Nutraceutical Composition Containing Natural Products Derivatives on the Modulation of the Endocrine Neuroimmune Axis

Study Overview
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Brief Summary
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The use of natural products in the prevention or treatment of chronic diseases is an emerging field in current medicine. And studying the mechanisms of actions by which natural products act in our bodies contributes to the rational use of these products. And the combination of different natural products such as prebiotics (FOS- fructooligosaccharides, GOS-Galactooligosaccharides and beta-glucans derived from yeast), herbal medicine (Silybum marianum), and minerals (Se-selenium, Zn-Zinc, and Mg-Magnesium), o which would result in a synergistic association between them can contribute to achieving not only preventive effects but treatment for chronic diseases such as diabetes and obesity. The present study aims to study the effects of a composition containing natural products on factors and markers that are part of mitochondrial biogenesis and the neuroimmune-endocrine system in healthy volunteers and that present grade 1 overweight/obesity.

Detailed Description
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Obesity and diabetes are chronic diseases characterized by increased visceral adipose tissue and sub-acute inflammation. These conditions can lead to insulin resistance, hyperglycemia, oxidative stress], and lipotoxicity, and lead to the development of metabolic syndrome. Previous work has shown that these comorbidities, as well as high levels of glucocorticoids, can modulate the hypothalamic-pituitary-adrenal (HPA) axis and inflammatory response. In some cases, these conditions and responses perturb neuroimmune endocrine system homeostasis. Another potential outcome arising from inflammation, insulin resistance, hyperglycemia, and lipotoxicity is a nonalcoholic fatty liver disease (NAFLD), a chronic liver disease that often coexists with obesity-related type 2 diabetes. Previous studies have demonstrated that NAFLD compromises glycemic and lipid homeostasis. The hyperglycemia and the lipotoxicity drive the metabolic pathways of the cell towards gluconeogenesis, glycolysis, and de novo lipogenesis, and the subsequent increase in free fatty acid (FFA) production can cause oxidative stress. Previous studies have shown that non-pharmacological interventions can be used as strategies for treating and/or preventing inflammatory and metabolic diseases. Furthermore, exploiting specific proteins and transcription factors that target the mitochondria represents potential mechanisms for these interventions. Along these lines, natural products and derivatives thereof, often referred to as natural medicines, have been gaining notoriety as sources of effective therapeutics. For example, polyphenols present in plants can be metabolized to form propionic acid, which is later transformed into another metabolite, butyrate. As reviewed by Juárez-Hernández et al., Na-butyrate and synthetic derivatives thereof increase prebiotic lactobacilli and bifidobacteria growth in the colon, consequently improving thermogenesis and energy expenditure, ultimately reducing hepatic lipid deposition, improving triacylglycerol content and insulin resistance and protecting against metabolic impairment, as well as liver inflammation and/or damage. Furthermore, butyrate can induce the expression of fibroblast growth factor 21 (FGF-21), which plays a vital role in stimulating hepatic fatty acid β-oxidation. In this sense, prebiotics may be a promising treatment for chronic inflammatory and metabolic diseases in humans. The efficacy of prebiotic compounds, such as fructooligosaccharides (FOS), galacto-oligosaccharides (GOS) and yeast β-glucans, minerals including magnesium, zinc, selenium, and plants like Silybum marianum (L.) Gaerth. (Silymarin), have been evaluated for the treatment of specific diseases. However, most of these studies only describe the effects of the supplements being administered alone. Thus, the present study monitored the synergic effects of a novel supplement formulation containing prebiotics, yeast β-glucans, minerals, and silymarin on lipid metabolism, inflammatory and mitochondrial proteins in eutrophic, and overweight/obesity I volunteer.

Official Title
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Evaluation of the Effects of a Nutraceutical Composition Containing Derivatives From Natural Products on the Modulation of the Endocrine Neuroimmune Axis - Translational Study.

