Randomized Controlled Trial Evaluating the Effect of Rosmarinus Officinalis Extract on Metabolic, Hepatic, Renal, Inflammatory, and Oxidative Stress Biomarkers in Patients With Type 2 Diabetes.
Phase: PHASE1
Conditions: Type 2 Diabetes Mellitus (T2DM), Oxidative Stress, Inflammation Chronic
Sponsor: Universidad Autonoma de Zacatecas

 
 
 
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Research Project 
Randomized Controlled Trial Evaluating the Effect of Rosmarinus Officinalis Extract on Metabolic, 
Hepatic, Renal, Inflammatory, and Oxidative Stress Biomarkers in Patients With Type 2 Diabetes.  
 
Responsible 
Dr. Blanca Patricia Lazalde Ramos 
Contributors 
M. Internist . Judisett Mirabal Rodríguez 
 Dr. Sol María Quirarte Báez 
Dr. Marisol Galván Valencia 
QFB. Diana Jazmín Soriano Gálvez 
M. Internist . Sandy Fidel Rodríguez Reyes 
 
 
 
 
 
 
Folio: CEI/ISSSTE/2025/043 
03/09/2025 

 
 
 
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INDEX 
LIST OF ABBREVIATIONS  ..................................................................................................................... iv 
TABLE OF CONTENTS  ..................................................................................................................... v 
INDEX OF FIGURES  .............................................................................................................................. vi 
SUMMARY  ......................................................................................................................................... vii 
1.  INTRODUCTION  .......................................................................................................................... 8 
2.  THEORETICAL FRAMEWORK  ..................................................................................................... 11 
2.1.  
Type 2 Diabetes Mellitus  ................................................................................................ 11 
2.1.1.  Metabolic effects of DM2  .......................................................................................... 11 
2.1.4 Risk factors for DM2  ........................................................................................................... 12 
2.1.2.  Association of DM2 with obesity  .............................................................................. 14 
2.1.3.  Main symptoms and signs of DM2 :  .......................................................................... 14 
2.1.4.  
Diagnostic criteria for DM2  ....................................................................................... 15 
2.1.5.  Complications of DM2  ............................................................................................... 16 
2.1.6.  Treatment ................................................................................................................... 17 
2.2.  
Rosmarinus officinalis L. (Romero)  ................................................................................ 19 
2.2.1.  Pharmacological Uses of Rosemary  .......................................................................... 19 
2.2.2.  Main secondary metabolites of Rosemary  ............................................................... 19 
2.2.3.  Hypoglycemic mechanisms of Romero  ..................................................................... 20 
7. METHODOLOGY  ............................................................................................................................ 29 
7.1.  
Type and design of the study  ......................................................................................... 29 
7.2.  
Stages of the study  ......................................................................................................... 29 
STAGE I. Preparation of the methanolic extract of Rosmarinus officinalis  .............................. 29 
STAGE II . Patient Recruitment  ................................................................................................. 30 
STAGE III. Intervention and follow-up of patients (  .................................................................. 30 
7.3.  
Operationalization of variables  ..................................................................................... 32 
Variables to be quantified  ....................................................................................................... 32 
Cardiovascular Assessment  ......................................................................................................... 41 
7.4.  
Clinical fieldwork  ............................................................................................................ 42 
7.5. Statistical Analysis  ................................................................................................................ 45 

 
 
 
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7.6. Writing 
and 
acceptance 
of 
protocol 
 
46 
7.7.  
Conflicts of interest  ........................................................................................................ 46 
7.8.  
Ethical considerations  .................................................................................................... 46 
7.9 .  
Human, physical and financial resources of the study  ................................................. 48 
Human Resources  .................................................................................................................... 48 
Equipment  ................................................................................................................................ 48 
7.10.  
Biosafety aspects  ............................................................................................................ 49 
9.  EXPECTED RESULTS AND IMPACTS.  .......................................................................................... 51 
10.  
BIBLIOGRAPHIC REFERENCES  .............................................................................................. 52 
11.  
ANNEXES .............................................................................................................................. 59 
Annex 1. Informed Consent  ......................................................................................................... 59 
Annex 2 .  
Initial clinical record  ............................................................................................... 61 
Annex 3.  
Laboratory sheet  .................................................................................................... 64 
Annex 4. Questionnaire on adverse effects  ................................................................................. 65 
Annex 5. Patient Instructions (Format for the proper administration of the assigned treatment) 
 ....................................................................................................................................................... 67 
 
 
 

 
 
 
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LIST OF ABBREVIATIONS 
CVD: Cardiovascular Disease 
DM 2: Type 2 Diabetes Mellitus 
RI: Insulin resistance () 
Carnosic Acid 
Rosmarinic Acid  
AGL: Free Fatty Acids 
TNF- α : Tumor necrosis factor alpha 
IL-6: Interleukin 6 
OBD: Obesity 
Tg : triglycerides 
Col: cholesterol 
MAD: Malondialdehyde 
SOD: Superoxide dismutase 
GSH: Reduced Glutathione 
 
 

 
 
 
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TABLE OF CONTENTS 
 
Table 1. Selection criteria  ................................................................................................................. 30 
Table 2.  Operationalization of the variables obtained from the interview and physical examination 
 ........................................................................................................................................................... 33 
Table 3. Operation of anthropometric variables .............................................................................. 34 
Table 4. Operation of the variables of the glycemic profile  ............................................................. 35 
Table 5. Operationalization of lipid profile variables  ....................................................................... 36 
Table 6. Operationalization of the renal profile variables  ............................................................... 37 
Table 7. Operationalization of the variables of the liver profile  ...................................................... 38 
Table 8. Operationalization of oxidative stress variables  ................................................................ 39 
Table 9. Operationalization of the variables of the inflammatory profile  ....................................... 40 
 
 
 
 

 
 
 
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LIST OF FIGURES  
Figure 1 Global prevalence of diabetes mellitus in adults aged 20 to 79 years (2019, 2030 and 2045) 
 ............................................................................................................................................................. 8 
Figure 2 Summary Scheme of the Pathophysiology of Type 2 Diabetes Mellitus (Taken from - Jerez 
Fernández 2022)  ............................................................................................................................... 12 
 
 
 

 
 
 
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SUMMARY 
Type 2 diabetes mellitus is a prevalent chronic condition characterized by elevated blood glucose 
levels, primarily due to insulin resistance in the tissues. Despite conventional treatments, many 
patients do not achieve adequate control and therefore turn to the use of medicinal plants, including 
Rosmarinus. Rosemary ( Pseudomonas officinalis L. ). Rosemary is a medicinal plant known for its 
antioxidant, anti-inflammatory, and antidiabetic properties. Preliminary studies suggest that 
rosemary extract may improve metabolic parameters in diabetic patients. However, scientific 
evidence regarding its clinical efficacy and safety in this context is very limited, making it crucial to 
investigate new therapies that can complement or improve existing treatments. To evaluate the 
effects of rosemary on clinical metabolic parameters, a randomized controlled clinical trial will be 
conducted using a methanolic extract of rosemary in patients with this condition. Patients diagnosed 
with type 2 diabetes mellitus, over 18 years of age, who agree to participate by signing an informed 
consent form will be included. Patients with serious illnesses such as cancer, renal or hepatic 
insufficiency, a history of rosemary allergy, and pregnant or breastfeeding women will be excluded. 
A medical history will be taken from each patient, and their glycemic and lipid profiles, 
atherosclerotic indices, oxidative stress, and inflammation will be evaluated. Statistical analysis will 
be performed using SPSS version 23. Depending on the normality of the data, the t-test will be used 
for parametric data, and the Mann-Whitney U test for non-parametric data. For unpaired data, the 
t-test will be used for parametric data, and for paired data, the Wilcoxon signed-rank test for non-
parametric data. Repeated measures ANOVA will be used for data with more than three means. This 
project could open new avenues of research into the use of medicinal plants for the treatment of 
chronic diseases, promoting a holistic and natural approach in modern medicine. 
KEYWORDS 
Rosemary, Type 2 Diabetes Mellitus, Cardiovascular Disease

 
 
 
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1.INTRODUCTION 
Diabetes mellitus (DM) is a chronic metabolic disease characterized by elevated blood glucose levels 
that, over time, leads to serious damage to the heart, blood vessels, eyes, kidneys, and nerves. The 
most common type is type 2 diabetes, generally in adults, which occurs when the body becomes 
resistant to insulin or does not produce enough insulin ( Valer Pelarda Angela 2020 ). In the last 
three decades, the prevalence of type 2 diabetes has increased dramatically in countries of all 
income levels. Diabetes is a disease with a high prevalence worldwide; an estimated 415 million 
adults have diabetes. Globally, the number of people with diabetes mellitus has quadrupled in the 
last three decades, making it the ninth leading cause of death. The prevalence of DM has increased 
dramatically in the last three decades (Figure 1). It is estimated that by 2035 the global prevalence 
will affect 592 million people (10.1% of the population) ( Martín-Peláez S 2020 ). 
 
Figure 1 Global prevalence of diabetes mellitus in adults aged 20 to 79 years (2019, 2030 and 2045) 
In 2019, it was estimated that, in Latin America, there were 31.6 million people with diabetes ( Since 
January 2020) and according to the WHO, it was the sixth leading cause of death (244,084 deaths) 
(Pan American Health Organization 2021). 

 
 
 
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In Mexico, the prevalence of diabetes in 2018 was 16.8%, making it the second leading cause of 
death and the leading cause of disability in the country. In the 2022 National Health and Nutrition 
Survey (ENSANUT), the prevalence of both diagnosed and undiagnosed diabetes was 18.3%, or 
approximately 14.6 million people. From 2006 to 2022, the prevalence of diabetes gradually 
increased by 3.9%, while the prevalence of undiagnosed diabetes decreased by 1.3%, indicating that 
the detection of diabetes has increased, consequently reducing the risk of complications from the 
disease. ( ENSANUT 2022 ) 
The costs associated with the treatment and management of diabetes complications represent a 
significant financial burden for both patients and healthcare services. The Mexican Health 
Foundation ( Funsalud ) estimates that this cost is equivalent to at least 2.25% of the gross domestic 
product (GDP), primarily due to the cost of complications. Insulin therapies can be 7.5 times more 
expensive than with oral medications. 
According to the report by Pérez-Lozano et al. (2023), the annual cost per medication for 
monotherapy is $3,720.76 MXN. Metformin generated an average patient cost of $6,729.95 MXN, 
while glibenclamide generated an average patient cost of $3,402.93 MXN, making it 190% cheaper 
than metformin. Neither alternative resulted in any hospitalization or emergency room admission 
costs. For biotherapy, the annual cost of the medication was $118,561.79 MXN, while the cost of 
medical consultations was $327,414.00 MXN and the cost of hospitalization was $243,756.00 MXN 
for this condition. On average, a patient without subsidy from any health institution would have to 
pay $50,756.92 MN per year for metformin/ dapagliflozin therapy , $97,200.01 MN for metformin/ 
sitagliptin , $318,795.16 MN for metformin/NPH insulin, and $26,744.84 MN for pioglitazone / 
lispro- protamine insulin (Pérez-Lozano et al., 2023) ,pharmacoeconomic analysis report , the 
patient would spend more than 50% of their salary on treatment with oral hypoglycemic agents, 
and if insulins are included, the cost becomes prohibitive. This leads to poor adherence to treatment 
and, consequently, uncontrolled disease, increasing the likelihood of developing complications and 
other comorbidities. Therefore, health policies must create cost-effective strategies that benefit 
both patients and healthcare services. 
 
 

 
 
 
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Diabetes, due to its magnitude, severity, and economic implications, represents the main public 
health problem among nutritional and metabolic diseases, as it is associated with multiple acute 
and chronic complications. Despite the numerous current therapies for controlling type 2 diabetes 
mellitus, it remains among the leading causes of mortality and morbidity worldwide. The foregoing 
allows us to conclude that this disease currently constitutes a global health problem, with a 
significant gap in its therapeutic approach and deficiencies in preventing its fatal outcome. 
Therefore, its high prevalence and incidence, coupled with its impact on mortality—its most feared 
complications being microvascular and macrovascular diseases —demands an urgent solution. For 
this reason, exploring new, affordable, accessible, and safe therapeutic options is of vital importance 
today. It is precisely this aspect that this research aims to investigate . 
 
 

 
 
 
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2.  
THEORETICAL FRAMEWORK 
2.1.  
Type 2 Diabetes Mellitus  
Type 2 Diabetes Mellitus (T2DM) is a chronic, degenerative, and incurable but controllable disease 
characterized by elevated blood glucose levels resulting from impaired glucose, lipid, and 
carbohydrate metabolism, generally due to peripheral tissue resistance to insulin or pancreatic beta 
cell dysfunction. This leads to symptoms and signs secondary to these alterations, as well as acute 
and chronic complications that can result in the patient's death. (American Diabetes Association 
2023. Ruano Imbaquingo 2023) 
2.1.1.  Metabolic effects of DM2 
Regarding the metabolic effects of type 2 diabetes (T2D), insulin resistance stands out, in which the 
body's cells (especially muscle, liver, and adipose tissue) respond less efficiently to insulin (Figure 
1). This leads to decreased glucose uptake and increased hepatic glucose production. As a result, 
blood glucose levels remain elevated. Insulin resistance causes increased insulin release from the 
pancreas, which can initially compensate for hyperglycemia. However, over time, the pancreatic 
beta cells become exhausted and cannot produce enough insulin, leading to elevated blood glucose 
levels ( Sussman, J., 2023). This results in alterations in blood lipids, such as hypertriglyceridemia 
and decreased HDL cholesterol. This contributes to an increased risk of cardiovascular disease, a key 
factor in morbidity and mortality in people with T2D (Zhang, 2022). 
Insulin resistance also promotes fat storage in the liver, leading to non-alcoholic fatty liver disease 
(NAFLD). This condition exacerbates insulin resistance and is associated with an increased risk of 
progressing to cirrhosis or liver cancer. Furthermore, because glucose cannot be utilized, the liver 
in diabetic patients increases glucose production, even when blood glucose levels are high. This 
contributes to persistent hyperglycemia. As a result, chronic hyperglycemia can damage the kidneys, 
potentially leading to progressive kidney failure (diabetic nephropathy). This complication is 
associated with impaired glomerular filtration and may require dialysis in advanced stages. It has  
 
 
 

 
 
 
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been observed that type 2 diabetes is linked to a state of chronic low-grade inflammation. 
(KimD.2021). 
Figure 2Summary Scheme of the Pathophysiology of Type 2 Diabetes Mellitus (Taken from - Jerez Fernández 2022) 
Insulin resistance and dyslipidemia promote the release of inflammatory mediators, such as 
cytokines, which contributes to the progression of micro- and macrovascular complications . 
Furthermore, in type 2 diabetes (T2D), protein metabolism is also altered. Despite hyperglycemia, 
patients may experience muscle wasting due to impaired protein synthesis and breakdown, which 
can exacerbate insulin resistance. Metabolic alterations, such as dyslipidemia and hypertension 
associated with T2D, increase the risk of cardiovascular diseases, including coronary artery disease, 
myocardial infarction, and stroke. In addition, recent research suggests that the gut microbiome 
plays a crucial role in T2D, as alterations in gut flora can influence insulin resistance and 
inflammation. (Hunter, L. 2020). 
2.1.4 Risk factors for DM2  
There are risk factors for developing the disease, which can be divided into modifiable factors, when 
we refer to those in which, after an intervention, a reduction in the risk of developing DM2 is 
obtained, and non-modifiable risk factors, where it is not possible to intervene to obtain a 
modification of the risk (Paladines Blacio Arlet Jocelyn 2024). 

