Rev Soc Esp Dolor [Internet]. 2010;17(6):286-96. doi: 
10.1016/j.resed.2010.06.002. 
51. 
Feldman EL, Stevens MJ, Thomas PK, et al. A practical two-step quantitative clinical and 
electrophysiological assessment for the diagnosis and staging of diabetic neuropathy. Diabetes 
Care [Internet]. 1994;17(11):1281-9. doi: 10.2337/diacare.17.11.1281. 

52. 
Mearin F, Montoro M. Síndrome de Intestino Irritable. Gastroenterology [Internet]. 
2006;130:S1040. 
URL: 
https://www.aegastro.es/sites/default/files/archivos/ayudas-
practicas/36_Sindrome_de_intestino_irritable.pdf. 
53. 
Mearin F, Ciriza C, Mínguez M, et al. Guía de práctica clínica: síndrome del intestino irritable 
con estreñimiento y estreñimiento funcional en adultos: concepto, diagnóstico y continuidad 
asistencial. 
(Parte 
1 
de 
2). 
Aten 
Primaria 
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2017;49(1):42-55. 
doi: 
10.1016/j.aprim.2016.11.003. 
54. 
Suverza A, Haua K. El ABCD de la evaluación del estado de nutrición. México: Mc Graw 
Hill; 2010. 331 p. 
55. 
Diagnóstico y Tratamiento del Sobrepeso y la Obesidad Exógena. Guía de Práctica Clínica, 
México, 
CENETEC; 
2018 
[01 
septiembre 
2020]. 
Disponible 
en: 
http://www.imss.gob.mx/sites/all/statics/guiasclinicas/046GER.pdf 
56. NORMA Oficial Mexicana NOM-030-SSA2-2009, Para la prevención, detección, diagnóstico, 
tratamiento y control de la hipertensión arterial sistémica. Diario Oficial de la Federación, (31-May-
2010). 
57. Diagnóstico y Tratamiento de la Hipertensión Arterial en el Primer Nivel de Atención. México: 
Instituto Mexicano del Seguro Social; 08/07/2014. 
58. Fuentes Arderiu X. Ciencias de laboratorio clínico. Conceptos esenciales. España: Grupo de 
Trabajo Iberoamericano de Nomenclatura y Traducciones; 2015. 214 p. 
59. Velázquez Salgado R. Manual de Prácticas Bioquímica Clínica. México; 2009. 160 p. 
60. Garmendia ML, Lera L, Sánchez H, et al. Valores normativos de resistencia a la insulina 
mediante HOMA-IR en adultos mayores de Santiago Chile. Rev Med Chile. [Internet]. 
2009;137:1409-16. doi: 10.4067/S0034-98872009001100001. 
61. Arancibia C, Galgani J, Valderas JP, et al. Evaluación de la insulinemia post carga oral de 
glucosa como método diagnóstico de resistencia a la insulina. Rev Med Chile [Internet]. 
2014;142:1106-12. URL: https://scielo.conicyt.cl/pdf/rmc/v142n9/art03.pdf 
62. Campuzano-Maya G, Latorre-Sierra G. La HbA1c en el diagnóstico y en el manejo de la 
diabetes. 
Med 
Lab. 
[Internet]. 
2010;16(5–6):211–41. 
URL: 
https://www.medigraphic.com/pdfs/medlab/myl-2010/myl105-6b.pdf 
63. Baez-Duarte BG, Zamora-Ginez I, González-Duarte R, et al. Triglycerides/high-density 
lipoprotein cholesterol (TG/HDL-C) index as a reference criterion of risk for metabolic syndrome 
(MetS) and low insulin sensitivity in apparently healthy 
subjects. 
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Med 
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[Internet]. 
2017;153:152-8. 
http://www.anmm.org.mx/GMM/2017/n2/GMM_153_2017_2_152-158.pdf 
64. López-Carmona J, Rodríguez-Moctezuma R. Adaptación y validación del instrumento de calidad de 
vida Diabetes 39 en pacientes mexicanos con diabetes mellitus tipo 2. Salud Publica Mex [Internet]. 
2006;48(3):200-11. URL: https://saludpublica.mx/index.php/spm/article/view/6689/8329. 
65. Dalama B, Mesa J. Nuevos hipoglucemiantes orales y riesgo cardiovascular. Cruzando la 
frontera metabólica. Rev Esp Cardiol. 2016;69(11):1088-97. doi: 10.1016/j.recesp.2016.07.029. 