Conditions
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Insulin Resistance, Inflammatory Bowel Diseases, Overweight and Obesity, Healthy

Intervention / Treatment
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* Dietary Supplement: Composition with Silymarin
* Dietary Supplement: Low-mineral composition without Silymarin


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Healthy (healthy) and sedentary volunteers: people without chronic use of drugs for chronic inflammatory diseases that require the use of corticosteroids or NSAIDs (Non-Steroidal Anti-inflammatories) for a period of more than 15 days. BMI (Body Mass Index) of 24.9 Kg / m2. Sedentary overweight/obesity grade 1 volunteer: overweight people, with a BMI of 25.0 to 29.9 kg / m2; and grade 1 obesity with a BMI of 30.0 and 34.9 kg / m2, with or without dyslipidemia and/or non-insulin-dependent type 2 diabetics. Exclusion Criteria: Volunteers who are allergic to some of the components of the nutraceutical formulation, who use insulin, corticosteroids, and NSAIDs for more than 15 days, AIDS, hepatitis, patients undergoing chemotherapy.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 80 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Other
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: Group 1 - composition with Silymarin (L5-Plus)<br>This group compound with healthy and overweight/obese-I will receive the composition with Silymarin (Silybum marianum), in a diary dose of 140 mg for 90 days. The formulation of the supplement/composition (Patent number: BR 10 2020 016156 3). | Dietary Supplement: Composition with Silymarin<br>* Volunteers will receive supplementation for 90 days. They took 4 capsules daily. Two capsules in the morning and two capsules at night. There will be blood and feces collection at zero time, before supplementation, after 30 and 90 days from the beginning of supplementation.<br>* Other names: Patent number: BR 10 2020 016156 3;Dietary Supplement: Low-mineral composition without Silymarin<br>* Volunteers will receive supplementation for 90 days. They took 4 capsules daily. Two capsules in the morning and two capsules at night. There will be blood and feces collection at zero time, before supplementation, after 30 and 90 days from the beginning of supplementation.<br>|
| Active Comparator: Group 2 - composition without Silymarin (L5-Plus)<br>This group compound with healthy and overweight/obese-I will receive the composition without Silymarin (Silybum marianum), in a diary dose for 90 days. The formulation of the supplement/composition (Patent number: BR 10 2020 016156 3). | Dietary Supplement: Composition with Silymarin<br>* Volunteers will receive supplementation for 90 days. They took 4 capsules daily. Two capsules in the morning and two capsules at night. There will be blood and feces collection at zero time, before supplementation, after 30 and 90 days from the beginning of supplementation.<br>* Other names: Patent number: BR 10 2020 016156 3;Dietary Supplement: Low-mineral composition without Silymarin<br>* Volunteers will receive supplementation for 90 days. They took 4 capsules daily. Two capsules in the morning and two capsules at night. There will be blood and feces collection at zero time, before supplementation, after 30 and 90 days from the beginning of supplementation.<br>|
| Active Comparator: Group3 - Low-mineral composition without Silymarin (L5)<br>This group compound with healthy and overweight/obese-I will receive the composition without Silymarin (Silybum marianum), in a diary dose for 90 days. The formulation of the supplement/composition (Patent number: BR 10 2020 016156 3). | Dietary Supplement: Composition with Silymarin<br>* Volunteers will receive supplementation for 90 days. They took 4 capsules daily. Two capsules in the morning and two capsules at night. There will be blood and feces collection at zero time, before supplementation, after 30 and 90 days from the beginning of supplementation.<br>* Other names: Patent number: BR 10 2020 016156 3;Dietary Supplement: Low-mineral composition without Silymarin<br>* Volunteers will receive supplementation for 90 days. They took 4 capsules daily. Two capsules in the morning and two capsules at night. There will be blood and feces collection at zero time, before supplementation, after 30 and 90 days from the beginning of supplementation.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change Lipid Profile Baseline after 4 and 12 weeks of intervention. | The serum levels of cholesterol (mg/dL), triglycerides (mg/dL), and high-density lipoprotein cholesterol (HDL-c) (mg/dL) will be measured with a commercially available kit (Bioclin, Belo Horizonte, MG, Brazil). | Baseline and 4 and 12 weeks. | 
