Sustainable use of coffee silverskin as a natural source of bioactive compounds for diabetes

Resumen

Introduction Coffee is the most consumed drink in the world (1). Therefore, large amounts of by-products are generated during the coffee industrial processing (2–4). Coffee silverskin (CS) is a thin tegument of the outer layer of the two beans forming the green coffee seed that is obtained as a by-product of the roasting process (5). The extraction of bioactive compounds from natural products is increasingly being used to prepare dietary supplements/nutraceuticals, food ingredients and some pharmaceutical products. Our research group patented an aqueous extract of coffee silverskin (CSE) (P201131128) that is rich in different bioactive compounds, especially chlorogenic acid (CGA) and caffeine. CGA formed by esterification of caffeic and quinic acids is one of the most abundant polyphenol in CSE (6,7). CGA has shown antioxidant (8), anti-inflammatory (9–11) and antiglycative (12,13) properties in vitro and in vivo. CSE is also a good source of caffeine (6,14) and it may improve the antioxidant status in humans (15,16). Antioxidants in general and phytochemicals in particular play an outstanding role in lowering chronic disease risk like type 2 diabetes (T2D) (17,18). T2D is very complex and multifactorial metabolic disease characterized by insulin resistance and beta cell failure leading to elevated blood glucose level. Hyperglycemia was estimated to be one major factor contributing to diabetic complications including accelerated non-enzymatic glycation (formation of advanced glycation end products (AGEs) (19,20), an increase in oxidative stress due to the imbalance between the generation of reactive oxygen species (ROS) and the organism's antioxidant potential, and the increase of reactive carbonyl compounds caused by their enhanced formation and/or decreased degradation or excretion (21). Coffee consumption has been associated to a wide variety of health beneficial effects, in particular the reduced risk of T2D (22). In our knowledge, the mechanism of action of CSE bioactive compounds in T2D complications is still unknown. To achieve this goal, the antidiabetic effect of CSE bioactive compounds was evaluated in vitro and in vivo. Rationale of the study The aim of this thesis is to obtain novel scientific evidences to demonstrate the effects of CSE in T2D. Since the health and wellness of diabetics is principally affected by complications associated to formation and accumulation of AGEs in the organisms, the first approach was to evaluate the antglycoxidative properties of the extract. These studies were performed in vitro and applying phytochemomics technologies. Studies of bioaccesibility and metabolism of caffeine and CGA present in CSE has been also performed. In vivo bioactivity is highly influenced by the bioaccesibility and metabolism of the food components. In vitro studies are not enough to demonstrate the feasibility of bioactive extracts for the reduction of the risk or treatment of chronic diseases. The effect of CSE on biomarkers of diabetes in cell culture and in vivo was also tested. Protein glycoxidation model systems were prepared containing bovine serum albumin (BSA) in the presence or absence of CGA or CSE for testing their effects on the glycoxidation reaction and potential in the prevention of complications of diabetes such as nephropathy, retinopathy and neuropathy. The glycoxidation reaction was started by the addition of methylglyoxal (MGO) and the mixtures were incubated at 37°C at different times. MGO was selected because its physiological relevance in the formation of in vivo AGEs and diabetic complications. To elucidate the mechanism of action of CSE in the pathogenesis of T2D, a beta beta cell line (INS-1E) was used. INS-1E cells were treated with different doses of CSE (1-10 μg/mL), CGA (1-10 µM) or caffeine (1-10 µM). In a second set of experiments beta cells were treated with the same concentrations of CSE, CGA or caffeine and streptozotocin (STZ; 5 mM) was added as a diabetogenic agent. The bioaccesibility of caffeine and CGA in CSE was determined by analysis of their content before and after in vitro gastrointestinal digestion. The processes were performed mimicking human digestion physiological conditions. Additionally, novel information regarding to the major contributors to the health benefits of CSE was obtained. The extract was fractionated and the chemical composition and bioactivity of the fractions containing high and low molecular weight coffee components were analysed in vitro. On the other hand, in order to evaluate CSE bioavailability, Wistar rats (n = 16) were housed singly in