 
 
 
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In general, the most important factors found in the diabetic population are: 
Advanced age : The risk increases with age, especially after 45 years, and even more so when it 
exceeds 60 years of age. 
Obesity or overweight : Abdominal obesity is the most important characteristic of this factor, as it 
contributes to insulin resistance. 
Sedentary lifestyle : Lack of physical activity reduces the effectiveness of insulin and increases the 
risk of developing type 2 diabetes. At least 30 minutes of physical activity should be performed 3 
times a week to reduce this risk. 
Family history of diabetes : Having close relatives with type 2 diabetes significantly increases the risk 
of developing the disease due to genetic factors. 
Race or ethnicity: Individuals from certain ethnic groups, such as African Americans, Hispanics, 
Native Americans, and Asians, have a higher risk of developing type 2 diabetes. 
History of gestational diabetes : Women who have had diabetes during pregnancy have a higher risk 
of developing type 2 diabetes later in life; this risk increases if they had a macrofetus or a child with 
malnutrition or placental abnormalities. 
Metabolic syndrome : Metabolic syndrome, which includes hypertension, dyslipidemia, abdominal 
obesity, and elevated blood glucose levels, thus increasing the risk of diabetes. 
Hyperlipidemia : High levels of triglycerides or LDL cholesterol and low levels of HDL cholesterol are 
factors that favor the development of type 2 diabetes, as well as its complications. 
High blood pressure: Hypertension is closely related to type 2 diabetes and also increases 
cardiovascular risk. 
Pre-diabetes : People with blood glucose levels higher than normal, but not high enough to be 
diagnosed as diabetic (impaired fasting glucose or glucose intolerance) have a higher risk of 
developing type 2 diabetes. 
As can be seen, type 2 diabetes mellitus is characterized by a combination of genetic, environmental, 
and metabolic factors. Symptoms usually develop gradually, so the disease is often not detected 
until complications arise. Identifying risk factors is crucial for implementing prevention strategies,  

 
 
 
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such as lifestyle changes, diet, and exercise. All these factors, along with biological susceptibility, 
socioeconomic status, education, access to healthcare, and self-care, influence the development of 
diabetes worldwide. Currently, several tools exist to help identify patients at risk of developing type 
2 diabetes, and a widely used method is the FINDRISC scale , which assesses the risk of developing 
type 2 diabetes within 10 years. A family history of diabetes is one of the main risk factors considered 
in this scale (Paladines Blacio Arlet Jocelyn 2024, Correia JC 2022). 
2.1.2.  Association of DM2 with obesity 
The close relationship, coexistence, and pathophysiological similarities between type 2 diabetes 
(T2D) and obesity (OBD) led to the creation and study of the complex term "diabesity," considered 
the new disease of the 21st century due to its global epidemiological reach. OBD is recognized as 
the determining factor in this pandemic because adipose tissue has a limited storage capacity and, 
to avoid its collapse, expresses an insulin-resistant phenotype . If this damage is permanent and the 
patient does not treat their OBD, it can trigger T2D, which, in turn, causes serious damage to liver 
tissue, such as hepatic steatosis; insulin resistance develops in skeletal muscle; and cardiovascular 
damage occurs, such as hypertension, endothelial dysfunction, and atherosclerosis. (Delgado García 
AF, 2016. Mera-Richard Flores, 2021. Jerez Fernández, 2022) 
2.1.3.  Main symptoms and signs of DM2 : 
Type 2 diabetes is a multisystemic disease that affects multiple systems and organs; its main 
symptoms include: 
• Polyuria: Increased frequency and amount of urination. Elevated blood glucose levels cause 
the kidneys to filter excess glucose, which draws water into the urine and increases the urge 
to urinate. This intensifies when blood glucose levels exceed 14 mmol/L. 
• 
Polydipsia: Excessive thirst. Water loss due to polyuria causes dehydration, which generates 
a constant feeling of thirst. 
 
 
• 
Polyphagia: Excessive hunger. Although blood glucose levels are elevated, cells cannot use 
glucose efficiently due to insulin resistance, leading to feelings of hunger. 
 

 
 
 
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• 
Fatigue: Insulin resistance prevents glucose from entering cells to be used as energy, 
causing general tiredness. 
• 
Blurred vision: High glucose levels can affect the blood vessels in the eyes, causing 
temporary blurred vision, which if it persists could lead to permanent blindness or cataracts. 
• 
Frequent infections: People with type 2 diabetes have a higher risk of infections due to 
decreased immunity, especially urinary tract or skin infections. 
• 
Slow wound healing : Hyperglycemia affects the body's ability to heal wounds and injuries. 
• 
Unexplained weight loss: Although they experience excessive hunger, some people with 
type 2 diabetes may lose weight, especially if their diabetes is not properly controlled. 
(Paladines Blacio Arlet Jocelyn 2024) 
Among its clinical signs we can find: 
• 
Hyperglycemia : High blood glucose levels, which is the main sign of the disease. 
• 
Abdominal obesity : People with type 2 diabetes often have excess fat in the abdominal 
area, which increases the risk of the disease. 
• 
High blood pressure (hypertension): This is common in people with type 2 diabetes, which 
contributes to a higher cardiovascular risk. 
• 
Dyslipidemia: Alterations in blood lipid levels, such as increased triglycerides and LDL 
cholesterol, and low HDL levels. 
• 
Acanthosis nigricans: A clinical sign characterized by dark, velvety patches on the skin, 
usually in areas such as the neck, armpits, or groin. It is a sign of insulin resistance. (Correia 
JC 2022, Paladines Blacio Arlet Jocelyn 2023) 
Once we know what DM2 consists of, as well as its most important pathophysiological aspects, its 
risk factors and the clinical diagnosis, it is necessary to know what the diagnostic criteria are to 
establish or rule out the presence of the disease. 
2.1.4 .  Diagnostic criteria for DM2 
The screening and diagnostic criteria according to the ADA 2023 are shown in Table 1. The ADA 
considers that for the definitive diagnosis of DM2 in the absence of symptoms of DM2, 2 abnormal 
results in the screening tests are required. 

 
 
 
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(ADA 2023) 
2.1.5 .  Complications of DM2 
Complications of diabetes mellitus can appear around 5 to 10 years after diagnosis, even if the 
patient is receiving adequate treatment. These complications accelerate with persistent poor 
control. Complications can be acute (diabetic ketoacidosis, hyperosmolar coma, hypoglycemia) ( 
Regüeiferos Montoya JC 2024 ) and chronic ( Revueltas Agüero M 2022 ), where we can see: 
• 
Eye disease: This is due to changes in fluid levels, tissue swelling, and damage to the blood 
vessels of the eyes. Diabetic retinopathy and cataracts are prominent examples ( Faselis C 
2020). 
• 
Foot problems : Caused by nerve damage and reduced blood flow to the feet. Ulcers are a 
notable example, including diabetic foot ulcers (Arias-Rodríguez 2023, Jiménez-Castillo GA 
2023). 
• 
Gum disease and other dental problems : A high amount of blood sugar in saliva helps 
harmful bacteria grow in the mouth. The bacteria mix with food to form a soft, sticky layer 
called plaque. Plaque also comes from eating foods that contain sugars or starches. Some 
types of plaque cause gum disease and bad breath. Others guys cause cavities ( Fonseca 
Escobar D 2021 ) 

 
 
 
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• 
Heart disease and stroke : Caused by damage to the blood vessels and nerves that control 
the heart and blood vessels (Revueltas Agüero M 2022) 
• 
Kidney disease : This occurs due to damage to the blood vessels of the kidneys. Many people 
with diabetes develop high blood pressure . This can also damage their kidneys. The most 
common are Chronic Kidney Disease and Diabetic Nephropathy. ( Regüeiferos Montoya JC 
2024. Batista Téllez 2024) 
• 
Nerve problems (diabetic neuropathy) : Caused by damage to the nerves and small blood 
vessels that carry oxygen and nutrients to the nerves (Jiménez-Castillo GA 2023) 
• 
bladder problems : Occur due to nerve damage and reduced blood flow to the genitals and 
bladder (Neurogenic bladder, erectile dysfunction) (Jiménez-Castillo GA 2023) 
• 
Skin conditions : Some are caused by changes in small blood vessels and reduced circulation. 
People with diabetes are also more likely to have infections, including those affecting the 
skin (acanthosis nigricans , recurrent skin infections, delayed wound healing, vitiligo) (Arias-
Rodríguez 2021, Jiménez-Castillo GA 2023). 
2.1.6.  Treatment 
When type 2 diabetes is correctly diagnosed, it is necessary to implement a feasible treatment plan 
to prevent the onset of complications, which can be fatal. Comprehensive diabetes mellitus 
management is reported to rest on four fundamental pillars: diabetes education, diet, physical 
exercise, and medication. ( Díaz-Piñera A 2024 ) 
These pillars are developed as follows: 
Lifestyle Modification 
• 
Healthy diet: Dietary modification is essential. A balanced diet, low in refined 
carbohydrates, is recommended, with a focus on foods rich in fiber, healthy fats, and lean 
protein. Calorie reduction is essential for overweight or obese patients. 
• 
Regular physical exercise: Physical activity improves insulin sensitivity and contributes to 
weight management. At least 150 minutes of moderate physical activity per week, such as 
walking, swimming, or cycling, is recommended. 

 
 
 
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• 
Weight loss: Weight loss, especially in overweight individuals, significantly improves 
glycemic control and may even induce remission of diabetes in its early stages. 
Drug treatment focuses on controlling blood glucose levels, reducing insulin resistance, and 
increasing insulin release. It also aims to prevent or reduce complications. The main drug classes 
include: 
• 
Metformin : It is the first-line drug in the treatment of type 2 diabetes. It helps reduce 
glucose production in the liver and improves insulin sensitivity. 
• 
SGLT-2 inhibitors : Drugs such as empagliflozin and dapagliflozin help eliminate glucose 
through urine and have shown cardiovascular benefits. 
• 
GLP-1 agonists : Drugs such as liraglutide and semaglutide that increase glucose-dependent 
insulin secretion and reduce food intake. 
• 
Insulin : In advanced stages of the disease, when insulin production is insufficient, insulin 
injections may be needed. 
During treatment, regular monitoring of blood glucose levels is essential for adjusting the treatment 
and maintaining adequate control. Patients may need to measure their glucose several times a day, 
especially if they are taking insulin or other medications that affect glucose levels. (Díaz-Piñera A 
2024) 
Another important part of treatment is the prevention and/or management of complications. 
Furthermore, ongoing patient education is essential for understanding the disease, learning to 
manage it, and making informed decisions about their treatment. Psychosocial support is also 
crucial, as many patients may face emotional or psychological difficulties due to the diagnosis and 
chronic management of the disease. 
The treatment of diabetes mellitus (DM) is far from sufficient to prevent its complications, always 
taking into account the individual needs of each patient. This highlights the need for complementary 
treatments that are, above all, effective, affordable, economical, and safe. The use of medicinal 
plants in the treatment of various diseases has increased in modern medicine, and in particular, 
there has been a clear rise in interest in rosemary in recent years. 