| Change Lymphocyte Phenotype at 4 and 12 weeks after intervention. | Lymphocytes will be isolated from 12 mL of whole blood. Anticoagulated blood is layered onto Histopaque-1077. During centrifugation, erythrocytes are aggregated by polysucrose and rapidly sediment. Lymphocytes and other mononuclear cells remain at the plasma/ Histopaque-1077 interface. Single-cell suspensions with lymphocyte will be incubated with the human antibodies indicated: T helper type 1 (Th1) cells are a subset of CD4+ effector T cells (cell surface expression of IL-12 R beta 2, IL-27 R alpha/WSX-1, IFN-gamma R2, IL-18 R, CCR5, and CXCR3); T helper type 2 (Th2) cells are a subset of CD4+ effector T cells (cell surface markers CCR3, CCR4, CCR8, CXCR4, and ST2/IL-1 R4). T helper type 9 (Th9) cells are a subset of CD4+ effector T cells [helper cell subsets including T-bet (Th1 cells), GATA-3 (Th2 cells), ROR gamma t (Th17 cells), or FoxP3 (regulatory T cells)]. Cells will be analyzed in a FACSCalibur (BD Biosciences, San Diego, CA). | Baseline and 4 and 12 weeks. | 
| Change Serum Cortisol Level Baseline after 4 and 12 Weeks of Intervention. | Serum cortisol (pg/ml) will be measured by Elisa Kit (Cayman Chemical, Ann Arbor, MI, USA), according to the recommendations of the manufacturer. | Baseline and 4 and 12 weeks. | 
| Change Gut Microbiota Baseline after 4 and 12 Weeks of Intervention. | For analysis of the gut microbiota, stool samples will be collected (approximately 15g) in a sterile plastic tube kit (ColOff®) and kept frozen at -80°C until the DNA extraction procedure. The material will not be collected in cases of watery stools or when the evacuation was not spontaneous (use of laxatives for evacuation). At the time of stool collection, none of the participants should be using or has used antibiotics for at least one month. Samples will be collected before bowel preparation for surgery, if necessary. Genomic DNA will be extracted from the fecal contents using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and the microbiota composition analysis will be performed by 16S rRNA gene sequencing on the Illumina MiSeq platform. | Baseline and 4 and 12 weeks. | 
| Change Haemogram Parameters Baseline after 4 and 12 Weeks of Intervention. | Anticoagulated blood will be used to do the complete blood count performed using basic laboratory equipment or an automated hematology analyzer, which counts cells and collects information on their size and structure. Total red blood count (RBC), Haemoglobin (HGB). Haematocrit (HCT), Mean Cell Volume (MCV), Mean Cell Haemoglobin (MCH), Mean Cell Haemoglobin Concentration (MCHC), and platelets (mm3). | Baseline and 4 and 12 weeks. | 
| Change Leucogram Parameters Baseline after 4 and 12 Weeks of Intervention. | Anticoagulated blood will be used to do the complete blood count performed using basic laboratory equipment or an automated hematology analyzer, which counts cells and collects information on their size and structure. Assessment of leukocyte numbers: Total WBC count (all cell types), relative (%) and absolute (cells/uL) differential leukocyte count (WBC separated by type). Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils. | Baseline and 4 and 12 weeks. | 
| Change Circadian Rhythms Over time of Intervention. | The first version of the Portuguese language questionnaire, Morningness-Eveningness-Stability Scale - improved by Horne e Ostberg (QMV-H&O), had 20 items (19 items translated from English language questionnaire and a 20th added by us). The total of the items is divided in three subscales, each one composed of five items: Morning Affect, Eveningness, and Distinctness. The items related to the Morning Affect subscale measure morningness preferences (early schedules), whereas the items of the Eveningness subscale assess evening preferences (late schedules). The remaining five items constitute the Distinctness subscale, that is, the amplitude dimension of this instrument. Each item is responded using a 5-points Likert scale and scored with 1-5 points, although some of them are reverse coded. The previous validation studies mentioned in the Introduction have revealed good indexes, such as Cronbach' alpha values for the three subscales ranging between 0.69 to 0.87. | Baseline and 4 and 12 weeks. | 
