FACULTAD DE CIENCIAS DE LA SALUD CARRERA PROFESIONAL DE NUTRICIÓN Y DIETÉTICA “ANÁLISIS DEL CONTENIDO DE CAFEÍNA, TEOBROMINA Y TEOFILINA POR HPLC-DAD EN BEBIDAS COMÚNMENTE COMERCIALIZADAS EN LIMA” Tesis para optar el título profesional de: Licenciado en Nutrición y Dietética Presentado por: Karolinhe Alicia Gonzales Yépez (0000-0001-6270-8053) Asesor: Oscar Reátegui Arévalo (0000-0001-9773-2176) Lima - Perú 2021 . . 1 Agradecimientos Le agradezco ante todo a Dios por haberme guiado y acompañado durante este proceso a lo largo de mi carrera, por ser mi camino y sostenerme para que siga adelante, gracias por cada día que me da lleno de alegría y salud. Agradezco a mis padres Edwin y Yolanda por siempre estar junto a mí y apoyarme, por haberme dado la vida, ser mi soporte en todo momento, brindarme su amor y ser mi ejemplo a seguir. A mis hermanos, Leonardo y Dominic por ser mis mejores amigos y llenarme de alegría cada día. A mis abuelos, por darme su guía, su apoyo, su fe, amor incondicional y protección. Y a toda mi familia, que me dio su confianza y apoyo cuando más lo necesitaba. Le agradezco la confianza, paciencia y dedicación de su tiempo al profesor Óscar Reátegui, por haber compartido conmigo sus conocimientos, brindarme la oportunidad de crecer profesionalmente, y brindarme su amistad. Gracias a mi universidad por haberme dado la bienvenida y las incomparables oportunidades para seguir con mis estudios y formarme para ser una buena profesional. 2 Resumen El propósito de este estudio fue determinar el contenido de cafeína, teobromina y teofilina por HPLC-DAD en las bebidas que se consumen comúnmente en Lima. Las muestras se dividieron en 6 grupos (bebidas a base de té, café en polvo, leches chocolatadas, gaseosas, bebidas rehidratantes y bebidas energéticas); cada uno con las cinco bebidas más consumidas. Analizadas por un método de cromografía líquida de alto rendimiento, rápido y selectivo (HPLC- DAD). Se pudo observar que entre los grupos gaseosas y bebidas energizantes se identificó principalmente la cafeína, siendo las bebidas energéticas el segundo grupo de mayor nivel de cafeína (10.38± 0.01 a 95.50±3.48 mg/L). En las bebidas a base de té, el contenido más alto identificado fue el de cafeína (0.47±0.01 a 4.91±0,05mg/L), a pesar de que la teofilina es un compuesto característico de la hoja de té. En las bebidas rehidratantes se obtuvo un nivel muy bajo de cafeína (0.03±0.01 a 0.05±0.01mg/L) y de teobromina (0.48±0.01 a 6.00±0.02mg/L). En las cinco marcas comerciales de leche con chocolate evaluadas se encontró cafeína (4.09±0.01 a 5.70±0.01mg/L) y teobromina (1.70±0.01 a 12.24±0.01mg/L), siendo esta última la de mayor concentración. Por último, en el grupo de bebidas de café en polvo se encontró el nivel más alto de contenido de cafeína (492.5±0.24 a 9644.0±4.93mg/L). Los resultados obtenidos en este estudio proporcionan información fiable sobre la composición y cuantificación de metilxantinas en las bebidas más consumidas en Lima. También, un impacto en el conocimiento de los consumidores. Palabras claves Cafeína, teobromina, teofilina, bebidas, metilxantinas. 3 Abstract The purpose of this study was to determine the content of caffeine, theobromine and theophylline by HPLC-DAD in beverages commonly consumed in Lima. The samples were divided into 6 groups (herbal teas, coffee powder, chocolate milks, soft drinks, sports drinks and energy drinks); each with the five most commonly consumed beverages. Analyzed by a high performance, fast and selective liquid chromatography method (HPLC-DAD). It could observed that among the groups of soft drinks and energy drinks, caffeine was mainly identified, with energy drinks having a more significant caffeine value (10.38± 0.01 to 95.50±3.48 mg/L). In herbal teas, the highest content identified was caffeine (0.47±0.01 to 4.91±0.05mg/L), despite the fact that theophylline is a characteristic compound of the tea leaf. With sports drinks, a very low level of caffeine (0.03±0.01 to 0.05±0.01mg/L) and theobromine (0.48±0.01 to 6.00±0.02) was obtained. In the five commercial brands of chocolate milk evaluated, caffeine (4.09±0.01 to 5.70±0.01mg/L) and theobromine (1.70±0.01 to 12.24±0.01mg/L) were found, being the last one with the highest concentration. And in the group of coffee powder samples had the highest level of caffeine content (492.5±0.24 to 9644.0±4.93mg/L). The results obtained in this study provide reliable information on the composition and quantification of methylxanthines in the most consumed beverages in Lima. Also, an impact on the knowledge of the consumers. Keywords Caffeine, Theobromine, Theophylline, Beverages, Methylxanthines. 