66. Tratamiento de la Diabetes Mellitus tipo 2 en el primer nivel de Atención. México: Instituto 
Mexicano del Seguro Social; 08/07/2014. 
67. Mifflin M, St Jeor S, Hill L, al e. A new predictive equation for resting energy expenditure in 
healthy individuals. 
 Am J Clin Nutr [Internet]. 1990;51:241-7. doi: 10.1093/ajcn/51.2.241. 
68. Joslin Diabetes Center. Clinical Guidelines United States2018 [Available from: https://joslin- 
prod.s3.amazonaws.com/www.joslin.org/assets/2020-07/2ebdm_joslin_chapter_2.pdf]. 
69. Asociación 
Médica 
Mundial. 
Declaración 
de 
Helsinki. 
1964; 
Disponible 
en: 
http://www.conamed.gob.mx/prof_salud/pdf/helsinki.pdf 
70. Tribunal Internacional de Nuremberg. Código de Nuremberg. 1947; Disponible en: 
http://www.conbioeticamexico.salud.gob.mx/descargas/pdf/normatividad/normatinternacional/2.INT
L._Cod_Nuremberg.pd f 
71. NORMA Oficial Mexicana NOM-012-SSA3-2012, Que establece los criterios para la ejecución 
de proyectos de investigación para la salud en seres humanos. Diario Oficial de la Federación, (04-
Ene-2013). 
72. Estados Unidos Mexicanos. Reglamento de la Ley General de Salud en Materia de 
Investigación para la Salud. 1984;1–23. 
73. NORMA Oficial Mexicana NOM-015-SSA2-2010, para la Prevención, Tratamiento y Control de 
la diabetes mellitus. Diario Oficial de la Federación, (23-Nov-2010) 
74. Browning LM, Hsieh SD, Ashwell M. A systematic review of waist-to-height ratio as a screening 
tool for the prediction of cardiovascular disease and diabetes: 0.5 could be a suitable global 
boundary value. Nutr Res Rev [Internet]. 2010;23(2):247–69. doi: 10.1017/S0954422410000144. 
75. Buonacera A, Stancanelli B, Colaci M, Malatino L. Neutrophil to Lymphocyte Ratio: An Emerging 
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2022;23. doi: 10.3390/ijms23073636. 
  
  

13. Appendices 
 
Appendix 1: Informed Consent  
 
Informed Consent 
“EVALUATION OF THE EFFECT OF COCOA SUPPLEMENTATION ON THE 
BIOCHEMICAL AND CLINICAL PROFILE AND SENSORY-MOTOR 
PROCESSING OF PERIPHERAL AND AUTONOMIC DIABETIC 
NEUROPATHY: RANDOMIZED CLINICAL TRIAL” 
 
 
Date:   
 
 
The study you are being invited to participate in will evaluate the effect of cocoa supplementation on diabetic 
peripheral and autonomic neuropathy over a 3-month period of treatment.  
The study will consist of the following: once a month—and until completing 3 months of treatment—you will 
be asked to come in for a consultation, where the following assessments will be performed: a) routine 
anthropometric measurements (weight, height, and abdominal circumference), b) blood pressure, c) a 
noninvasive study—called the paired H-reflex test—to assess changes in your nerves, d) a questionnaire 
about your quality of life, and f) a scale for the shape and consistency of your bowel movements—called 
the Bristol scale. None of the above assessments will cause you any side effects or pain.  
In addition, laboratory tests will be performed at the start of treatment and at the end of the three months, 
during which glycated hemoglobin, glucose, insulin, blood lipids, as well as markers of inflammation and 
oxidative stress will be assessed. 
Once these evaluations are completed, you will be provided with an individualized dietary plan and given 
the capsules to take daily for the duration of the three months. At this point, you should be aware that you 
may be randomly assigned to receive either the active ingredient capsules or placebo capsules (you won't 
be able to tell because they both look the same); this information will be disclosed only at the end of the 
study. 
It should be noted that your participation in the study and the tests and treatments provided as part of it will 
not represent any cost to you; however, you will not receive any payment. Your participation in this study is 
of great importance to us, but it is not mandatory. You have the right to withdraw this consent at any time, 
without affecting the care and attention provided at the Institution. 