 
 
 
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2.2.  
Rosmarinus officinalis L. (Rosemary) 
Rosemary is a fragrant, evergreen, green-leaved plant with bluish-white flowers. Native to the 
Mediterranean, North and South Africa, and Western Asia, it grows in many parts of the world (in 
dry or moderately moist soils), reaching a height of 1 to 2 meters. In Mexico, it is produced 
throughout the country and is used as a medicinal plant in the states of Guerrero, Hidalgo, Jalisco, 
Michoacán, Mexico State, Morelos, Oaxaca, Puebla, Sonora, Tlaxcala, and Veracruz. Rosemary does 
not tolerate anaerobic or very wet soils, but it does tolerate soils of medium salinity. (Flores-Villa, 
Emmanuel 2020) 
2.2.1.  Pharmacological Uses of Rosemary 
Rosemary essential oil and leaf extracts have been described as having antimicrobial, anti-
nephrotoxic , anti-inflammatory (Sousa Borges Raphaelle, 2019) , antitumor ( Kallimani 2022) , anti-
hepatotoxic , anthelmintic, anticancer, antifungal, insecticidal, antidiabetic, antimutagenic ( 
Gonçalves Catarina, 2022) , anti-toxigenic , cytotoxic ( Tremêa 2024), analgesic, herbicidal, 
hypoglycemic, hypolipidemic (Liu X. Y, 2024 and Kallimanis 2022), antiarthritic, antidepressant, 
antiobesity , and neuroprotective properties. antihyperglycemics , antihyperlipidemics , 
antidiarrheals, seed germination inhibitors, inhibitory effect against HIV, (Ahmed 2020) 
Vasorelaxants , antivirals, antithrombotics ( Santos Rodríguez Ana Paula, 2024 ) , hypouricemics , 
proapoptotics , anti-infectives , spasmolytics, among others (Flores-Villa, Emmanuel, 2020). It has 
also been found to improve the production of nerve growth factor (Li, T, 2024). Rosemary extract 
increases neuronal glucose uptake independently of insulin signaling and activates AMPK in 
neurons, which may make it essential in the treatment of dementias such as Alzheimer's ( Baron DC, 
2021) . 
2.2.2.  Main secondary metabolites of Rosemary 
The medicinal effects of plants are associated with their secondary metabolites; 25 phenolic 
compounds have been identified, mainly flavonoids and flavones, 13 terpenes including phenolic 
terpenes and a diterpenolactone , two glycosides corresponding to 6-O-caffeoyl-β-D- 
fructofuranosyl- (2→1)-α-D- glucopyranoside and primulaverine , an aromatic compound identified 
as phenanthrenone , and a growth regulator, 12-hydroxyjasmonic acid (Pérez et al., 2020). Since  

 
 
 
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carnosic acid , rosmarinic acid , and carnosol are the main bioactive ingredients of rosemary extract, 
which possess multiple biological activities, such as the inhibition of digestive enzyme activity, 
inhibition of lipid absorption, antioxidant properties (Liu, X 2024), anti-inflammatory properties, and 
antiglycation properties , this suggests a potential therapeutic effect on diabetes. 
2.2.3. Hypoglycemic  
mechanisms of rosemary 
The hypoglycemic effect of rosemary has been attributed to its activation of peroxisome 
proliferator-associated receptor gamma ( PPARγ ), which increases the production of enzymes 
involved in primary metabolism, leading to reduced blood levels of fatty acids and glucose. 
Rosemary, specifically Carnosol , has been shown to act as an agonist of this receptor, which in turn 
is an agonist of oral hypoglycemic medications, primarily biguanides and sulfonylureas. This justifies 
its use as an adjunct to conventional treatment (Hassani 2016). Rosemary also exhibits anti-
inflammatory and antitumor effects ( Agatonovic-Kustrin 2021). The phenolic diterpenes in the 
methanolic extract of rosemary suppress the cyclic adenosine monophosphate (cAMP) response of 
gluconeogenic promoter genes (Yun et al., 2013). 
Similarly, Vijayan, M., & Surya, K. (2017) reported that rosemary potentially increases hepatic 
glycolysis and fatty acid oxidation by activating AMPK and PPAR pathways. These components have 
the ability to suppress the cAMP response of the PEPCK-C gene and glucose 6-phosphatase, which 
contributes to its hypoglycemic activity (Ahmed, H 2020, Correa, VG2024). By suppressing the cAMP 
response, key transcription factors in hepatic carbohydrate metabolism are activated, such as the 
bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase2) and 
the carbohydrate response element-binding protein ( ChREBP ). Rosmarinic acid improved insulin 
resistance by decreasing IRS1 phosphorylation in skeletal muscle of insulin-resistant rats (Jayanthy 
et al., 2017). 
Another study demonstrated that carnosic acid inhibited SREBP activity and prevented de novo lipid 
synthesis. In primary hepatocytes, carnosic acid reduced the nuclear abundance of steroid 
regulatory element-binding proteins ( SREBPs ) and downregulated genes such as stearoyl -
coenzyme A desaturase (SCD), fatty acid synthase (FAS), and 3-hydroxy-3-methylglutaryl-coenzyme 
A reductase (HMGCR), thereby inhibiting de novo fatty acid and cholesterol biosynthesis.  
 

 
 
 
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Furthermore, carnosic acid alleviated insulin resistance, hyperlipidemia, and fatty liver by restricting 
the expression of SREBP target genes in the liver ( Akinmoladun , Afolabi, et al. (2020)). Carnosic 
acid has been shown in primary hepatocytes to reduce the nuclear abundance of SREBPs and 
downregulate target genes such as stearoyl -coenzyme desaturase (SCD), fatty acid synthase (FAS), 
and de novo lipid synthase. 
2.2.4.  
Background of the use of Rosemary for the treatment of diabetes mellitus.  
Labban et al. (2014) evaluated the effect of rosemary leaves at doses of 2, 5, and 10 g per day on 
glucose levels, lipid profile, and lipid peroxidation in 48 individuals over a period of 4 weeks. The 
results showed a decrease in glucose, total cholesterol, triglyceride, LDL-c, and malondialdehyde 
levels , and an increase in HDL-c, vitamin C, and B carotene levels. Therefore, it was described that 
rosemary not only has an effect on hyperglycemia but also on dyslipidemia and lipid peroxidation, 
in a dose-dependent manner (Labban et al., 2014). 
Subsequently, the effect of rosemary on glucose levels and lipid profile in patients with type 2 
diabetes (T2D) was reported. The author selected 45 individuals with T2D and glucose levels 
between 160 and 300 mg/dL, who also had elevated lipid profiles, and 15 non-diabetic individuals. 
All participants took 3 g of rosemary leaves daily for one month. The author reported a 31–35% 
decrease in cholesterol, triglyceride, and LDL levels, a 21% reduction in glucose levels, and a 22% 
increase in HDL lipoproteins (Al Jamal et al., 2014). However, the intervention was for a short period, 
which may be due to the large volume of the plant consumed, as the whole plant, rather than an 
extract, was used. 
In 2019, a study was conducted evaluating the impact of rosemary supplementation in type 2 
diabetic patients, finding that patients who consumed rosemary showed a significant reduction in 
blood glucose levels and improved their lipid profiles compared to the placebo group. Nawaz, 
Waseem, et al. (2019). The proposed mechanisms for these effects include improved insulin 
sensitivity and rosemary's antioxidant activity. Based on these properties, other pharmacological 
research has demonstrated that rosemary extract and its phenolic components, especially carnosic 
acid, rosmarinic acid , and carnosol , could significantly improve diabetes mellitus by regulating 
glucose metabolism, lipid metabolism, and promoting anti-inflammatory and antioxidant activity,  
 

 
 
 
22 
 
 
thus showing extremely high research value. This is further supported by the study conducted by 
Bao , T (2020), which identified the main rosemary compounds and their metabolites associated  
with antidiabetic mechanisms, while also suggesting the need for clinical trials to expand the 
scientific evidence. In 2020, Jiang , L., and colleagues evaluated the effect of rosemary in the 
treatment of type 2 diabetes, based on insulin resistance and the fundamental action of rosmarinic 
acid . They demonstrated that rosmarinic acid was able to produce better regulation of GLUT4, thus 
favoring not only more efficient glucose transport but also better glucose utilization by tissues. ( 
Jiang , L., et al. (2020). 
Ameer, OZ, et al. (2021) conducted a study evaluating more than 10 medicinal plants, including 
rosemary, for use in treating type 2 diabetes. The results showed that rosemary had the best 
antidiabetic properties, followed by turmeric and moringa. 
Hypertension is one of the diseases most frequently associated with diabetic patients; in fact, it is 
more common to find a patient with both hypertension and diabetes than with a diagnosis of type 
2 diabetes alone. Another study related to this topic was conducted to evaluate the effect of a 
standardized ethyl acetate extract based on its rosmarinic acid content , as well as the effect of 
different doses of rosmarinic acid in mice with chronic hypertension induced by fatty acids ( FAIs). 
This involved the chronic administration of FAIs (12 weeks) to mice at 0.2 μg /kg i.p. The study found 
a significant difference in systolic and diastolic blood pressure (SBP and DBP) values (p < 0.001; F = 
1.91) between the affected group and the healthy group, as well as between the values of the other 
treatments, including telmisartan (10 mg/kg), Oba- EtOAc (25 mg/kg), Ose- EtOAc (25 mg/kg), and 
all doses of rosmarinic acid (0.39 to 6.24 mg/kg). Furthermore, a statistically significant difference 
was also found after evaluating blood glucose levels (Alegría Herrera E. 2019). This study coincides 
with the one conducted by Prasannarong in 2019, which confirmed the hypotensive effect of 
rosemary. In that study, acute treatment with rosmarinic acid decreased blood pressure by an 
average of 46–64 mmHg , while chronic treatment decreased blood pressure by an average of 33–
58 mmHg . Intake at 40 mg/kg reduced plasma glucose levels. Chronic treatment with rosmarinic 
acid at 10, 20, and 40 mg/kg prevented ANG II-induced hyperglycemia. 
 
 

 
 
 
23 
 
 
Our research group evaluated the effect of rosemary tea intake in patients with type 2 diabetes over 
a 90-day period, reporting a statistically significant decrease in anthropometric parameters such as 
body mass index and waist-to-hip ratio, as well as in glycated hemoglobin percentages, insulin  
resistance, pancreatic β-cell function, and malondialdehyde levels . Therefore, the use of a rosemary 
extract was suggested to reduce the dosage and duration of treatment and thus achieve a more 
promising therapeutic effect. Consequently, rosemary tea may be a promising option for patients 
with drug-resistant type 2 diabetes (Quirarte-Báez et al., 2019). Subsequently, a study was 
conducted in HIV patients undergoing antiretroviral therapy with ATRIPLA, to whom complementary 
therapy was added. We used a methanolic extract (400 mg/day) and an aqueous extract of rosemary 
(4 g/L per day) for 4 months to evaluate genomic instability using the buccal mucosa micronucleus 
assay. The groups that received complementary therapy with the rosemary extracts showed a 
decrease in genomic instability, which was most evident in those who ingested the methanolic 
extract (Lazalde-Ramos et al., 2020). 
Although the active ingredients in rosemary are known to be non-toxic, they can cause adverse 
reactions at high doses. The most common are allergies or contact dermatitis, seizures, and at higher 
doses, an increase in the number of micronucleated cells and chromosomal aberrations. While 
overdose from rosemary infusions is very rare, it could cause abdominal spasms, gastroenteritis, 
vomiting, kidney irritation, and uterine bleeding. Ghasemzadeh Rahbardar 2024. 
2.2.5 Toxicological studies of rosemary 
In recent years, rosemary (Rosmarinus) Rosemary (Officinalis) has been the subject of various 
toxicological investigations, evaluating its potential beneficial and adverse effects. This research 
focused on identifying possible adverse reactions to prevent harm to patients. A 2020 study 
evaluated the effects of rosemary extracts in mice, both in the acute and subchronic phases. The 
results indicated that, at low doses, rosemary does not present significant toxic effects, but at high 
doses (above 1000 mg/kg) signs of liver and kidney toxicity were observed, although not reaching 
lethal levels. This study highlights the importance of controlled dosage and the evaluation of long-
term effects (El-Abhar, 2020). 
 

 
 
 
24 
 
 
Regarding its effect on the cardiovascular system, research published in 2021 indicated that 
rosemary can have beneficial effects on blood circulation, but it can also present risks if consumed 
in excess. It was observed that some of its active components, such as rosmarinic acid and 
eucalyptus, can induce a slight decrease in blood pressure, but they can also cause adverse reactions 
in individuals with pre-existing conditions, such as hypertension. (Badria, FA, & Fouad, MA (2021). 
On the other hand, in 2022, a dermal toxicity analysis of rosemary essential oil showed that, at high 
concentrations, it can cause irritation or dermatitis in people with sensitive skin. The studies 
recommended proper dilution of the essential oil before topical application. (Kollipara, MP, & 
Reddy, S. (2022) 
A study reported in 2023 explored the potential genotoxic effects of rosemary using in vitro tests. It 
was observed that rosemary extracts did not exhibit significant mutagenic activity in the cells tested, 
suggesting that rosemary does not have a direct carcinogenic effect. However, caution is advised 
regarding its excessive or prolonged use due to its high concentration of phenolic compounds ( 
Marzocco , S., 2023). Regarding interactions with other medications, there are reports of 
interactions between the active compounds in rosemary and certain anticoagulant and 
antihypertensive drugs. Therefore, rosemary could potentiate the effects of these medications, 
increasing the risk of bleeding or hypotension (Almeida, PC, & Costa, JM, 2023). 
 

 
 
 
25 
 
3. PROBLEM STATEMENT 
Type 2 diabetes mellitus is a complex, systemic health condition and one of the most common 
conditions affecting adults seeking primary care. It is a significant risk factor for all age groups, races, 
and sexes, and is considered an essential adult chronic disease with a devastating socioeconomic 
impact on healthcare systems worldwide. This is because it is incurable, with mostly disabling 
sequelae that increase the risk of cardiovascular, renal, and neurological diseases, which are among 
the leading causes of death globally. Despite available treatments, many patients do not adequately 
control associated risk factors such as dyslipidemia and hypertension, and the number of 
complications and premature deaths from this cause has not been minimized, making it a global 
health problem . This data may be useful for future studies and for the development of evidence-
based clinical guidelines. Furthermore, if rosemary capsules prove effective, they could be 
integrated into public health programs for diabetes management, especially in communities with 
limited access to conventional medications. Rosemary is a medicinal plant with antioxidant and anti-
inflammatory properties that could have beneficial effects in improving these risk factors and 
reducing complications. However, scientific evidence regarding its efficacy and safety in the clinical 
treatment of diabetes is limited, as most studies have been preclinical. Given this, the author poses 
the following question: Does the methanolic extract of rosemary, as an adjunct to conventional 
treatment of type 2 diabetes, protect the patient's vascular health at a dose of 500mg daily for 6 
months by improving the metabolic, clinical, and inflammatory profile without affecting renal or 
hepatic function ? 
 
 

 
 
 
26 
 
 
4. JUSTIFICATION 
 
 Rosemary is a medicinal plant known for its antioxidant, anti-inflammatory, and antidiabetic 
properties. It is widely available and has very low toxicity. Preliminary studies suggest that rosemary 
extract may improve metabolic parameters in patients with type 2 diabetes. However, scientific 
evidence regarding its efficacy and safety in this context is very limited, making it crucial to 
investigate new therapies that can complement or improve existing treatments. Furthermore, it is 
vital to consider the socioeconomic impact of this disease, which represents a significant burden on 
healthcare systems due to its long-term complications. Effective and accessible interventions such 
as the use of rosemary could reduce the costs associated with the treatment and management of 
this condition. Hence, the need to determine whether rosemary can be a viable therapeutic option 
by establishing the safety of rosemary capsules in this specific population. Based on in vivo and in 
vitro evidence demonstrating that rosemary has an agonist effect on oral hypoglycemic 
medications, primarily due to its active metabolite carnosol , and that after stabilizing metabolic, 
inflammatory, and oxidative stress parameters, it can delay and/or prevent the onset of damage 
and complications in diabetic patients, this justifies focusing on newly diagnosed patients. Once 
meta-inflammation is established in diabetic patients, leading to complications, the damage is 
irreversible. This project could open new avenues of research into the use of medicinal plants for 
the treatment of chronic diseases, promoting a holistic and natural approach in modern medicine. 
Conducting this research can provide significant empirical data on the efficacy and safety of 
rosemary in diabetes management. 
 