4 Determination of caffeine, theobromine, and theophylline by HPLC-DAD in beverages commonly consumed in Lima-Peru Karolinhe A. Gonzales1 1School of Nutrition and Dietetics, Universidad Científica del Sur, Lima, Perú 1. Introduction Over the last decades, some research has focused on the study of biologically active ingredients, particularly alkaloids in beverages, for their possible beneficial effects on human health (1). One of those alkaloids are methylxanthines, compounds commonly present in food (2). Montero et al. (3) noted that both animals and plants naturally produce these compounds, being caffeine (CF), theobromine (TB) and theophylline (TF) the most studied ones. These compounds are mainly found in food such as coffee beans, cocoa beans and tea leaves (4). These three methylxanthines are chemically very similar (5). According to De Sena et al. (6), they share stimulating effects on the central nervous system, as well as on other systems such as the gastrointestinal, cardiovascular, renal and respiratory systems. Caffeine, for example, one of the best known and most studied substances, has a stimulating effect on the central nervous system, increasing alertness, improving long-term memory and concentration, as well as may improve physical performance in athletes (7, 8 and 9). There is even a debate about the neuro- protective effects against certain types of degenerative diseases such as Alzheimer's and Parkinson's (10). According to data reviewed by Canadian scientists (11), they concluded that for the healthy adult population, a moderate daily caffeine intake of up to 400 mg per day is not associated with adverse health 5 effects. Likewise, it was indicated by the FDA (Food and Drug Administration) as a safe dose of up to 400mg of caffeine per day in the healthy adult population, varying according to the sensitivity of the person or vulnerable group (pregnant or breastfeeding or those with any special health condition) (12). However, excessive consumption may be related with hypertension, anxiety, hyperactivity and headaches (7). Theobromine, which is present in high concentrations in cocoa, has not been studied as much (13). According to Martinez-Pinilla, the main mechanisms of action are inhibition of phosphodiesterases and blockade of adenosine receptors (13). Another clinical study suggests that theobromine could be the main active component of cocoa responsible for the effect of increasing HDL cholesterol (14). As for their safe or lethal daily dose, there is no specific and/or conclusive information. And theophylline, which has similar pharmacological and toxicological properties as caffeine (2). According to Dolder (15), can be found mainly in tea, and in asthma medication used as a bronchodilator. Although there is no precise information on a recommended dose, Corey et al.(16) point out that inadequate consumption may cause toxic effects, and may cause tachycardia, hypertension, nausea, vomiting, diarrhea. Last years, it can observe that there is a greater scope and access to a huge variety of products (17), such as the group of beverages (ready-to-drink, coffee powders, soft drinks, energy drinks, sport drinks, herbal teas). Products that are commonly marketed in our locality, most of which in their nutritional labeling do not indicate with certainty their concentration in certain compounds that they contain. So, this information is often unknown to the consumer public. 6 Therefore, the aim of this study was to determine the content of caffeine, theobromine and theophylline by HPLC-DAD and antioxidant capacity in beverages commonly consumed in Lima. 2. Material and methods 2.1. Chemicals All solvents and reagents were analytical grade with deionized water (Mili-Q quality) (4). The standards of caffeine, theobromine and theophylline were purchased from Sigma-Aldrech (Merck Peruana, Lima). Acetonitrile and methanol (HPLC Grade) were also obtained from Sigma-Aldrech (Merck Peruana, Lima). 