Relación entre la infusión de Stevia rebaudiana Bertoni (Ka'a He'ê) y la concentración de alfa-amilasa salival

• Autores: Ponce, Jorge Orlando
• Año de publicación: 2022
• Idioma: español castellano
• Tipo de recurso: tesis doctoral
• Colaborador/a o director/a de tesis: Juárez, Rolando Pablo Alejandro; Mazza, Silvia Matilde
• Institución: Universidad Nacional del Nordeste

Fil: Ponce, Jorge Orlando. Universidad Nacional del Nordeste. Facultad de Odontología; Argentina.
Fil: Juárez, Rolando Pablo Alejandro. Universidad Nacional del Nordeste. Facultad de Odontología; Argentina.
Fil: Mazza, Silvia Matilde. Universidad Nacional del Nordeste. Facultad de Odontología; Argentina.
Dentro de los componentes de la saliva, la -amilasa salival (AAs) ha sido estudiada, en grupos de individuos que poseen una condición específica, como caries, asma, diabetes, con el fin de encontrar su relación tanto con enfermedades orales como enfermedades sistémicas. Se conoce desde hace tiempo la relación estrecha y directa entre el consumo de azúcares y la presencia de caries, también que dicha relación se debe al efecto que la presencia de azúcares libres en la boca tiene sobre el desarrollo de las bacterias causantes de la caries. La AAs cumple un papel importante en la digestión del almidón, el glucógeno y otros polisacáridos, por lo que su inhibición se considera una estrategia para el tratamiento de trastornos en la absorción de hidratos de carbono, tales como la diabetes y la obesidad, así como las enfermedades relacionadas con la placa bacteriana, caries dental y enfermedad periodontal. El papel potencial de vegetales inhibidores de la α-amilasa ha sido revisado por varios autores, pero no se ha descripto dicha actividad para Stevia rebaudiana (Bertoni). El objetivo de esta tesis ha sido determinar la relación entre el consumo de una infusión de estevia (S. rebaudiana, Bertoni) y la concentración de AAs. Con los objetivos específicos de: 1) relacionar los contenidos de -amilasa salival con la edad de los sujetos; 2) evaluar la asociación entre los contenidos de AAs en los diferentes momentos de medición; 3) analizar los contenidos de AAs basal y después de 60 y 120 minutos posteriores al consumo de líquidos; 4) comparar los contenidos de AAs entre los sujetos que consumieron infusión de estevia, jugo artificial de naranja y agua. Las hipótesis de investigación que se plantearon son: 1) los contenidos de -amilasa salival se encuentran relacionados con la edad de los sujetos; 2) existe asociación entre los contenidos de -amilasa salival en los diferentes momentos de medición; 3) los contenidos de -amilasa salival basal difieren de los valores obtenidos después de 60 y 120 minutos posteriores a la ingestión de líquidos; 4) los contenidos de -amilasa salival presentan variaciones entre los sujetos que consumieron infusión de estevia, jugo artificial de naranja y agua. El enfoque de la investigación fue de tipo cuantitativo, con un diseño experimental transversal, de ensayo controlado. La población estuvo constituida por estudiantes de las Clínicas Integradas de la Facultad de Odontología dependiente de la Universidad Nacional del Nordeste, en los meses abril-noviembre del año 2019. Los sujetos experimentales fueron incluidos considerando criterios de inclusión y de exclusión, además se seleccionaron mediante un muestreo aleatorio simple del 20% de la población, los estudiantes que pasarían a integrar la muestra, asignando a cada uno de los grupos experimentales siguientes: Grupo 1, ingesta de infusión de estevia (n1 = 20); Grupo 2, ingesta de bebida artificial sabor naranja (n2 = 20); Grupo 3, ingesta de agua (n3 = 20). Los grupos 2 y 3 son considerados controles (grupo 2, control con edulcorantes artificiales y grupo 3, blanco). La infusión de Stevia se preparó haciendo hervir en 250 ml de agua por 10 minutos 2.0 g de hojas secas de estevia trituradas, dejando luego reposar por otros 10 minutos. Para la preparación de jugo artificial de naranja se utilizaron jugos en polvo de la marca comercial BC La Campagnola®, siguiendo las instrucciones del envase. A los integrantes del tercer grupo se les suministró 250 ml de agua corriente de red. A los sujetos de todos los grupos experimentales se les realizaron extracciones de saliva y determinaciones de AAs en tres momentos, siguiendo los tiempos de las pruebas funcionales de absorción intestinal modificada: a. Toma inicial de una muestra de saliva, con ayuno de al menos 3 horas antes. b. Toma de muestra de saliva luego de ingerir la bebida correspondiente a su rupo experimental y un reposo de 60 minutos. c. Toma de muestra de saliva luego de ingerir la bebida correspondiente a su grupo experimental y un