All information you provide for the study will be strictly confidential in accordance with the General Health Law on 
health research. It will be used only by the project's research team and will not be made available for any other 
purpose. You will be identified by a number, not your name. The results of this study will be published for scientific 
purposes, but will be presented in a manner that prevents you from being identified. 
We are committed to answering any questions you may have regarding the study procedure at any time (such as 
evaluation procedures, risks, benefits, etc.), as well as providing you with any information obtained during the 
study. Finally, all information related to this project will be kept accessible only to the participating team as 
researchers. The personal information of each participating patient will be available at all times, but will be 
provided only to you. Therefore, please be assured that your data will be handled confidentially, respecting your 
anonymity. 
We thank you in advance for your cooperation and remain at your disposal for any questions or comments you 
may have. 
Carlos Alberto Cuéllar Ramos 
Senior researcher 
Phone number: 5548774277 
 
Gabriela Gutiérrez Salmeán 
Senior researcher 
Phone number:  55 28986740 
 
Rebeca Kababie Ameo 
PhD candidate in Nutritional Sciences  
Phone number:  55 85502972 
 
I,  
I have read and understood the above 
information, and my questions have been answered satisfactorily. I have been informed and understand the 
procedures that will be performed, as well as that the results obtained may be published and disseminated for 
scientific purposes.

Appendix 2:  Cocoa capsules 
 
 
 
 

Appendix 3 “The Michigan Neuropathy Screening Instrument” (MNSI) Instrument 
 
 

Appendix 4: Toronto Clinical Scoring System (TCSS) 
 
 
 
 

 
Appendix 5: BEST questionnaire 
 

Appendix 6: Bristol Scale 
 
 
 

Appendix 7: Quality of Life Questionnaire “Diabetes 39” 
La calidad de vida de las personas está afectada por muchas causas. Estas causas pueden incluir el estado de 
salud, la oportunidad para vacacionar o divertirse, los amigos, la familia o el trabajo. El siguiente cuestionario se 
diseñó para ayudar a conocer lo que afecta la calidad de vida en las personas con diabetes. 
Las siguientes preguntas se relacionan con el grado de afectación que la diabetes le ocasionó en su calidad 
de vida durante el último mes. Se le agradecerá que lea cuidadosamente las siguientes preguntas y conteste 
colocando una cruz (X) en el cuadro del número que refleje mejor el grado de afectación en su vida respecto a 
cada una de las preguntas señaladas, tomando en cuenta que el número 1 indica falta de afectación y, al 
avanzar la numeración, aumenta el grado de afectación en forma progresiva hasta llegar al máximo, que es el 
número 7, que indica afectación extrema. Si tiene alguna duda, con gusto se le prestará ayuda. 
Se le suplica responder todas las preguntas. 
 
Durante el último mes, ¿en qué medida se vio afectada la calidad de su vida por las siguientes causas? 
 
1. El horario de los medicamentos para su diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
2. Preocupaciones por problemas económicos 
Nada afectada en absoluto 
Sumamente afectada 
 
3. Limitación en su nivel de energía 
Nada afectada en absoluto 
Sumamente afectada 
 
 
4. Seguir el plan indicado por su médico para el tratamiento de la diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
 
5. No comer ciertos alimentos para poder controlar su diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
6. Estar preocupado(a) por su futuro 
Nada afectada en absoluto 
Sumamente afectada 
 
 
7. Otros problemas de salud aparte de la diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
 
8. Tensiones o presiones en su vida 
Nada afectada en absoluto 
Sumamente afectada 
 
 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 

9. Sensación de debilidad 
Nada afectada en absoluto 
Sumamente afectada 
 
 
10. Restricciones sobre la distancia que puede caminar 
Nada afectada en absoluto 
Sumamente afectada 
 
11. Los ejercicios diarios que ha de hacer por su diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
 
12. Visión borrosa o pérdida de la visión 
Nada afectada en absoluto 
Sumamente afectada 
 
 
13. No poder hacer lo que quisiera 
Nada afectada en absoluto 
Sumamente afectada 
 
 
Durante el último mes, ¿en qué medida se vio afectada la calidad de su vida por las siguientes causas? 
 