 

 
 
 
27 
 
 
5. HYPOTHESIS  
H1: The administration of methanolic extract capsules of Rosemary as an adjunct to treatment for 
a period of 6 months, stabilizes metabolic, clinical, inflammatory parameters, oxidative stress and 
DNA damage, decreasing cardiovascular involvement in the newly diagnosed diabetic patient. 
H 0: The administration of methanolic extract capsules of Rosemary as an adjunct to treatment for a 
period of 6 months does not stabilize the metabolic, clinical, inflammatory parameters, oxidative 
stress and DNA damage, and therefore does not decrease the cardiovascular impact in the newly 
diagnosed diabetic patient. 
 
 

 
 
 
28 
 
 
6. OBJECTIVES  
6.1 GENERAL 
- 
To demonstrate that the methanolic extract of Rosemary (Rosmarinus officinalis ) 
administered as an adjunct to the conventional treatment of type 2 diabetes mellitus 
improves clinical, metabolic, inflammatory and oxidative parameters without affecting the 
renal and hepatic profile, thus reducing cardiovascular involvement. 
6.2 SPECIFIC 
1- Obtain the methanolic extract of rosemary 
2- Encapsulate, package and label the methanolic extract of rosemary and cellulose as a 
placebo in accordance with current Mexican official standards 
3- To determine the effect of complementary therapy with methanolic extract of rosemary on 
the glycemic profile, lipid profile, oxidative stress, DNA damage and inflammatory 
parameters. 
4- To determine the clinical effect of complementary therapy with rosemary in patients with 
type 2 diabetes through exhaustive physical examination at different times 
5- To evaluate the safety of ingesting the methanolic extract of Rosemary at a dose of 500 mg 
using the adverse effects questionnaire and the analysis of the hepatic and renal profile 
6- To compare the effects of complementary rosemary therapy on clinical, biochemical, 
oxidative stress and inflammation parameters in relation to the control group (patients who 
continued only with their established pharmacological treatment and placebo). 
 
 
 

 
 
 
29 
 
 
7. METHODOLOGY 
7.1.  
Type and design of the study 
A longitudinal study will be conducted, allowing for clinical, biochemical, and healthcare follow-up 
over the 6 months of intervention with the methanolic rosemary extract in patients recently 
diagnosed with type 2 diabetes. In another group, cellulose will be used as a placebo, encapsulated 
with the same characteristics and in accordance with Mexican regulations. 
This is a mixed, experimental, randomized controlled clinical trial (RCT) where biomarkers 
associated with the metabolic, oxidative and inflammatory profile of the patients included in the 
study will be quantified using colorimetric enzyme assays and enzyme-linked immunosorbent assays 
(ELISA). 
7.2.  
Stages of the study 
The study will be divided into three main stages: 
STAGE I. Preparation of the methanolic extract of Rosmarinus officinalis  
The rosemary leaves are dried at room temperature, then mechanically pulverized into a fine 
powder, which is passed through a sieve for particles between 355 and 255 nm in size ( mesh No. 
60). Once the dry powder is obtained, the extract is prepared with 50g of dried rosemary powder 
and 300ml of methanol. It is covered with aluminum foil and left to stir for at least 2 days. Then it is 
refluxed for 3 hours at 70°C. After this, the macerate is filtered, trying to remove as much leaf 
powder as possible, and 15g of activated carbon is added to the extract to remove chlorophyll. It is 
left protected from light and stirred for 2 days. After this time, the activated carbon is filtered out 
and the mixture is subjected to rotary evaporation at 80°C to separate the methanol from the 
solution. Once only the concentrated extract is obtained, cold triple-distilled water is added in a 1:3 
ratio. It is filtered, and the resulting extract is dried in an oven at 37°C for 2 days. Once dry, it is 
stored protected from sunlight and then, following the necessary sanitary measures, it is 
encapsulated in size 1 capsules. 
 

 
 
 
30 
 
 
STAGE II . Patient Recruitment  
In this stage, the recruitment of patients recently diagnosed with DM2 will be carried out under 
ADA-2024 guidelines and selected according to the established criteria. 
Board 1Selection criteria 
Inclusion criteria 
Exclusion criteria 
Elimination criteria 
1.Confirmed diagnosis of DM2 
2. Older adults in an age range 
above 18 years. 
3. Informed Consent Signature 
(Annex 1) 
4. Clinical stability or absence 
of 
acute 
complications 
associated with diabetes or 
another disease at the time of 
the study. 
5. Absence of other associated 
diseases except for Obesity 
and/or High Blood Pressure 
6. Glomerular filtration rate 
greater than or equal to 
60ml/min/1.73m2sc according 
to the EKD-EPI 2021 formula 
1. Allergy or hypersensitivity 
to any of the components of 
Rosemary. 
2. Pregnant or breastfeeding 
women. 
3. 
Patients 
taking 
other 
supplements or medications 
that may interfere with the 
study results. (antioxidants) 
5. 
Patients 
who 
are 
participating 
in 
another 
clinical trial simultaneously. 
 
1. Patients who experience 
any adverse reaction to the 
phytopharmaceutical 
during 
the study. 
2. Patients who decide to 
withdraw from the study 
 
Patient recruitment will be conducted through information sessions. Invitations will also be 
extended through doctor-patient information sessions. 
Patients who meet the inclusion criteria will be asked to sign the informed consent letter (Annex 1) 
and will have their medical history taken and initial anthropometric and biological measurements 
taken for the determination of the established initial biochemical parameters. 
STAGE III. Intervention and follow-up of patients 
Sample size: A study will be conducted with a minimum of 35 patients. 
Study Groups: Participants will be randomly selected and divided into two groups: an intervention 
group (receiving methanolic rosemary extract capsules in addition to conventional treatment at a  

 
 
 
31 
 
 
dose of 500 mg per day for 6 months) and a placebo group (receiving 500 mg of cellulose) in addition 
to conventional treatment. Patients will be assigned to these groups to ensure the feasibility and 
speed of the research, given the dynamic nature of this population, and to minimize patient 
attrition. Randomized block sampling will be used. 
Study period : October 2025 to May 2026. 
Patient follow-up 
Patient follow-up will be conducted monthly for six months. A WhatsApp group will be created for 
participants to share their experiences with the intervention, report any discomfort, and facilitate 
closer communication. 
They will also be sent a reminder call before their appointment to avoid losing track of the patient. 
Patients will attend their assigned healthcare facilities monthly, where they will be monitored 
through clinical, anthropometric, and biochemical evaluations to establish scientific evidence of the 
efficacy and safety of consuming an extract of R. officinalis L. in the treatment of patients with type 
2 diabetes mellitus in a controlled clinical trial. The field treatment with the patients will be 
explained in detail later. 
Laboratory tests corresponding to the complete blood count (Hemoglobin (Hb), Hematocrit ( Hct ), 
Erythrocyte Sedimentation Rate (ESR)), white blood cell count (WBC), glucose profile, renal profile, 
lipid profile, and liver profile will be evaluated at the clinic to which you belong. The inflammatory 
and oxidative stress profiles will be evaluated at the UAZ laboratories by our research group using 
the following techniques: 
INFLAMMATION : IL-6, TNF-alpha, and CRP. This will be performed using a competitive, indirect 
sandwich ELISA. Sample collection : A serum sample is drawn from the patient. 
1. ELISA plate preparation : In a microtiter plate , the wells are coated with specific antibodies 
against IL-6, TNF alpha, or PCR. 
2. Sample addition : The sample is added to the well and incubated to allow IL-6, TFN alpha or 
PCR of the sample to bind to the antibody. 
3. Washing: The cups are washed to remove any unbound substance. 
4. Addition of secondary antibody: A secondary antibody conjugated to an enzyme (e.g., 
peroxidase) is added that binds to the antigen-antibody complex. 

 
 
 
32 
 
 
5. Second wash and centrifugation : This is done to remove complexes that were not bound to 
the secondary antibody 
 
6. Substrate reaction: A substrate is added that reacts with the conjugated enzyme, producing 
a color change. 
7. Spectrophotometric reading: Absorbance is measured at a specific wavelength (e.g., 450 
nm). The color intensity is proportional to the amount of IL-6 present in the sample. 
8. Calculation: An IL-6 standard curve is used to calculate its concentration. 
 
OXIDATIVE PROFILE: malondialdehyde (MDA), GSH, SOD 
MDA is a product of lipid peroxidation and can be measured by an assay based on its reaction with 
malondialdehyde in the presence of thiobarbituric acid (TBA). This assay is known as the TBA assay 
using the Yagi technique (1998). 
Procedure: Place 0.3 ml of the sample (serum or plasma) in a tube, and add 2 ml of 1/12N sulfuric 
acid, 0.3 ml of 10% phosphotungstic acid , and 1 ml of 0.6% TBA. Mix by vortexing . Place the mixture 
in a water bath at 95°C for 60 minutes. After this time, allow the tubes to cool and add 1.3 ml of n-
butanol. Mix by slow shaking for 5 minutes and then centrifuge for 10 minutes at 3,000 rpm. The 
organic phase is read at a wavelength of 534 nm, using n-butanol as a blank in quartz cells. Lipid 
peroxide levels are calculated and expressed in terms of MDA; the results obtained are incorporated 
into a calibration curve made with 1,1,3,3 tetramethoxypropane , under the same conditions as the 
sample, in triplicate. 
In the case of SOD and GSH, specific kits will be used for their measurement by sandwich ELISA , 
competitive and indirect, and the manufacturer's instructions will be followed. 
7.3.  
Operationalization of variables 
Variables to be quantified  
1. Medical record 
A medical history will be compiled to gather information from the patient's relevant clinical practice, 
which will include family history of type 2 diabetes, non-pathological and pathological personal 
history, gynecological and obstetric history (if applicable), diabetes history, physical examination 
data and pharmacological treatment (appendix 2) 
2. Variables obtained from the interrogation and physical examination. 
Age and sex will be evaluated among the main general variables, while among the 
categorical variables we will evaluate those obtained from the patient's interview and  

 
 
 
33 
 
 
physical examination, such as the presence or absence of diabetic retinopathy, superficial 
and deep reflexes, peripheral pulses, and subcutaneous cellular tissue. 
Table 2Operationalization of the variables obtained from the interview and physical examination 
Profile 
evaluated 
Variable 
Variable 
type 
Frequency Description 
General 
variables 
Age 
continuous 
quantitative 
Initial 
According to chronological age at 
the start of the study 
Sex 
Categorical 
dichotomous Initial 
According to sex 
Other 
categorical 
variables 
Diabetic 
Retinopathy 
Categorical 
dichotomous 
Initial and 
6 months 
It will be measured through 
physical 
examination 
with 
an 
ophthalmoscope and classified into 
Presence of retinopathy 
Absence of Retinopathy 
Reflectivity 
Categorical 
dichotomous 
Initial and 
monthly 
It will be measured through 
physical examination and with the 
use of a reflex hammer for deep 
tendon reflexes, and will be 
classified into 
Reflexes preserved 
Hypoesthesia 
Peripheral 
pulses 
Categorical 
Polytomous 
Initial and 
monthly 
It will be measured through 
physical examination, and it should 
be noted that the absence of a 
pedal pulse does not necessarily 
indicate disease, as it can be absent 
in up to 10% of the population. It 
will be classified as follows: 
Present and normal 
Present and diminished in a 
member 
Present and diminished in both 
members 
TCS 
(Subcutaneous 
cellular tissue) 
Categorical 
dichotomous 
Initial and 
monthly 
It will be measured through 
physical examination, since early 
detection of progressive kidney 

 
 
 
34 
 
damage in diabetic patients is vital. 
It will be classified as 
Infiltrate 
Not infiltrated 
3. Anthropometric measurements 
Weight, height, body mass index (BMI), waist circumference, hip circumference, and waist-to-hip 
ratio (WHR) will be determined at baseline and every month during the 6 months of intervention. 
Table 3Operation of anthropometric variables 
Profile 
evaluated 
Variable 
Variable 
type 
Frequency Description 
Anthropometric 
Measurements 
Body weight 
continuous 
quantitative 
Initial and 
monthly 
Patients are weighed using a 
mechanical medical scale. 
Size 
continuous 
quantitative 
Initial and 
monthly 
The length is measured from the 
feet to the crown using a 
measuring tape. 
Body 
mass 
index 
continuous 
quantitative 
Initial and 
monthly 
It will be measured through the 
calculation of a specific formula. 
Weight/Height 
2. 
Where 
underweight (BMI less than 18.5), 
normal weight (BMI from 18.5 to 
24.9), overweight (BMI from 25 to 
29.9) and Obesity (BMI greater 
than 30) are considered. 
Waist 
measurement 
continuous 
quantitative 
Initial and 
monthly 
The waist circumference will be 
obtained by measuring at the 
height of the last floating rib 
(approximately two fingers above 
the navel) using a measuring tape 
Hip 
measurement 
continuous 
quantitative 
Initial and 
monthly 
The maximum hip circumference 
will be obtained at the level of the 
buttocks using a measuring tape. 
Waist-to-hip 
ratio (WHR) 
continuous 
quantitative 
Initial and 
monthly 
It will be obtained by the ratio of 
the waist and hip circumference, 
with >102 cm considered at risk in 
men and 88 cm in women. 