reposo de 120 minutos. Se solicitó a los participantes que se abstuvieran de cepillarse los dientes y hacer ejercicio al menos una hora antes de la recolección. Se advirtió no beber bebidas alcohólicas 18 horas antes de las muestras matutinas. Durante el día de estudio, se aconsejó evitar el consumo de cafeína y mateína, las situaciones estresantes y las emociones. Los días de recolección y medición fueron entre semana con horarios de clases normales, se evitaron los días de exámenes o con exigencias académicas no habituales. Todas las muestras de saliva se recogieron en un rango horario comprendido entre las 07:00 am y las 10:00 am. El método de recolección de saliva fue no estimulado mediante salivación pasiva, se instruyó a los sujetos para que se enjuaguen bien la boca antes del ensayo de recolección y que vacíen la boca de saliva, se les indicó luego que se sentaran cómodamente con los ojos abiertos, la cabeza inclinada ligeramente hacia adelante y que descansen durante 5 minutos. Las muestras fueron recolectadas en sesiones por cada individuo, en horario prefijado, utilizando un recipiente de propileno desechable, estéril y con tapa a rosca de 15 ml de capacidad, donde los individuos experimentales depositaron la saliva, luego de acumularla por un período de dos minutos en la boca. En el laboratorio, cada muestra fue depositada en tubos milimetrados estériles de 15 ml, rotulados con el grupo de estudio, la edad y género del sujeto, y fueron procesadas de manera inmediata. Antes de las determinaciones, las muestras de saliva se centrifugaron a 3000 rpm durante 10 m. La actividad de AAs se determinó mediante un kit comercial (Amilasa 405 ®, Wiener lab, Rosario, Argentina), que se basa en la hidrólisis del sustrato 2-cloro-pnitrofenil-α-D-maltriosido por la alfa amilasa presente en la muestra para producir 2- cloro-p-nitrofenol el cual se determinó espectrofotométricamente a 405 nm, empleando un Espectrómetro marca Spectrum. os valores se expresan en unidades amilolíticas. Amilasa 405 línea liquida AA, es un método cinético a 405 nm para la determinación de amilasa en suero, plasma u orina y saliva, sustrato CNPG3.
Within the components of saliva, salivary a-amylase (sAA) has been studied, in groups of individuáis who have a specific condition, such as caries, asthma, diabetes, to find its relationship with both oral diseases and systemic diseases. It has long been known the cióse and direct relationship between the consumption of sugars and the presence of caries, also that this relationship is because that the presence of free sugars in the mouth has on the development of bacteria that cause caries. The sAA play an important role in the digestión of starch, glycogen, and other polysaccharides, so its inhibition is considered a strategy for the treatment of disorders in carbohydrate absorption, such as diabetes and obesity, as well as diseases related to bacterial plaque, dental caries and periodontal disease. The potential role of a-amylase inhibitor plants has been reviewed by several authors, but no such activity has been described for Stevia rebaudiana (Bertoni). The objective of this thesis has been to determine the relationship between the consumption of an infusión of stevia (S. rebaudiana, Bertoni) and the concentraron of sAA. With the specific objectives of: 1) relating the contents of salivary a-amylase with the age of the subjects; 2) evalúate the association between the contents of salivary a-amylase at different times of measurement; 3) analyze the contents of salivary a-amylase baseline and after 60 and 120 minutes after fluid consumption; 4) compare the contents of salivary a-amylase among subjects who consumed stevia infusión, artificial orange juice and water. The research hypotheses that were proposed are: 1) the contents of salivary a-amylase are related to the age of the subjects; 2) there is an association between the contents of salivary a-amylase at different times of measurement; 3) the contents of basal salivary a-amylase differfrom the valúes obtained after 60 and 120 minutes after the ingestión of liquids; 4) the contents of salivary a-amylase present variations between the subjects who consumed infusión of stevia, artificial orange juice and water. The research approach was quantitative, with a cross-sectional, controlled trial experimental design. The population was constituted by students of the Integrated Clinics of the Faculty of Dentistry dependent on the National University of the Northeast, in the months April-November of the year 2019. The experimental subjects were included considering inclusión and exclusión criteria and the students who would join the sample were selected by means of a