2.2. Sample collection The samples were divided into six groups: herbal teas (HT), sports drinks (SD), chocolate milk (CM), soft drinks (SOD), energy drinks (ED) and coffee powder (CP). Each one with the five most consumed beverages, obtained by non- probabilistic convenience sampling, based on a survey of consumers in Lima (Anexo 5). Samples were obtained through their purchase in three supermarkets in Lima Metropolitana. The nutritional labels of each sample analyzed were reviewed to corroborate their declared composition. In the case of herbal teas, they are ready-to-drink cold drinks that are marketed as herbal infusions with an added value (e.g. fruit flavors or various properties), which can be highly appreciated by a certain sector of the national and international market (18). Sports drinks provide carbohydrates, electrolytes and liquids to the body, helping the body hydrate before, during and after physical activity (19). 7 Chocolate milks is a mixture based on milk, sucrose, cocoa and/or cocoa powder and some hydrocolloids, which are added to improve the consistency and avoid the sedimentation of the cocoa particles (20). On the other hand, soft drinks are flavored drinks, which are produced by adding CO2 gas directly to the drink, diluted with sucrose, and depending on the drink may or may not contain caffeine (21). Energy drinks are used to provide an extra burst of energy, as well as to promote wakefulness, increase attention span, maintain alertness, and improve athletic performance (22). In the case of coffee powder, is a drink prepared with coffee beans in powder form (23), having caffeine as its main compound. All coffee powder samples were prepared. For this research, there were no samples sweetened with non-caloric sweeteners. 2.3. Total polyphenol measurement The content of total phenolics was determined according to Singleton and Rossi (24). Briefly, 1 mL of 10% Folin-Ciocalteu reagent was mixed with 0.1 mL of sample for 5 min at room temperature, then 1 mL of 5% sodium carbonate was added and the mixture was placed in a water bath at 45°C for 15 min. All samples were analyzed in triplicate. Absorbance was read on a spectrophotometer at 725 nm. The results were expressed in mg galic acid equivalent/100mL (mg GAE/100mL). 8 2.4. Total flavonoid measurement Total flavonoid measurement was performed according to Wolfe et al. (25). To 0.250 mL of sample was added 0.075 mL of 5% sodium nitrite and allowed to react for 5 min, then 0.150mL of aluminum chloride was added, and the mixture could stand for 5min. Finally, 0.275 mL sodium hydroxide (1 M) was added to the mixture and it was left to react for 15 min. All samples were analyzed in triplicate. The reading was performed at 510 nm in a spectrophotometer. The results were expressed in µg catechin equivalent/100mL (µg CE/100mL). 2.5. Antioxidant capacity by ferric reducing antioxidant power (FRAP) Antioxidant activity evaluated by the FRAP assay was performed according to the methodology proposed by Benzie and Strain (26). Briefly, 1 mL of distilled water and 1 mL of the FRAP reagent were added to 0.1mL of sample, the mixture was placed in a 37 °C water bath and allowed to react for 10 min. The reading was made in a spectrophotometer (Pharo 300, Spectroquant, USA) at 593 nm. A standard curve was prepared using different concentrations of Fe2+ ranged from 15 to 75 mM. All samples were analyzed in triplicate. The results were expressed in µM Fe2+/100mL (µM Fe2+/100mL). 2.6. Chromatographic conditions HPLC analyses of caffeine, theobromine and theophylline were performed according to Srdjenovic et al. (4), with slight modifications. Briefly, a VWR HITACHI Chromaster 600 HPLC with a diode array detector (DAD CM 5430), autosampler and a reversed phase purospher STAR RP-8 column (5 µm particle size, i.d. 4.6 x 150 mm) was performed in a isocratic elution mode with the mobile phase water-THF(A) (0.1% THF in water, pH 8)-acetonitrile (B) (90:10, v/v). The pH was adjusted with 0.1 M NaOH. The mobile phase and all the solutions were 9 filtered (0.45-µm x 47mm Millipore nylon filter), and the run time was 5 min, with a flow rate of 0.8 mL/min. The column temperature of 25°C and the analytes were detected at 273nm. All samples were run in triplicate for each analysis. 