| Change Quality of Life Assessments Over time of Intervention. | One of the instruments that is regularly used to measure QOL, is the World Health Organization Quality of Life instrument - 100 items (WHOQOL-100; WHOQOL Group, 1998b), a cross-culturally developed generic instrument. It was created by the World Health Organization Quality of Life Group (WHOQOL Group), that defined QOL as an individual's perception of his/her position in life in the context of the culture and value systems in which he/she lives and in relation to his/her goals, expectations, standards and concerns (WHOQOL group, 1995). The WHOQOL-BREF contains 26 items; one item from each of the 24 facets contained in the original WHOQOL-100 and two items were retrieved from the 'Overall QOL and General health' facet. The WHOQOL-BREF covers four domains: Physical health, Psychological health, Social relationships, and Environment. | Baseline and 4 and 12 weeks. | 
| Change Sleep Patterns Over time of Intervention. | Sleep patterns will be evaluated using the Brazilian Portuguese Version of the Mini-Sleep Questionnaire (MSQ-BR). It is composed of a ten-item self-report scale that measures frequencies of sleep difficulty. Insomnia is evaluated by four questions which assess difficulty in falling asleep, mid-sleep awakenings, early awakening in the morning, and hypnotic medication use (MSQ-BR insomnia). For evaluation of hypersomnia, the questions include snoring, feeling tired upon awakening, excessive daytime sleepiness, and restless sleep (MSQ-BR hypersomnia). Answers are given on a seven-point scale ranging from 1 (never) to 7 (always). The total sum of scores is divided into four levels of sleep difficulties: 10-24 points, good sleep quality; 25-27 points, mild sleep difficulties; 28-30 points, moderate sleep difficulties; and ≥31 points, severe sleep difficulties. The total score offers an estimate of sleep quality, with higher scores reflecting more sleeping problems. | Baseline and 4 and 12 weeks. | 
| Change Sleep Quality Over Time of Intervention. | The Pittsburgh Sleep Quality Index (PSQI) assesses sleep quality over a 1-month period. The questionnaire consists of 19 self-rated questions and 5 questions that should be answered by bedmates or roommates. The latter questions are used only for clinical information. The 19 questions categorized into 7 components, graded on a score that ranges from 0 to 3. The PSQI components are as follows: subjective sleep quality (C1), sleep latency (C2), sleep duration (C3), habitual sleep efficiency (C4), sleep disturbances (C5), use of sleeping medication(C6), and daytime dysfunction (C7). The sum of scores for these 7components yields one global score, which ranges from 0 to 21, where the highest score indicates the worst sleep quality. A global PSQIscore greater than 5 indicates major difficulties in at least 2 components or moderate difficulties in more than 3 components. | Baseline and 4 and 12 weeks. | 
| Change Sleepiness Patterns Over time of Intervention. | Excessive daytime sleepiness (EDS) is defined as an increased propensity to fall asleep under circumstances that the affected individual and others would consider inappropriate. A Portuguese-language version of the Epworth sleepiness scale (ESS) will be applied to volunteers. The ESS is based on questions referring to eight such situations, some known to be very soporific; others less so. The questionnaire, which is self-administered. Subjects are asked to rate on a scale of 0-3 how likely they would be to doze off or fall asleep in the eight situations, based on their usual way of life in recent times. A distinction is made between dozing off and simply feeling tired. If a subject has not been in some of the situations recently, he is asked, nonetheless, to estimate how each might affect him. | Baseline and 4 and 12 weeks. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change Body Mass Index (BMI) Over time of Intervention. | Body Mass Index (BMI) - weight in kilograms divided by the square of height in meters (kg/m^2) . | Baseline and 4 and 12 weeks. |