 
14. Tener diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
 
15. El descontrol de su azúcar en sangre 
Nada afectada en absoluto 
Sumamente afectada 
 
 
16. Otras enfermedades aparte de la diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
 
17. Hacerse análisis para comprobar sus niveles de azúcar en sangre 
Nada afectada en absoluto 
Sumamente afectada 
 
 
18. El tiempo requerido para controlar su diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
 
19. Las restricciones que su diabetes impone a su familia y amigos 
Nada afectada en absoluto 
Sumamente afectada 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 

 
20. La vergüenza producida por tener diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
 
21. La interferencia de su diabetes en su vida sexual 
Nada afectada en absoluto 
Sumamente afectada 
 
 
22. Sentirse triste o deprimido 
Nada afectada en absoluto 
Sumamente afectada 
 
 
23. Problemas con respecto a su capacidad sexual 
 
Nada afectada en absoluto 
Sumamente afectada 
 
 
24. Tener bien controlada su diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
 
25. Complicaciones debidas a su diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
 
26. Hacer cosas que su familia y amigos no hacen 
Nada afectada en absoluto 
Sumamente afectada 
 
 
27. Tener que anotar sus niveles de azúcar en sangre 
Nada afectada en absoluto 
Sumamente afectada 
 
 
28. La necesidad de tener que comer a intervalos regulares 
Nada afectada en absoluto 
Sumamente afectada 
 
 
29. No poder realizar labores domésticas u otros trabajos relacionados con la casa 
Nada afectada en absoluto 
Sumamente afectada 
 
 
30. Menor interés en su vida sexual 
Nada afectada en absoluto 
Sumamente afectada 
 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 

 
Durante el último mes, ¿en qué medida se vio afectada la calidad de su vida por las siguientes causas? 
31. Tener que organizar su vida cotidiana alrededor de la diabetes 
Nada afectada en absoluto 
Sumamente afectada 
 
 
32. Tener que descansar a menudo 
Nada afectada en absoluto 
Sumamente afectada 
 
 
33. Problemas al subir escaleras 
Nada afectada en absoluto 
Sumamente afectada 
 
 
 
34. Dificultades para sus cuidados personales (bañarse, vestirse o usar el sanitario) 
Nada afectada en absoluto 
Sumamente afectada 
1 
2 
3 
4 
5 
6 
7 
 
35. Tener el sueño intranquilo 
 
Nada afectada en absoluto 
Sumamente afectada 
1 
2 
3 
4 
5 
6 
7 
 
36. Andar más despacio que otras personas 
Nada afectada en absoluto 
Sumamente afectada 
1 
2 
3 
4 
5 
6 
7 
 
 
37. Ser identificado como diabético 
Nada afectada en absoluto 
Sumamente afectada 
1 
2 
3 
4 
5 
6 
7 
 
 
38. Interferencia de la diabetes con su vida familiar 
Nada afectada en absoluto 
Sumamente afectada 
1 
2 
3 
4 
5 
6 
7 
 
 
39. La diabetes en general 
Nada afectada en absoluto 
Sumamente afectada 
1 
2 
3 
4 
5 
6 
7 
 
 
 
 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 
1 
2 
3 
4 
5 
6 
7 

Calificación global 
 
1. 
Por favor, marque con una cruz (X) el cuadro que indique la calificación de su calidad de vida 
Mínima calidad 
Máxima calidad 
1 
2 
3 
4 
5 
6 
7 
 
2. 
Por favor, marque con una cruz (X) el cuadro que indique lo que usted piensa de la gravedad de su diabetes 
Ninguna gravedad 
Extremadamente grave 
1 
2 
3 
4 
5 
6 
7 
 
Gracias por sus respuestas