2.7. Sample preparation The herbal teas and sports drinks were filtered through a 0.22 µm nylon filter. Ten milliliters of the filtrate, were adjusted to pH 8 with 0.1 M NaOH (4). The chocolate milks (25mL) containing suspended particles were filled up to 200mL with water in a separate container and extracted for 30 minutes at 60 ° C in the ultrasonic bath. The extracted sample was filtered through filter paper to remove solids. Then, 10 mL of the filtrate was adjusted to pH 8 with a 0.1M NaOH solution (4). The soft drinks and energy drinks samples were degassed for 15 minutes to release CO2. Before analysis, the samples were adjusted to pH 8 with a 0.1M NaOH solution and filtered through a 0.22-µm x 33mm Millipore Millex-GN nylon filter. The coffee powder samples were weighed (5g) and extracted with boiling water (200 mL), mixed in a thermal flask for 5 min on the magnetic stirrer (4). The extracts were then filtered and 10 mL of the filtrate was adjusted to pH 8 with 0.1M NaOH. 2.8. Cleanup procedure According to Srdjenovic et al. (4), the Supelclean LC-18 SPE cartridges were conditioned with 2 x 6 mL of methanol, followed by 2 x 6 mL of ultra-pure deionized water. The extracts from each sample were then passed through the SPE cartridges, followed by washing with 6 mL of ultra-pure deionized water. They were air dried under vacuum for 10 min and the fluids were discarded. Last, 10 CF, TB and TF were extracted from the SPE cartridges with 10 mL of chloroform. The solution was evaporated until dryness. The residue of all samples was reconstituted in 1 mL of pH 8 water, with the exception of chocolate milk, which was reconstituted in 2 mL. Samples were filtered through a 0.22 µm nylon filter and injected into the HPLC (4). 2.9. Statistical analysis The results of each analysis were presented in tables and the statistical analyses of each test were presented as “mean ± standard deviation” using SPSS version 22 for Windows (SPSS, Inc., Chicago, IL, USA). 3. Results and Discussion 3.1. Bioactive compounds and Antioxidant capacity Total polyphenols content, total flavonoids content and FRAP of the six groups of beverages are presented in Table I. As can be seen, the total polyphenols content in beverages varies according to the group. Decreases, according to the results found, in the following order CP ˃ ED ˃ HT ˃ CM ˃ SOD ˃ SD (Table II). This is because coffee contains numerous phenolic compounds, 3.57 ± 0.17 to 19.97 ± 0.17 mg GAE/100mL, compared to the other samples studied. In fact chlorogenic acid is attributed more potential benefits of coffee, which has been shown to contribute to the prevention of Alzheimer's disease-induced cognitive dysfunction (27). However, can be noted that in the group of herbal teas, a notable difference in polyphenol content was found between the infusions based on black tea (HT-3, 4, and 5): 0.06 ± 0.01 to 0.40 ± 0.01 mg GAE/100mL, and green tea (HT-1 and 11 HT-2): 4.08 ± 0.27 to 4.74 ± 0.18 mg GAE/100mL. Consistently with Horžić et al. (1), the green tea is the richest source of total phenols. In the results of total flavonoids content, it was found that the coffee powder (CP) and chocolate milks (CM) presented the highest levels of concentration in these results (Table II): 57445.07 ± 25969.47 µg CE(+)/100mL and 20785.92 ± 7315.11 µg CE(+)/100mL respectively, compared to the other groups. Besides that, according to the results, the group with the lowest flavonoid content was the herbal teas. There are no similar studies comparing these groups of beverages, as to establish a factor explaining why teas have lower flavonoid content. On the other hand, within this group of tea beverages, it is conceivable that the catechin content may vary according to tea variety, origin, time of harvest, and sun exposure (1), which would be factors to be considered in future research. Further, for FRAP, it can be observed that coffee powder (CP) have a high antioxidant capacity, 29.51 ± 0.29 to 228.88 ± 1.60 µmoles Fe(2+)/100mL, well above the other samples, such as soft drinks (SOD), which show a very low or almost no antioxidant capacity. As can be seen, based on these results, the group of powdered coffee drinks predominates in the majority of the tests carried out, being the group of drinks that has the greatest antioxidant capacity, due to its bioactive compounds such as chlorogenic acid. The other groups vary, in relation to the ingredients of each sample. 12 Table I. Total polyphenols, flavonoids and FRAP results of most consumed beverages from Lima* FOUND VALUES BEVERAGE Total Total flavonoids (µg FRAP (µmoles SAMPLES polyphenols CE(+)/100mL) Fe2+/100mL) (mgGAE/100mL) SOD-1 1.12 ± 0.04 2718.31 ± 70.42 3.59 ± 0.10 SOD-2 0.19 ± 0.01 915.49 ± 70.42