simple random sampling of 20% of the population, assigning to each of the following experimental groups: Group 1, stevia infusión intake (m = 20); Group 2, intake of orange-flavored artificial drink (n2 = 20); Group 3, water intake (m = 20). Groups 2 and 3 are considered Controls (group 2, control with artificial sweeteners and group 3, white). The Stevia infusión was prepared by boiling 2.0 g of crushed dry stevia leaves in 250 mI of water for 10 minutes, then letting stand for another 10 m¡ñutes. For the preparation of artificial orange juice, powdered juices of the BC La Campagnola® trademark were used, following the ¡nstructions on the package. The members of the third group were given 250 mi of mains running water. Subjects from all experimental groups underwent saliva extractions and determinations of sAA at three times, following the times of modified intestinal absorption functional tests: a. Initial collection of a saliva sample, fasting at least 3 hours before. b. Saliva sampling after ingesting the drink corresponding to the experimental group and a rest of 60 minutes. c. Saliva sampling after ingesting the drink corresponding to the experimental group and a rest of 120 minutes. Participants were asked to refrain from brushing their teeth and exercising at least one hour before collection. Caution was advised not to drink alcoholic beverages 18 hours before morning samples. During the study day, it was advised to avoid caffeine and mateine consumption, stressful situations, and emotions. The days of collection and measurement were during the week with normal class schedules, exam days were avoided or with unusual academic demands. All saliva samples were collected in a time range between 07:00 am and 10:00 am. The saliva collection method was not stimulated by passive salivation, subjects were instructed to rinse their mouths thoroughly before the collection trial and empty their mouths of saliva and were then instructed to sit comfortably with their eyes open, their heads tilted slightly forward, and to rest for 5 minutes. The samples were collected in sessions by everyone, at a predetermined time, using a sterile disposable propylene container with a screw cap of 15 ml capacity, where the experimental individuals deposited the saliva, after accumulating it for a period of two minutes in the mouth. In the laboratory, each sample was deposited in sterile millimeter tubes of 15 ml, labeled with the study group, the age and gender of the subject, and were processed immediately. Prior to determinations, saliva samples were centrifuged at 3000 rpm for 10 m. The activity of sAA was determined by a commercial kit (Amylase 405, Wiener lab, Rosario, Argentina), which is based on the hydrolysis of the substrate 2-chloro- pnitrophenyl-a-D-maltrioside by the alpha amylase present in the sample to produce 2-chloro-p-nitrophenol which was determined spectrophotometrically at 405 ® nm, using a Spectrum Spectrometer. Valúes are expressed ¡n amylolytic units. Amylase 405 liquid line AA ¡s a kinetic method at 405 nm for the determination of amylase ¡n serum, plasma or uriñe and saliva, substrate CNPG3. The speclflcatlons of the commercial kit (Amylase 405 ®) were followed for the measurement procedure and the concentration of sAA was expressed ¡n units per liter (U/L). The determinations were made ¡n the Sclentlflc Research Laboratory of the Faculty of Dentistry of the UNNE. The following variables were recorded ¡n the clinical history file and subsequently turned to a database: the experimental or independent variable was constituted by the experimental group (¡ntake of stevia infusión, artificial orange juice or plain water); the response or dependent variables were the contents of sAA baseline (prior to consumption of beverages), at the first hour and at the second hour; these variables were categorized into three ranges, taking into account the valúes of the first (Q1) and third (Q3) quartile, in low valúes of sAA (< Q1), means of sAA (between Q1 and Q3) and high (> Q3). In addition, age was recorded as the intervening variable because ¡t was considered a factor that could ¡nfluence the response variable. To study the general behavior of the data, an exploratory analysis was carried out, graphically using box and point density graphs, and analytical through the calculation of descriptive measures of position and dispersión. The associations between the variables sAA and age and the correlations between the valúes of sAA baseline, in the first and second hour, were studied using the Pearson correlation coefficient (r) with a level of significance a = 0.05. To graphically visualize the relationships between variables, scatter plots