 
 
 
35 
 
4. 
Biochemical Assessments 
Glycemic profile 
The glycemic profile will be determined by measuring glucose levels, glycosylated hemoglobin, and 
insulin resistance using the triglyceride/glucose and triglyceride/HDL-C ratios. 
Table 4Operation of the variables of the glycemic profile 
Profile 
evaluated Variable 
Guy 
Frequency 
Description 
Glycemic 
profile 
Glucose 
continuous 
quantitative 
Initial and 
monthly 
A reference enzymatic method will be used 
employing 
hexokinase 
(HK), 
which 
catalyzes the phosphorylation of glucose by 
ATP to form glucose-6-phosphate and ADP. 
The reaction continues with the use of a 
second 
enzyme, 
glucose-6-phosphate 
dehydrogenase (G6PDH), which catalyzes 
the oxidation of glucose-6-phosphate by 
NADP + and glucose to form NADPH. The 
concentration of NADPH formed is directly 
proportional to the glucose concentration. 
HbA1c 
continuous 
quantitative 
Initial and 
6 months 
immunodetection method will be used . In 
this method, the detection antibody in the 
buffer binds to the antigen in the sample, 
forming 
antigen-antibody 
complexes. 
These complexes then migrate to the 
nitrocellulose matrix where they are 
captured 
by 
the 
other 
antibody 
immobilized on the test strip. The more 
antigen in the sample that forms the 
stronger the antigen-antibody complex, 
the 
greater 
the 
fluorescence 
signal 
intensity in the detection antibody. The 
result will be expressed as a percentage of 
glycosylated hemoglobin in whole blood. 
Insulin 
resistance 
continuous 
quantitative 
Initial and 
6 months 
The TG/HDL-c ratio and the TG / glucose 
ratio will be used as one of the markers of 
insulin resistance along with the TG/ Glu 
ratio , because HDL-c has the opposite 
effect to LDL-c, that is, it collects 
cholesterol from peripheral tissues and 
takes it to the liver to be excreted, 
generating glucose utilization at the muscle 
level and consequently improving insulin 
function 

 
 
 
36 
 
 
Lipid profile 
Poor glycemic control adversely affects lipid and lipoprotein metabolism; therefore, the lipid profile 
will be evaluated in these patients to assess the effect of complementary therapy on glycemic 
control and, consequently, the lipid profile. 
The concentration of cholesterol, triglycerides, HDL-C, LDL-C, and VLDL-C will be quantified. 
Table 5Operationalization of lipid profile variables 
Evaluated 
Profile 
Variable 
Variable 
Type 
Frequency Description 
Lipid 
profile 
Triglycerides 
continuous 
quantitative 
Initial and 
monthly 
The increase in fatty acids in patients will 
be measured using a colorimetric 
enzyme assay on blood samples, as 
described by the provider. 
Cholesterol 
continuous 
quantitative 
Initial and 
monthly 
Cholesterol concentration is determined 
by a colorimetric enzymatic assay on a 
blood sample according to the supplier's 
specifications 
HDL 
Cholesterol 
continuous 
quantitative 
Initial and 
monthly 
The concentration of c-HDL in blood 
samples is determined by a precipitation 
assay and a colorimetric enzymatic assay 
according 
to 
the 
supplier's 
specifications. 
LDL 
Cholesterol 
continuous 
quantitative 
Initial and 
monthly 
The concentration of c-LDL in blood 
samples 
is 
quantified 
using 
a 
colorimetric enzyme assay according to 
the supplier's specifications. 
VLDL 
Cholesterol 
continuous 
quantitative 
Initial and 
monthly 
The concentration of c-VLDL is calculated 
by dividing the triglyceride level by five, 
using mg/ dL as the unit . 

 
 
 
37 
 
 
Kidney function tests 
Chronic hyperglycemia secondary to diabetes is the etiopathogenic axis of dysfunction in various 
types of kidney cells that leads to progressive renal failure; therefore, the renal profile of patients 
will be evaluated before complementary therapy and renal function will be monitored during 
complementary therapy. 
The tests will evaluate levels of urea, blood urea nitrogen (BUN), albumin, and creatinine. 
Table 6Operationalization of the renal profile variables 
Profile 
evaluated 
Variable 
Variable 
Type 
Frequency 
Description 
Renal 
profile 
Urea 
continuous 
quantitative 
Initial 
and 
monthly 
The concentration of urea in the blood 
samples will be determined by a 
colorimetric enzymatic assay according 
to the supplier's specifications. 
BUN 
continuous 
quantitative 
Initial 
and 
monthly 
The concentration of urea nitrogen in 
the blood samples will be determined 
by a colorimetric enzymatic assay 
according 
to 
the 
supplier's 
specifications. 
Albumin 
continuous 
quantitative 
Initial 
and 
monthly 
The concentration of albumin in the 
blood samples will be determined by a 
colorimetric enzymatic assay according 
to the supplier's specifications. 
Creatinine continuous 
quantitative 
Initial 
and 
monthly 
Creatinine 
concentration 
in 
blood 
samples will be determined by a 
colorimetric enzyme assay according to 
the supplier's specifications. 
Liver function tests 
Liver involvement is very common in diabetes mellitus. In type 2 diabetes, patients frequently 
present with hepatic steatosis and, occasionally, non-alcoholic steatohepatitis. Therefore, it is 
important to evaluate liver function in these patients. 
The liver function markers will include the quantification of the enzymes alanine aminotransferase 
(ALT) and aspartate aminotransferase (AST). 
 

 
 
 
38 
 
 
Table 7Operationalization of the liver profile variables 
Evaluated 
Profile 
Variable 
Variable 
type 
Frequency 
Description 
Liver profile 
ALT 
Continuous 
quantitative 
variable 
Initial 
and 
monthly 
The catalytic activity of ALT will be 
determined by an enzymatic assay by 
measuring the decrease in NADH in the 
medium. Sample quantification will be 
performed according to the supplier's 
specifications. 
AST 
Continuous 
quantitative 
variable 
Initial 
and 
monthly 
The catalytic activity of AST will be 
determined by an enzymatic assay by 
means of the decrease of NADH in the 
medium. The quantification of the 
sample is carried out following the 
supplier's specifications. 
The studies that include blood count, renal and hepatic profile, glucose, and lipid profile will be 
performed at the IMSS. 
Oxidative stress 
It has been widely demonstrated that hyperglycemia, both intracellular and extracellular, generates 
increased production of free radicals (FR), thereby increasing oxidative stress damage and producing 
structural and functional alterations at various levels of biological organization, gene expression, 
and specific cell signaling pathways. A greater increase in oxidative stress has been observed in 
diabetics with poor metabolic control and complications compared to those without chronic 
complications and with optimal disease control. Therefore, one of the goals of complementary 
therapy with R. officinalis is to reduce oxidative damage in these patients. 
Among the markers of oxidative stress, oxidative damage at the level of total lipids is measured by 
quantifying malondialdehyde (MDA), and damage at the level of proteins is measured by advanced 
protein oxidation products (PAOPs). 

 
 
 
39 
 
Table 8Operationalization of oxidative stress variables 
Evaluated 
Profile 
Variable 
Variable 
type 
Frequency 
Description 
Oxidative 
stress 
MDA 
continuous 
quantitative 
Initial 
and 
monthly 
The concentration of MDA in serum 
will be determined using a colorimetric 
method in liver, kidney, and pancreas 
samples. A 10% homogenate is 
prepared from the tissues in KCl 
(1.15%), and the blood serum in H₂SO₄ 
( 12 N ). The addition of TBA (0.3%) 
creates colored compounds that are 
measured 
photometrically 
at 
a 
wavelength of 534 nm. The calibration 
curve of MDA of known concentration 
allows for the determination of MDA 
levels in the samples (Uchiyama & 
Mihara, 1978). 
GSH 
continuous 
quantitative 
Initial and 6 
months 
Blood, urine, or tissue samples are 
collected and must be stored correctly 
to preserve the stability of the 
biomarkers. The concentration of 
reduced 
glutathione 
(GSH) 
is 
measured by a colorimetric assay with 
dinitrobenzene 
sulfonate 
(DNBS), 
where the intensity of the color is 
related to the amount of GSH. 
SOD 
continuous 
quantitative 
Initial and 6 
months 
Blood, urine, or tissue samples are 
collected and must be stored correctly 
to preserve the stability of the 
biomarkers. Superoxide dismutase 

 
 
 
40 
 
(SOD) activity is measured by a 
colorimetric assay that evaluates the 
inhibition 
of 
nitrobluetetrazolium 
reduction, using a spectrophotometer. 
 
Inflammation Assessment 
Type 2 diabetes mellitus (T2DM) is characterized by a state of chronic inflammation where immune 
cell activation and pro-inflammatory cytokines play a role in the development, progression, and 
pathogenesis of its complications. Therefore, the effect of complementary therapy on the 
inflammatory state in patients with T2DM will be evaluated to determine whether complementary 
therapy with *R. officinalis* will have an effect on the progression and pathogenesis of diabetic 
complications associated with inflammation. 
C-reactive protein, interleukin 6, and tumor necrosis factor alpha (TNF-α) will be determined. 
Table 9Operationalization of the variables of the inflammatory profile 
Profile 
evaluated 
Variable Guy 
Frequency Description 
Inflammation 
C-
reactive 
protein 
continuous 
quantitative 
Initial and 
monthly 
dL is determined after agglutination 
occurs , and subsequently, a series of 
dilutions are performed to determine the 
relevant concentration. 
IL-6 
continuous 
quantitative 
Initial and 
6 months 
A sandwich enzyme immunoassay will be 
used for the quantitative measurement of 
human IL-6 in serum. The plate is coated 
with an IL-6-specific antibody. The sample 
and standards are added to the wells, and 
the IL-6 present in each sample binds to 
the wells via the immobilized antibody. 
After incubation, the wells are washed 
and then incubated with biotinylated 
anti-IL-6 antibody , which binds to the 
captured IL-6 present in each well. After 
incubation, the unbound biotinylated 
detection antibody is removed by 
washing, an HRP- streptavidin conjugate 
is added to the wells, and the microtiter 
plate is incubated . After incubation and 
washing, TMB substrate solution is used 

 
 
 
41 
 
to visualize the enzymatic reaction of HRP 
catalysis, producing a blue product that 
turns yellow after the addition of an acidic 
stop solution. The density of yellow is 
proportional to the amount of IL-6 
captured in each well. 
 
TNF-α 
continuous 
quantitative 
Initial and 
6 months 
Quantitative measurement of TNF-α will 
be performed using a sandwich enzyme 
immunoassay in serum. The microtiter 
plate of the kit is coated with a TNF-α-
specific antibody. The corresponding 
standards or samples from the microtiter 
plate containing a biotin-conjugated 
antibody preparation specific for TNF-α 
are added to the wells. Horseradish 
peroxidase-conjugated avidin (HRP) is 
then added to each well of the microplate 
and incubated. After adding the TMB 
substrate 
solution, 
only 
the 
wells 
containing TNF-α, the biotin-conjugated 
antibody, and the enzyme-conjugated 
avidin will exhibit a color change. The 
enzyme-substrate reaction is terminated 
by adding a sulfuric acid solution, and the 
color 
change 
is 
measured 
spectrophotometrically at a wavelength 
of 450 nm ± 10 nm. 
 
Cardiovascular Evaluation 
Cardiovascular involvement will be assessed by measuring Troponin T levels , given that this 
troponin has been most strongly associated with asymptomatic cardiovascular damage in type 2 
diabetic patients. 
 
Profile 
to 
be 
evaluated 
Variable 
guy 
Frequency 
Description 
Cardiovascular 
Troponin T 
Continuous 
quantitative 
Initial 
and 
6 
months 
Troponin 
T 
measurement 
will 
be 
performed using 
the 
enzyme 
immunoassay 
technique 
with 
the use of a kit, 

 
 
 
42 
 
and following the 
manufacturer's 
instructions. 
7.4.  
Clinical fieldwork 
Patients will be recruited at the assigned research clinic, with prior authorization from the Director, 
with the goal of obtaining at least 30 volunteers. These volunteers will be randomly assigned to two 
groups: one for the experimental treatment (Rosemary Capsules) and another for the control 
treatment. Consultations for patients enrolled in the study will take place at the Research Clinic on 
Wednesdays from 11:00 AM to 5:00 PM (to cover both regular medical appointment times). If a 
participant is unable to attend during these hours, they will be provided with a telephone number 
to contact the researcher and schedule an appointment at their convenience. 
First date 
Once patients have been referred to our clinic following the inclusion and exclusion criteria, a 
comprehensive interview will begin, where we will assess the patient's personal and family medical 
history, lifestyle, reported symptoms, and medications they take for diabetes. 
A physical examination will then be performed, respecting the patient's modesty, but thorough in 
search of important signs in the diabetic patient. 
The next step will be the anthropometric measurements, in which weight will be taken using an 
OMRON HBF-514C digital scale with a reading accuracy of 0.1 kg (0.220 lb) and a weighing capacity 
of 149.69 kg (330 lb). Without shoes or heavy clothing, the participant should stand in the center of 
the scale, placing their feet in the designated footrests and remaining still during the measurement. 
The weight will be recorded when the numbers on the digital scale stabilize, along with the time the 
measurements are taken. Height will be measured using a SECA wall-mounted mechanical 
stadiometer , which has a measuring range of 0 to 220 cm. The participant, barefoot, will stand with 
their heels together, legs straight, and shoulders relaxed. Their heels, hips, shoulder blades, and the 
back of their head should be in contact with the vertical surface on which the stadiometer is 
mounted . The head should be positioned in the Frankfort horizontal plane, which is represented by 
a line between the lowest point of the eye socket and the tragus (the cartilaginous prominence in 
front of the opening of the external auditory canal). Just before the measurement is taken, the 
individual should inhale deeply, hold their breath, and maintain an erect posture. The hair will then  

 
 
 
43 
 
 
be compressed, and the measurement taken. Any hair ornaments that could interfere with the 
measurement should be removed. 
Using the weight and height data obtained, BMI will be calculated (using the formula Height / 
Weight²) based on the WHO tables (2007) to stratify each participant into the appropriate BMI 
category according to the data obtained (underweight, normal weight, overweight, obese). 
Participants will be given a request form for laboratory tests, and the appointment will be scheduled 
by the Researcher (Master's student) within a maximum of 10 days. The samples will be collected 
and processed at the IMSS facilities by laboratory personnel, with prior authorization. The results  
will be entered into the CILAB system, which is accessible only to medical personnel with a 
password. 
The data is recorded on the Clinical form (Annex 2) 
At this first appointment, each patient will be informed about the study, its objectives, and, of 
course, any possible adverse reactions they might experience with the use of the herbal medicine. 
Informed consent will also be obtained (Appendix 1). A follow-up appointment will be scheduled for 
14 days later. 
Second appointment (day 0) 
Laboratory results will be collected and reported in the corresponding section of Appendix 3. Blood 
pressure, anthropometry, and a buccal mucosal sample will be taken. If patients meet the criteria, 
they will be admitted to the research study. They will be assigned the initial treatment with a 
consecutive number corresponding to the same number as their questionnaire. Subsequently, the 
questionnaire, the clinical record, and the questionnaire on possible adverse effects (Appendix 4) 
will be completed. This questionnaire will record any symptoms present before the start of 
treatment that are not attributable to the treatment itself, and patients will be asked at each visit 
about possible side effects, covering all organ systems (tolerability). 
They will be randomly assigned the treatment to follow (first allocation), giving verbal and written 
instructions: (Annex 5) 
 

 
 
 
44 
 
 
1) Regarding taking the medication, schedules and dosage. 
2) In the case of amenorrhea in women of childbearing age, they will be instructed to immediately 
discontinue the medication and consult with the responsible physician. 
3) Procedure to follow in case of severe side effects (immediately stop treatment and seek medical 
advice). 
4) Be alert for the presence of side effects. 
5) Attend each of the following appointments on time. 
A follow-up appointment will be scheduled in 7 days. 
Third appointment (30 days): 
Following the fourth week of treatment, a physical examination, blood pressure measurement, 
anthropometric measurements, and oral swab will be taken. The follow-up questionnaire and 
questionnaire on potential adverse effects will be completed, gathering information in the specific 
sections of the respective questionnaire. Any questions will be answered. A second supply of the 
assigned treatment will be provided, along with a further explanation of how to identify warning 
signs. An open appointment will be scheduled for emergency care if needed, and patients will be 
monitored for any side effects. During this appointment, the effectiveness, tolerability, and 
adherence to the treatment will be evaluated (by counting ingested doses). Subsequent 
appointments will be scheduled every 30 days. 
Appointments at 2, 3, 4, 5 and 6 months of intervention  
Patients will be comprehensively re-evaluated, with a thorough physical examination, the presence 
or absence of adverse effects will be recorded, oral samples will be taken every month, and 
biochemical studies corresponding to the month of evaluation will be indicated. 
At the final appointment, each patient will be informed of the test results and their progress with 
the complementary treatment. Any questions will be addressed, and the form will be completed 
with the information gathered at this appointment. 
We must bear in mind that weekly consultations will be held to recruit new patients, as well as 
educational talks about self-care for diabetic patients, questions will be answered, and capillary  

 
 
 
45 
 
 
blood glucose tests will be performed sporadically and randomly using reagent strips and 
glucometers. 
7.5. Statistical Analysis 
A database will be created in the EXCEL 2019 program with the results of the questionnaires, 
proceeding to the validation of the data and subsequent statistical analysis. 
Sample size calculation: The calculated sample size for this study is N = 30 participants. This value 
was obtained using G Power software, considering a 95% confidence level, 80% statistical power, 
and a large effect size (assessed based on the glucose variable with an expected decrease of 180 
mg/dL). This sample size is adequate to detect significant differences in the variables of interest,  
ensuring the validity and reliability of the results. Block random sampling will be used to ensure that 
both groups are adequately represented, following the aforementioned inclusion and exclusion 
criteria. The sample will be representative of the target population. 
Descriptive statistics: Qualitative variables will be reported with absolute frequencies, relative 
frequencies and cumulative relative frequencies, in addition to measures of significance of 
proportions; quantitative variables will be reported with measures of central tendency and 
dispersion as appropriate (parametric variables will be evaluated with means and standard 
deviation and non-parametric variables with medians and interquartile ranges). This normality 
analysis will be performed with a Shapiro test. 
Intergroup Comparison Analysis 
To compare the variables between the intervention group and the control group, the following 
statistical tests will be used depending on the type of variable: 
• 
Quantitative variables: The independent samples t-test will be used if the variables follow a 
normal distribution. If the variables do not follow a normal distribution, the Mann-Whitney 
U test will be applied. This analysis will be performed initially, that is, when collecting clinical 
and laboratory data before the ingestion of the extract, in order to establish our initial 
comparisons between the control group and the case group. Once we have the data from 
the second laboratory measurements, as well as the monthly clinical observations, we will 
establish the comparisons using paired data, employing t-tests for parametric variables and  

 
 
 
46 
 
 
• 
Wilcoxon signed-rank tests for non-parametric variables. rank test. In the case of variables 
where we have 3 or more measurements, a repeated measures ANOVA will be performed 
if they are parametric, and if they are not, a Friedman test will be performed. 
• 
Categorical variables: The chi-square test will be used to compare the proportions between 
the two groups. If the expected frequencies are less than 5, Fisher's exact test will be used. 
For repeated measures categorical variables, McNemar's test will be performed. 
For the reliability index, a p-value of 0.05 will be used to reject null hypotheses and evaluate whether 
or not there are significant differences after using the extract. All data will be analyzed using SPSS 
version 23. 
 
7.6.  
Writing and acceptance of the protocol 
The protocol will be written by the internist Judisett Mirabal Rodríguez using Microsoft Word 
software, to be submitted for consideration to the National Research Commission of the IMSS and 
to the Research Ethics Committee of the Autonomous University of Zacatecas Francisco García 
Salinas. 
7.7.  
Conflicts of interest 
There are no conflicts of interest. 
7.8.  
Ethical considerations 
This research project will be conducted in accordance with the guidelines established in the 
Regulations of the General Health Law on Health Research, Title Two, Ethical Aspects of Research in 
Human Beings, Chapter II, Article 65, Pharmacological Research. Pharmacological research is 
defined as activities for which there is no prior experience in the country, that have not been 
registered by the Ministry of Health, and therefore are not commercially distributed, as well as 
registered and approved medications when their use is investigated with modalities, indications, 
doses, or routes of administration different from those established, including their use in 
combinations. According to Article 66, this is considered a Phase II clinical pharmacology drug 
research study. Furthermore, we will follow the guidelines of Article 67, where our research group 
has conducted preclinical studies in different species (dog, rat, and mouse). We are also awaiting 
any other guidelines that the aforementioned Regulations may require. 

 
 
 
47 
 
 
As part of the ethical considerations, an information session will be held with participants, and the 
project's objectives and scope, as well as its potential health implications and adverse effects, will 
be provided in writing. If individuals agree to participate in the project, they will be asked to provide 
their informed consent, which they must sign (See Annexes). 
Informed consent will be obtained in accordance with the Nuremberg Code, which clarifies the basic 
principles governing the ethical conduct of research, such as the capacity to give consent, absence 
of coercion, and understanding of the risks and benefits involved. The Code also establishes the 
fundamental ethical principles of respect for persons, justice, and beneficence. Participants will not 
receive any payment for their participation in the study, their personal data will be handled 
confidentially, and the results will be used solely for research purposes. 
The protocol will be registered at the Autonomous University of Zacatecas and submitted to the 
ethics and research committees of IMSS and UAZ 
Therefore, it is considered that the procedures to be carried out comply with the ethical standards 
and the Regulations of the General Health Law on Health Research, adhering to the 
recommendations for biomedical research in human beings, dictated in the Declaration of Helsinki 
in 1964 and revised in Tokyo in 1975, respecting the principles contained in the Nuremberg Code, 
the Belmont Report and the United States Code of Federal Regulations (Common Rule); prior 
authorization by the Ethics Committee of the National Coordination of Research of the IMSS. 
Regarding ISO Standards, we mention that our protocol complies with ISO 19609 (Parts 1, 2 and 3) 
which governs matters related to botanical products used as medicines: 
• 
Part 1: General quality requirements. 
• 
Part 2: Identification and testing. 
• 
Part 3: Pollutants and waste. 
Also with ISO 9001 on quality management systems: being applicable to laboratories or companies 
that produce the extract, it guarantees traceability, control and continuous improvement. 
Furthermore, we mention those ISO standards that regulate clinical studies involving human 
intervention: 
 

 
 
 
48 
 
 
• 
ISO 14155:2020 On clinical trials of medical devices in humans – Good clinical practice (GCP): 
Widely used as an ethical and technical basis for any RCT. 
• 
ISO 20916:2019 Relevant in our case since laboratory tests are used to measure the 
response of patients (such as glucose levels or HbA1c) and it governs the protocols for these 
tests. 
• 
ISO/IEC 
27001 
Information 
Security 
Management:  
This is of vital importance in our environment for the security of sensitive patient data and 
requires confidentiality and protection of clinical information. 
For conducting laboratory studies and techniques such as ELISA, our protocol will follow the 
following ISO standards to maintain the quality of the results of these studies: ISO 
15189:2022 on particular requirements for quality and competence applicable directly to 
medical laboratories that process human samples; ISO 13485:2016 Quality management 
systems for medical devices; in this case, we will use a kit for performing ELISA, ensuring 
that the kit manufacturer complies with good practices for in vitro diagnostic devices. 
7.9 .  
Human, physical and financial resources of the study 
Human resources 
The execution of this project relies on intra-institutional and inter-institutional collaboration 
networks. 
Within the Autonomous University of Zacatecas there is a collaboration network between the 
Academic Groups, Experimental Models for Diagnosis and Therapeutics (CA-UAZ-235) and 
Pharmacology in Molecular Biomedicine (CA-UAZ-175) 
Equipment 
The Laboratory of Pharmacology in Molecular Biomedicine and Neuroscience at the Autonomous 
University of Zacatecas has the equipment for obtaining the methanolic extract of R. officinalis. 
(rotary evaporators, water baths, irons, scales, -20°C refrigerator, mill, among others) as well as an 
ultra-freezing unit (-70°C) for the preservation of plasma samples until processing 
The acquisition of a semi-automatic encapsulator is necessary, which will be used to encapsulate 
the methanolic extract of R. officinalis  

 
 
 
49 
 
 
This research project aims to provide information on the benefits of complementary therapy with 
R. officinalis as an antidiabetic treatment, which will be reflected in the reduction of treatment costs 
and comorbidities 
7.10.  Biosafety aspects 
The study will be conducted in accordance with institutional biosafety regulations, taking into 
account the Official Mexican Standard that establishes the characteristics, identification procedures, 
classification, and list of hazardous waste (NOM-052-SEMARNAT-2005). All liquid or solid Biological 
Infectious Hazardous Waste (RPBI) generated in the laboratory will be decontaminated by 
laboratory personnel before collection and disposal by qualified personnel, in accordance with the 
procedures of NOM-087-ECOL-SSA1-2002 (Environmental Protection, Environmental Health,  
Biological-Infectious Hazardous Waste, Classification and Handling Specifications). The handling of 
biological waste will take into account that all material in contact with human samples (tubes, 
pipettes, ELISA plates) must be inactivated (e.g., with hypochlorite) and then disposed of as 
infectious biological waste according to local regulations. 
The waste will be delivered to the person in charge of the warehouse in the Health Sciences area, 
who is responsible for delivering it to the contracted collection company for its final disposal in 
accordance with the provisions of the official Mexican standard NOM-087-ECOL-SSA1-2002 
Furthermore, the laboratories at the Medical Units are equipped with the necessary equipment for 
sample evaluation, storage, and processing, all in accordance with the institution's regulations. 
Safety measures such as the use of gloves, gowns, and face masks will be in place to prevent contact 
and aspiration during handling. Appropriate aseptic techniques will also be followed for the 
preparation and encapsulation of the methanolic extract. The students involved have received prior 
training on working with the material and the proper technique for preparing the rosemary 
methanolic extract. 
Regarding biosafety requirements directly related to patients, an Informed Consent form will be 
signed, which includes an explanation of the risks, even the minimal ones associated with the 
supplement. Medical monitoring will be carried out, including tracking for possible adverse effects 
of the rosemary extract (for example, interactions with hypoglycemic medications). A thorough pre-
treatment evaluation for allergies or possible adverse reactions to rosemary compounds will be  

 
 
 
50 
 
 
performed through a comprehensive interview. We will also ensure that the encapsulated product 
is free of microbiological contaminants (microbiological standards for ingestible products ). 
A follow-up of possible adverse reactions to complementary therapy reported by patients will also 
be carried out (Annex 4). 
Regarding international biosafety standards, our study will be guided by ISO 10993, which 
establishes the biological evaluation of medical devices and is widely used to assess 
biocompatibility, toxicity, and dermal or systemic sensitization. Furthermore, the collection, 
storage, and analysis of human biological samples (blood, serum) will be conducted in accordance 
with ISO 20387:2018, respecting ethical consent, sample traceability, and storage and processing 
conditions. 

 
 
 
51 
 
 
9.  
EXPECTED RESULTS AND IMPACTS. 
Among the expected results of this project, we hope to generate scientific knowledge. We will have 
a validated method for the simultaneous quantification of rosmarinic acid , carnosol , and carnosic 
acid . Likewise, we will obtain the metabolic and inflammatory profile of the methanolic extract of 
Rosmarinus. The study will also examine the bioavailability of R. officinalis secondary metabolites ( 
rosmarinic acid , carnosol , and carnosic acid) in patients with type 2 diabetes mellitus. 
Undergraduate and postgraduate students will be trained. Researchers will participate in national 
and international forums and publish their findings in indexed journals. Meetings will be held at 
healthcare centers to disseminate the results to the public. 
By carrying out the intervention and clinical, biochemical, and healthcare follow-up of patients with 
type 2 diabetes included in the study, implementing support, counseling, and continuing education 
strategies to ensure their continued participation, the aim is to improve the health of these patients 
by contributing to the treatment of metabolic and inflammatory alterations present in patients with 
type 2 diabetes through the administration of a standardized extract of R. officinalis, thus having a 
positive impact on the quality and life expectancy of the patients. 
Type 2 diabetes (T2D) has a direct economic impact on healthcare spending. Diabetic patients die 
prematurely or live with the disease daily, experiencing significant economic consequences that 
represent a social cost. This economic burden affects a country's economic and human 
development, the capacity of its workforce, and conditions of equity and poverty. This project aims 
to reduce the economic burden of T2D by positively impacting the quality of life and life expectancy 
of patients with the disease. This will be reflected in both direct economic costs (outpatient care 
and treatment of complications) and indirect costs (premature death, absenteeism, work disability, 
invalidity, and presenteeism). 