and scatter plot matrix were used. To visualize the behavior of the subjects and the variables at different times, a Principal Components analysis was carried out and subsequently the individuáis and variables were plotted on the first two components using a Biplot graph. To study the curve of sAA over time, Regression analysis was performed between the moments of saliva extraction and the contení of sAA in each of the experimental groups. To check if there is an effect of the experimental group on the valúes of sAA, Analysis of Variance and subsequent F Tests were performed, with a level of significance a = 0.05. To determine if the frequencies with which the categories of sAA were presented in the three moments of measurement are linked to the experimental group, Independence tests were performed using and Chi-Square statistics, with a level of significance a = 0.05. Statistical analyses were performed using InfoStat Software versión 2020. The ages of the subjects ¡n the sample ranged from a minimum of 20 to a máximum of 33 years, with an average of 22.32 years, with a standard deviation of 1.94 years and a coefficient of variability of 8.67%, indicating low variability. It has been found that there are no differences between the ages of the subjects assigned to the 3 groups, confirming the ¡nternal validity of the sample in relation to the homogeneity of the subjects assigned to each experimental group. Pearson's Correlation coefficients between salivary a-amylase contents and the age of the subjects were at very low valúes and were not significant. This has allowed to establish that the levels of basal salivary a-amylase, at the first and second hour after fluid intake are not associated with the age of the subjects. The associations between the contents of salivary a-amylase at different measurement moments showed cióse associations and were statistically significant. These associations were all direct or positive, which indicates that as the basal salivary amylase contení increases, so do the salivary amylase contents at 60 and 120 minutes. The strength of the association is greater between the contení of (basal salivary a-amylase and at 60 minutes, followed by the contení of salivary a-amylase at 60 minutes with that of 120 minutes and, finally, the least cióse association occurs between the contení of basal salivary a-amylase and at 120 minutes, however it continúes to be statistically significant. By analyzing all the variables studied together, it has been possible to represent the subjects and the variables in two dimensions, preserving 90.8% of the information. The subjects studied are located separating on the horizontal axis, mainly by the contents of salivary a-amylase of first hour and second hour, placing cióse, with similar profiles, to the subjects who ingested infusión of stevia and running water, while those who ingested artificial orange juice are mostly concentrated at the other end of the graph. Basal salivary a-amylase contents ranged from a mínimum of 8,414 U/L to a máximum of 50,896 U/L, with an average of 39,034.55 U/L. The observations showed high variability, with a range of 42,482 U/L, a standard deviation of 11,767.74 U/L and a coefficient of variability of 30.15%. The contents of salivary a-amylase first hour ranged from a mínimum of 8,337 U/L to a máximum of 53,340 U/L, with an average of 38,303.05 U/L. The variability of the observations is high, with a range of 45,003 U/L, a standard deviation of 13,010.05 U/L and a coefficient of variability of 33.97%. Second hour salivary a- amylase levels ranged from a low of 7,341 U/L to a high of 50,653 U/L, with an average of 32,630.07 U/L. The variability of the observations is high, with a range of 43,312 U/L, a standard deviation of 11,832.88 U/L and a coefficient of variability of 36.26%. All these valúes are below the ranges described as normal by other authors. The analysis over time of salivary a-amylase levels indicates that the contents decrease over time and more sharply between the first and second hour after fluid intake. It has been possible to establish Linear Regression Models between salivary amylase content and the minutes elapsed after fluid intake. When the 60 subjects of the sample were considered, a linear model of the shape was fitted: y = 43.060,37 - 3.202,24 x Both coefficients were statistically significant, indicating that at time zero the content of salivary amylase has a non-zero valué, and that the content of salivary amylase decreases over time. When the functions linking salivary amylase contents and minutes elapsed since fluid intake by experimental group were studied, the following linear models could be adjusted: ....