 
 
 
52 
 
10 .  
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https://doi.org/10.35663/amp.2023.403.2731  
25. Díaz-Piñera, A., Rodríguez-Salvá, A., Achiong-Estupiñán, F., Cardona-Garbey, D., Maldonado-
Cantillo, G., Londoño-Agudelo, E., & Vander Stuyft, P. (2024). Therapeutic adherence of patients 
with type 2 diabetes mellitus in four health areas of the country. Finlay Journal, 14(1). 
Available from: https://revfinlay.sld.cu/index.php/finlay/article/view/1367  
26. Tremêa, G.T.F., Kleibert, K.R.U., Krause, L.S., Fell, A.P.W., Scapini, A.R., Marschall, K.W., Baiotto, 
C.S., da Silva, M.H.T., da Silva, J.A.G., & Colet, C.F. (2024). Aesthetic radiofrequency associated 
with Rosmarinus officinalis supplementation is safe and reduces oxidative stress in women: A 
randomized, double-blind clinical trial. Journal of Evidence-Based Integrative Medicine, 29, 
2515690X241246293. https://doi.org/10.1177/2515690X241246293  
27. Flores-Villa, E., Sáenz-Galindo, A., Castañeda-Facio, A.O., & Narro-Céspedes, R.I. (2020). 
Rosemary (Rosmarinus officinalis L.): Its origin, importance, and general aspects of its 
secondary metabolites. TIP: Specialized Journal in Chemical-Biological Sciences, 23, e20200266. 
https://doi.org/10.22201/fesz.23958723e.2020.0.266  
28. Huang, M., Wang, H., Xu, X., Lu, X., Song, X., & Zhou, G. (2020). Effects of nanoemulsion-based 
edible coatings with a composite mixture of rosemary extract and ε-poly-L-lysine on the shelf 
life of ready-to-eat carbonado chicken. Food Hydrocolloids, 102, 1–9. 
https://doi.org/10.1016/j.foodhyd.2019.105576  
29. Hendel, N., Sarri, D., Sarri, M., Napoli, E., Palumbo Piccionello, A., & Ruberto, G. (2024). 
Phytochemical analysis and antioxidant and antifungal activities of powders, methanol 
extracts, and essential oils from Rosmarinus officinalis L. and Thymus ciliatus Desf. Benth. 
International Journal of Molecular Sciences, 25(14), 7989. 
https://doi.org/10.3390/ijms25147989  
30. Li, T., Wang, W., Guo, Q., Li, J., Tang, T., Wang, Y., Liu, D., Yang, K., Li, J., Deng, K., Wang, F., Li, H., 
Wu, Z., Guo, J., Guo, D., Shi, Y., Zou, J., Sun, J., Zhang, X., & Yang, M. (2024). Rosemary (Rosmarinus 
officinalis L.) hydrosol based on serotonergic synapse modulation for insomnia. Journal of 

 
 
 
55 
 
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Ferreira L., Barbosa Evora PL, Kise Capellini V., et al. Effect of Rosmarinic acid on the arterial blood 
pressure in normotensive and hypertensive rats: role of ACE. Phytomedicine [Internet]. 2017 
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31. Prasannarong M., Saengsirisuwan V., Surapongchai J., et al. Rosmarinic acid improves 
hypertension and skeletal muscle glucose transport in angiotensin II-treated rats. BMC 
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Available at: https://doi.org/10.1186/s12906-019-2579-4 
32. Zhang, Y., & Li, X. (2022). "Impact of Type 2 Diabetes on Lipid Metabolism and Cardiovascular 
Risk." Frontiers in Endocrinology, 13, 657-664. 
33.  Hassani, F.V., Shirani, K., & Hosseinzadeh, H. (2016). Rosemary (Rosmarinus officinalis) as a 
potential therapeutic plant in metabolic syndrome: a review. Naunyn-Schmiedeberg's archives of 
pharmacology, 389(9), 931–949. https://doi.org/10.1007/s00210-016-1256-0 
34.  Borges RS, Ortiz BLS, Pereira ACM, Keita H, Carvalho JCT. Rosmarinus officinalis essential oil: A 
review of its phytochemistry, anti-inflammatory activity, and mechanisms of action involved. J 
Ethnopharmacol . 2019 Jan 30; 229:29-45. doi : 10.1016/j.jep.2018.09.038. PMID: 30287195. 
35. Quirarte-Báez SM, Zamora-Pérez AL, Reyes-Estrada CA, Gutiérrez-Hernández R, Sosa-Macías M, 
Galaviz-Hernández C, Manriquez GGG, Lazalde-Ramos BP. A shortened treatment with rosemary 
tea (Rosmarinus officinalis) instead of glucose in patients with type 2 diabetes mellitus (T2D). J 
Popul Ther Clin Pharmacol . 2019 Dec 3;26(4): e18-e28. doi : 10.15586/ jptcp.v26i4.634 . PMID: 
31909576. 
36. Bao, T.Q., Li, Y., Qu, C., Zheng, Z.G., Yang, H., & Li, P. (2020). Antidiabetic Effects and Mechanisms 
of Rosemary (Rosmarinus officinalis L.) and its Phenolic Components. The American journal of 
Chinese medicine, 48(6), 1353–1368. https://doi.org/10.1142/S0192415X20500664 
37. Ahmed, H.M., & Babakir -Mina, M. (2020). Investigation of rosemary herbal extracts (Rosmarinus 
officinalis) and their potential effects on immunity. Phytotherapy research : PTR, 34(8), 1829–
1837. https://doi.org/10.1002/ptr.6648 
38. Lazalde-Ramos, BP, Zamora-Pérez, AL, Ortega-Guerrero, AI, Quintero-Fraire, SZ, Palacios-Lara, O., 
Quirarte-Báez, SM, Galaviz-Hernández, C., Sosa-Macías, M., Ortiz-García, YM, & Morales-
Velázquez, G. (2020). Genomic Instability Decreases in HIV Patients by Complementary Therapy 

 
 
 
56 
 
with Rosmarinus officinalis Extracts. Journal of Medicinal Food, 23(10), 1070–1076. 
https://doi.org/10.1089/jmf.2019.0199 
39. Jayanthy G, Roshana Devi V, Ilango K, Subramanian SP. Rosmarinic Acid Mediates Mitochondrial 
Biogenesis in Insulin Resistant Skeletal Muscle Through Activation of AMPK. J Cell Biochem . 2017 
Jul;118(7):1839-1848. doi :10.1002/jcb.25869 
40. Labban L, Mustafa US, Ibrahim YM. The effects of Rosemary (Rosmarinus officinalis) leaves 
powder on glucose level, lipid profile and lipid peroxidation. Inte J Clin Med 2014;5:207 –304. 
41. . Jiang, L., et al. (2020). " Rosmarinic acid in rosemary: A potential remedy for improving insulin 
resistance in type 2 diabetes." Pharmacological Research . ( Accessed September 20, 2024) 
42. Ameer, OZ, et al. (2021). "A review of medicinal plants used for diabetes management: Focus on 
rosemary." Journal of Medicinal Plants Studies. ( Accessed September 20, 2024) 
43. Baron, DC, Marko, DM, Tsiani, E., & MacPherson, REK (2021). Rosemary extract increases 
neuronal cell glucose uptake and activates AMPK. Applied physiology, nutrition, and metabolism  
44. = 
Physiologie 
appliquee 
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46(2), 
141–147. 
https://doi.org/10.1139/apnm-2020-0014 
45.  Agatonovic-Kustrin , S., Balyklova , K.S., Gegechkori , V., & Morton, D.W. (2021). HPTLC and 
ATR/FTIR Characterization of Antioxidants in Different Rosemary Extracts. Molecules (Basel, 
Switzerland), 26(19), 6064. https://doi.org/10.3390/molecules26196064 
46. Tu Z, Moss-Pierce T, Ford P, Jiang TA. Rosemary (Rosmarinus officinalis L.) extract regulates 
glucose and lipid metabolism by activating AMPK and PPAR pathways in HepG2 cells. J Agric Food 
Chem. 2013 Mar 20;61(11):2803-10. doi :10.1021/jf400298c. 
47. Gonçalves C, Fernandes D, Silva I, Mateus V. Potential Anti-Inflammatory Effect of Rosmarinus 
officinalis in Preclinical In Vivo Models of Inflammation. Molecules. 2022 Jan 18;27(3):609. doi 
:10.3390/molecules27030609. PMID: 35163873; PMCID: PMC8840442. 
48. Kallimanis , P., Chinou, I., Panagiotopoulou, A., Soshilov, AA, He, G., Denison, MS, & Magiatis , P. 
(2022). Rosmarinus officinalis L. Leaf Extracts and Their Metabolites Inhibit the Aryl Hydrocarbon 
Receptor ( AhR ) Activation In Vitro and in Human Keratinocytes: Potential Impact on 
Inflammatory Skin Diseases and Skin Cancer. Molecules (Basel, Switzerland ), 27(8), 2499. 
https://doi.org/10.3390/molecules27082499 
49. Santos Rodríguez AP, Faria E Souza BS, Alves Barros AS, de Oliveira Carvalho H, Lobato Duarte J, 
Leticia Elizandra Boettger M, Barbosa R, Maciel Ferreira A, Maciel Ferreira I, Fernandes CP, Cesar 

 
 
 
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Matias Pereira A, Tavares Carvalho JC. The effects of Rosmarinus officinalis L. essential oil and its 
nanoemulsion on dyslipidemic Wistar rats. J Appl Biomed. 2020 Dec;18(4):126-135. doi : 
10.32725/jab.2020.016. Epub 2020 Nov 6. PMID: 34907765. 
50.  Zhang, L., & Lu, J. (2024). Rosemary (Rosmarinus officinalis L.) polyphenols and inflammatory 
bowel diseases: Major phytochemicals, functional properties, and health effects. Fitoterapia, 177, 
106074. https://doi.org/10.1016/j.fitote.2024.106074 
51.  Ghasemzadeh Rahbardar, M., & Hosseinzadeh, H. (2024). Toxicity and safety of rosemary 
(Rosmarinus officinalis): a comprehensive review. Naunyn-Schmiedeberg's archives of 
pharmacology, 
10.1007/s00210-024-03336-9. 
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https://doi.org/10.1007/s00210-024-03336-9 
52. Akinmoladun , Afolabi, et al. (2020). "Rosmarinus officinalis L. (rosemary) as a potent natural 
source for managing hyperlipidemia: A review of its molecular mechanisms". Environmental 
Toxicology and Pharmacology . 
53. González Madariaga1 Yisel, Boffill Cárdenas María de los Ángeles. Metabolic syndrome in basic 
research: Its potential treatment from a plant species. Medicent Electrón 2024;28: e4057 ISSN 
1029-3043 
54. Liu, XY, Wang, WZ, Yao, SP, Li, XY, Han, RM, Zhang, D., Zhao, Z., Wang, Y., & Zhang, JP (2024). 
Antioxidation Activity Enhancement by Intramolecular Hydrogen Bond and Non-Browning 
Mechanism of Active Ingredients in Rosemary: Carnosic Acid and Carnosol. The journal of physical 
chemistry. B, 128(31), 7627–7638. https://doi.org/10.1021/acs.jpcb.4c02949 
55. Vijayan, M., & Surya, K. (2017). "The role of herbal remedies in managing diabetes: Focus on 
rosemary." Journal of Ethnopharmacology . Consulted September 20, 2024 
56. Alegría Herrera E., Herrera Ruiz M., Román Ramos R., et al. Effect of Ocimum basilicum , Ocimum 
selloi , and Rosmarinic Acid on Cerebral Vascular Damage in a Chronic Hypertension Model. Biol 
Pharm Bull. (2019) [Internet]. 2019 [ cited 2024 Sep 27]; 2019;42(2):201-211. Available from: 
https://www.ncbi.nlm.nih.gov/pubmed/30713252 
57. Nawaz, Waseem, et al. (2019). "Effects of Rosmarinus officinalis (rosemary) on glucose and lipid 
levels in patients with type 2 diabetes: A randomized clinical trial." Journal of Ethnopharmacology  
58. Pérez-Mendoza MB, Llorens-Escobar L, Vanegas-Espinoza PE, Cifuentes A, Ibáñez E, Villar-
Martínez AAD. Chemical characterization of leaves and calli extracts of Rosmarinus officinalis by 
UHPLC-MS. Electrophoresis. 2020 Oct;41(20):1776-1783. doi :10.1002/elps.201900152. 

 
 
 
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59. Al Jamal A. Effect of Rosemary (Rosmarinus officinalis) on lipid profiles and blood glucose in 
human diabetic patients (type-2). Afr J Biochem Res 2014;8:147 –50 
60. El-Abhar, H.S., & El-Masry, T. (2020). Acute and sub-chronic toxicity evaluation of Rosmarinus 
officinalis L. in mice. Toxicology Reports, 7, 287-296. doi :10.1016/j.toxrep.2020.04.001 
61. Badria , FA, & Fouad, MA (2021). Cardiovascular effects of Rosmarinus officinalis: Beneficial or 
harmful Phytomedicine, 82, 153436. doi : 10.1016/j.phymed.2020.153436 
62. Kollipara, M.P., & Reddy, S. (2022). Dermatological safety of essential oils from Rosmarinus 
officinalis. Journal of Dermatological Science, 106(1), 65-72. doi : 10.1016/j.jdermsci.2021.09.004 
63. Marzocco, S., & Greco, F. (2023). Genotoxicity and carcinogenicity testing of Rosmarinus officinalis 
L. extracts in vitro. Environmental Toxicology and Pharmacology, 81, 103538. doi : 
10.1016/j.etap.2022.103538 
64. Almeida, PC, & Costa, JM (2023). Interactions of Rosmarinus officinalis with pharmaceutical drugs: 
A review of potential risks and benefits. Phytotherapy Research, 37(7), 2228-2237. 
doi:10.1002/ptr.7714 
65. Sussman, J., & Xie, H. (2023). "Metabolic Effects of Type 2 Diabetes: Beyond Glucose Control." 
Journal of Clinical Endocrinology & Metabolism, 108(6), 1072-1085 
66. Kim, D., & Lee, Y. (2021). "Insulin Resistance and Liver Metabolism in Type 2 Diabetes." Diabetes 
Research and Clinical Practice, 176, 108737. 
67. Hunter, L.J., & Bailey, C.J. (2020). "Chronic Inflammation and Insulin Resistance in Type 2 
Diabetes." Diabetes & Metabolism Journal , 44(4), 485-494. 
68. effectiveness of type 2 diabetes mellitus treatment in Mexico of treatment of type 2 diabetes 
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59 
 
1. ANNEXES 
 
Annex 1. Informed Consent 
Informed consent form for participation in research protocols 
Study name : 
Evaluation of the clinical metabolic effects of the methanolic extract of Rosemary 
(Rosmarinus officinalis L.) in patients with Type 2 Diabetes Mellitus . An interventional 
study 
Sponsor external : 
Not applicable . 
Place and date : 
October - November 2025 
Registration number 
institutional : 
CEI/ISSSTE/2025/043 
Justification 
and 
objective 
of 
the 
study: 
Type 2 diabetes mellitus is among the top nine causes of death in Mexico. The primary 
goal of treating type 2 diabetes is to normalize blood glucose levels to prevent or avoid 
associated complications, regardless of the medications used. However, this is 
sometimes insufficient to stop these complications, which is why patients turn to 
medicinal plants. Natural products made from plants remain an important part of 
traditional medicine, and there has been a recent increase in interest in their use. One 
of the plants used worldwide for treating various ailments, as well as in food 
preparation, is rosemary (Rosmarinus ). Rosemary officinalis contains substances that 
can be used to treat various diseases, such as type 2 diabetes, and also has 
hypoglycemic and hypotensive effects. This study will be conducted to determine the 
antidiabetic activity of a phytomedicine based on methanolic extract of rosemary. 
Procedures : 
Participants will be randomly assigned to two groups: one receiving only conventional 
treatment and the other receiving complementary therapy with 1.5g of methanolic 
rosemary extract for 6 months. An initial medical history will be taken, and blood 
samples will be collected before starting the rosemary complementary treatment and 
again at 3 and 6 months of treatment. A 10 ml blood sample will be taken on each 
occasion. 

 
 
 
60 
 
Possible 
risks 
and 
inconveniences : 
The treatment generally does not cause discomfort. If any side effects occur, they 
should be mild, although a hypersensitivity reaction (a minor or major allergic 
reaction), vomiting, or diarrhea may develop. If you experience any discomfort, you 
should stop the treatment and go to your Family Medicine Unit or, if necessary, to the 
Emergency Room for medical attention. 
Potential 
benefits 
you will receive by 
participating in the 
study: 
You will not receive any payment for your participation. The benefits lie in the 
improvement of clinical and laboratory parameters, as well as offering another 
therapeutic alternative for your condition. 
Information 
on 
results and treatment 
alternatives: 
Your progress will be explained to you in a simple and clear way, and if any 
complications arise, you will receive the necessary medical support. Referrals to other 
specialists will be provided if needed. 
Participation 
or 
withdrawal : 
The decision to participate in the study is voluntary, and if at any time you decide to 
leave the study, you may or may not inform us of your reason, which will not be judged 
negatively and your decision will be respected at all times. 
Privacy 
and 
confidentiality : 
At all times, the information obtained will be treated with respect and handled with 
the utmost discretion and secrecy by the research team. Furthermore , you will not be 
identified in any potential [unclear/unclear]. publications . 
Declaration of consent: 
After having read and having all my doubts about this study explained to me: 
I do not agree to participate in the study. 
 
I agree to participate and give my authorization for laboratory tests to be taken only for this study. 
If you have any questions or require clarification regarding the study, you may contact Dr. Judisett Mirabal 
Rodríguez, Internist, at 8180269497 or judisettm@gmail.com ; Dr. Sol María Quirarte Báez, at 4929490914 or 
sol.quirarte@imss.gob.mx 
; 
or 
Dr. 
Blanca 
Patricia 
Lazalde 
Ramos, 
at 
84921702977 
or 
lazalderamos@uaz.edu.mx .  

 
 
 
61 
 
 
 
Patient 's name and signature 
 
 
Name and signature of the person obtaining consent 
 
Witness 1 
Name, address, relationship and signature 
 
 
Witness 2 
Name, address, relationship and signature 
 
Annex 2 .  
Initial clinical record 
 
Evaluation of the clinical metabolic effects of the methanolic extract of rosemary (Rosmarinus officinalis 
L.) in patients with type 2 diabetes mellitus. An interventional study 
Instructions: 
The purpose of this clinical record is to identify the patients participating in the study. 
Please fill in each and every one of the following points in pencil. 
1 ) GENERAL DATA: 
Patient's name: ____________________________________________________________ 
Membership Number: ______________________________________ Age: _________________ 
Affiliated clinic: ____________________________________________________________ 
Home: ______________________________________________________________________ 
Telephone: _________________ Telephone 2: _____________________ 
Work address: ________________________________________________________________ 
Date: ___________________________________________ 
Folio Number: __________________________________ 
Consultation number: _______________________________ 
Assigned treatment: ______________________________ 
Sex: 

 
 
 
62 
 
Female ______ 
Male_____ 
Schooling: 
1) Primary: 
Complete____ 
Truncated____ 
2) Secondary: 
Complete____ 
Truncated____ 
3) Preparatory: 
Complete____ 
Truncated____ 
4) Degree: 
Complete____ 
Truncated____ 
5) Specialty: 
Complete____ 
Truncated____ 
6) Mastery: 
Complete____ 
Truncated____ 
7) Doctorate: 
Complete____ 
Truncated____ 
Occupation: 
3) PERSONAL BACKGROUND 
• 
Gynecological and obstetrical (if female): 
Are you currently pregnant? 
Yeah____ 
No____ 
Are you currently breastfeeding? 
Yeah____ 
No____ 
 
• 
Past medical history 
Do you suffer from any serious illness? 
No____ 
If Yes, Please Specify _____________ 
Heart disease? 
No____ 
Yeah____ 
Nephropathy? 
No____ 
Yeah____ 
Liver disease? 
No____ 
Yeah____ 
Cancer? 
No____ 
Yeah____ 
Diabetes mellitus? 
No____ 
If ____ Specify type _________ 
Do you suffer from other illnesses? 
No____ 
If Yes, Please Specify _____________ 
Do you suffer from any mental illness? 
No____ 
If Yes, Please Specify _____________ 
Do you smoke? 
No____ 
Yeah____ 
Cigarettes per day__________________ 
Do 
you 
drink 
alcohol? 
No____ 
Yeah____ 
Type and frequency_________________ 
Do you use or have you used 
prohibited substances? 
No____ Yeah____ 
Type 
and 
frequency_______________ 
Examination with an ophthalmoscope: 

 
 
 
63 
 
 
  
 
 

 
 
 
64 
 
Annex 3.  
Laboratory sheet  
 
IDENTIFICATION SHEET 
 
 
Name:_ ___________________________________________________ 
Folio:_ _________ 
Number : _________________________________ 
Date:_ __________________ 
Age._ ______ 
Sex:_ ______ 
FUM:_ __________________ 
MPF._ ___________________ 
Initial 
Weight:_ ______ 
Size:_ _____ 
BMI:_ ___________________ 
3 
months 
Weight:_ ______ 
Size:_ _____ 
BMI:_ ___________________ 
6 
months 
Weight:_ ______ 
Size:_ _____ 
BMI:_ ___________________ 
 
 
 
 
Parameter 
Initial 
3 months 
6 months 
Hb 
 
 
 
Hto 
 
 
 
Erythrocyte 
 
 
 
Leukogram 
 
 
 
AST 
 
 
 
ALT 
 
 
 
Tg 
 
 
 
Cholesterol 
 
 
 
Fasting glucosa 
 
 
 
Postprandial glucosa 
 
 
 
Glycosylated Hemoglobin 
 
 
 
Creatinine 
 
 
 
Urea 
 
 
 
Uric Acid 
 
 
 
Final Degree Project 
 
--------- 
 
MDA 
 
--------- 
 
SOD 
 
--------- 
 
IL-6 
 
--------- 
 
Triglyceride/glucose index 
 
--------- 
 
Triglyceride/HDL-C ratio 
 
--------- 
 
 
 
 

 
 
 
65 
 
Annex 4. Questionnaire about effects adverse 
General Patient Information 
Age:_ _________ 
Sex._ _________ 
 
 
 
Have you previously used rosemary 
extract or related products? 
No:_ _____ Yeah:_ 
______ 
Please specify:_ ____ 
______________ 
Are you currently taking any medication 
or supplements? 
No:_ _____ Yeah:_ 
______ 
Please specify:_ ____ 
______________ 
General Symptoms 
Have you experienced any general 
discomfort after starting treatment with 
rosemary extract? 
No:_ _____ Yeah:_ 
______ 
Please 
specify:_ 
_____________ 
______________ 
Have you noticed excessive fatigue or unusual 
drowsiness? 
No:_ _____ Yeah:_ ______ 
Have you experienced fever or chills? 
No:_ _____ Yeah:_ ______ 
Digestive Effects 
Have you had stomach problems, such 
as nausea, vomiting, or abdominal 
discomfort? 
No:_ _____ Yeah:_ 
______ 
Intensity:_ ______________ 
______________ 
Have you experienced diarrhea or constipation? 
No:_ _____ Yeah:_ ______ 
Have you had any digestive problems, such as 
heartburn or acid reflux? 
No:_ _____ Yeah:_ ______ 
Skin Effects 
Have you noticed any skin reactions, 
such as rashes, redness, or itching? 
No:_ _____ Yeah:_ 
______ 
Area:_ ____________________ 
______________ 
Have you experienced any allergic reaction, such as 
swelling of the face, lips, or tongue? 
No:_ _____ Yeah:_ ______ 
Respiratory Effects 
Have you had breathing difficulties or a feeling of 
shortness of breath? 
No:_ _____ Yeah:_ ______ 
Have you experienced persistent coughing or 
wheezing? 
No:_ _____ Yeah:_ ______ 
Effects on the Nervous System 
Have you noticed frequent or severe headaches? 
No:_ _____ Yeah:_ ______ 
Have you experienced dizziness or vertigo? 
No:_ _____ Yeah:_ ______ 
Have you had trouble concentrating or felt confused? 
No:_ _____ Yeah:_ ______ 
Cardiovascular Effects 
Have you noticed any changes in your heart rate, such 
as palpitations or irregular heartbeats? 
No:_ _____ Yeah:_ ______ 
Have you experienced high or low blood pressure after 
taking the extract? 
No:_ _____ Yeah:_ ______ 
Other Effects : ________________________________________________________ 
Have you had any adverse reactions that were 
not mentioned in the previous questions? 
No:_ 
____ 
Yeah:_ 
_____ 
Describe 
it:_ 
___  
__________________________ 

 
 
 
66 
 
Have you noticed any changes in your 
emotional state, such as anxiety, depression, 
or irritability? 
No:_ 
____ 
Yeah:_ 
_____ 
Please specify: ____ 
_______________ 
Frequency and Duration of Symptoms 
How long after starting treatment with rosemary 
extract did you notice adverse effects? 
At the moment:_ _____________________ 
After 1-2 days:_ ______________ 
After 1 week or more:_ ________ 
How often do you experience the adverse effects 
mentioned? 
Diary._ ____________ 
Weekly:_ __________ 
Occasionally:_ ____ 
Only once :_ _______ 
Have the symptoms improved, worsened, or remained 
the same since treatment began? 
Improved:_ _________ 
Worsened:_ ________ 
Equal:_ ______________ 
Action Taken 
Have you stopped using rosemary extract due to 
adverse effects? 
No:_ ____ 
Yeah:_ _____ 
Have you consulted a healthcare professional about the 
adverse effects? 
 
No:_ ____ 
Yeah:_ _____ 
Have you received any treatment to alleviate the 
adverse effects? 
No:_ ____ 
Yeah:_ _____ 
Which._ 
______________________ 
 
 
 

 
 
 
67 
 
Annex 5. Patient Instructions (Format for the proper administration of the assigned treatment) 
 
Aimed at patients with type 2 diabetes who voluntarily begin the study to receive treatment with 
methanolic extract of rosemary 
Patient's name: ________________________________________________ 
Membership number: ________________________________________________ 
Date: ____________________________ 
Please read the following instructions carefully before starting your prescribed treatment: 
1) Do not take any other medication, natural product or remedy for your illness. 
2) Your treatment lasts 6 months from today, concluding on the next day: ___________________. 
3) You must not discontinue treatment unless instructed to do so by the responsible physician. 
4) Please take the medication at the indicated time. 
5) The treatment is taken three times a day, morning, afternoon and evening. 
6) Make sure that the medication is always taken at the same time. 
7) During treatment you can eat your normal diet and do physical activity. 
8) During treatment you can do all your usual tasks. 
9) At each appointment, you will be given the necessary amount of medication to complete your 
treatment until your next appointment. 
10) Please attend an evaluation with the responsible physician on the following dates and times: 
Appointment 1: Date: ______________________; Time: ________________________. 
Appointment 2: Date: ______________________; Time: ________________________. 
Appointment 3: Date: ______________________; Time: ________________________. 
Appointment 4: Date: ______________________; Time: ________________________. 
Appointment 5: Date: ______________________; Time: ________________________. 
Appointment 6: Date: ______________________; Time: ________________________. 
Appointment 7: Date: ______________________; Time: ________________________. 
If you have any questions, you can visit the research office from 11:00 AM to 5:00 PM, Monday through 
Friday, or call 8180269497. You can also email judisettm@gmail.com 
 
 

 
 
 
68 
 
Annex 6. MN Questionnaire 
 
Data general 
No. of record: 
Name: 
Date birthdate : 
Age: 
Sex: 
M 
F 
Weight and size: 
 
In case of be women 
Last date of menstruation : 
Duration of the period menstrual: 
Place birthdate : 
Home: 
Schooling: 
Company where he /she works: 
Guy of job that performs : 
Years working in bliss company: 
Jobs Previous: 
Background of depression familiar 
Yeah 
No 
Background of diabetes 
Yeah 
No 
Background of diseases cardiovascular 
Yeah 
No 
Background of arthritis rheumatoid 
Yeah 
No 
Background of lupus erythematous systemic 
Yeah 
No 

 
 
 
69 
 
 
Background of cancer 
Yeah 
No 
Background of disorders food 
Yeah 
No 
Consumption 
tobacco : 
Yeah 
No 
Guy: 
Exposure to the tobacco (passive either asset): 
Age of start ( exposure either consumption): 
Time of consume tobacco: 
consumption : 
Packs consumed to the anus : 
Consumption 
of 
drugs: 
Yeah 
No Which is it: 
Frequency of consumption: 
Consumption 
of 
alcohol: 
Yeah 
No 
Guy: 
Frequency of consumption: 
Age to start: 
Amount of alcohol (weekly): 
Consumption of vitamins I accessories food: 
Yeah 
No 
Guy: 
Vitamin 
(to) 
(b) 
(c) 
(and) 
( Acid ) pholic ) 
Others: 
Yeah remember the brand write bullet 
Consumption 
of
medications: 
Yea
h 
No 
Specify (time, guy and dose): 
Employment 
of 
contraceptives
hormonal: 
Yeah 
No 
Guy: 
Contact with solvents, fertilizers, gasoline: 
Yeah 
No 
Please specify: 

 
70