metabolic cages with free access to food and water, and 24 h urine samples were collected from untreated rats as a control (n = 4). Then, the animals were divided into three groups: CSE group (n = 4) receiving one single dose of CSE (adjusted to provide 2.2 mg caffeine/kg body weight), CGA group (n = 4) receiving pure CGA (providing 1.5 mg CGA/kg body weight) and caffeine group (n = 4) receiving pure caffeine (providing 5 mg/kg body weight). After the administration of each treatment, urine samples were serially collected at different times during 24 h. The bioavailability experiments were repeated with the same animals after 3 days of clearance. The evaluation of CSE bioactivity in STZ-nicotinamide (NA) diabetic rats was conducted using Wistar rats (n = 32) divided into four groups (n = 8). Daily, the animals were supplemented by gavage with CSE (providing 2.2 mg caffeine/kg body weight, 0.8 mg CGA/kg body weight), pure CGA (providing 1.5 mg CGA/kg body weight) or pure caffeine (providing 5 mg caffeine/kg body weight) during 42 days. The fourth group (STZ group) was treated similarly with sterile water. At day 35, all rats were induced T2D by the intraperitoneal injection of STZ (60 mg/kg body weight) and NA (200 mg/kg body weight), and blood glucose levels were monitored daily in the following days. Rats were considered diabetic when blood glucose levels were above 200 mg/dL. At that moment (day 42), fasting rats were anaesthetised with Ketamine-Xylazine and sacrificed. Plasma samples and pancreas were obtained and frozen at -80 ºC until further analysis. Methodology To evaluate the in vitro inhibition of AGEs formation by CSE bioactive compounds using the glycoxidation model systems mimicking physiological conditions, we analysed free amino groups, fluorescence AGEs formation and total AGEs formation. Data on protein structure were obtained by mass spectrometry, Folin reaction and UV-Vis spectral analysis. In addition, to determine changes in protein functionality, the antioxidant capacity of the protein fraction was evaluated by ABTS assay. Pancreatic cell oxidative status was determined by measuring ROS, reduced glutathione (GSH) and glutathione peroxidase (GPx) and glutathione reductase (GR) activities in cell lysates after treatments with CSE, CGA and caffeine. Besides, glucose-induced insulin content and insulin secretion was quantified by using enzyme-linked immunosorbent assay (ELISA) kit. In the second experiment, the protective effect of treatments against STZ-induced damage was evaluated by analysing markers of oxidative stress (ROS, GSH, GPx and GR) and cell death (crystal violet assay) in INS-1E beta cells. The bioaccesibility of components of CSE was estimated by analysis of total phenolic compounds, CGA and caffeine before and after the in vitro digestion. The overall antioxidant capacity (ABTS and ORAC) was also determined. The bioavailability of CGA and caffeine in CSE was determined by measuring the excretion of their principal metabolites after providing a single dose of CSE, CGA or caffeine. Hippuric acid and paraxanthine were determined in 24 h urines of rats by UPLC-MS/MS analysis as the main CGA and caffeine metabolites, respectively. Novel information regarding to the effects of CSE, caffeine and CGA on biomarkers of T2D were obtained in INS-1E beta cells and in vivo using as experimental model STZ-NA-induced T2D rats. In vivo protective effect of CSE, CGA and caffeine was evaluated using an experimental model of STZ-NA T2D rats. Thirty-two rats were daily pre-treated with the CSE, CGA or caffeine during 34 days. At day 35, diabetes was induced by intraperitoneal injection of STZ-NA and blood samples were collected in the fasting state at day 42. After blood centrifugation, plasma was separated and frozen at -80 ºC. The pancreas were removed promptly, weighted, divided into three parts and then stored at −80 °C until required. Plasma glucose was measured using a colorimetric kit. Plasma and pancreas insulin content was analysed using an ELISA kit. Fructosamine, as a biomarker of plasma protein glycation, was evaluated by nitroblue tetrazolium (NBT) colorimetric assay. Carbonyls content, as a biomarker of protein glycoxidation, was measured in plasma and pancreas homogenates using a colorimetric assay. Biomarkers of antioxidant defence, GSH and GPx and GR activities were analysed in the pancreas of diabetic rats. Results Novel findings were obtained during the development of this investigation. For the first time, it has been associated the inhibitory capacity of AGEs formation of CGA free and in CSE to its ability to form protein-phenol complexes. Reactive adducts of arginine and lysine were found in glycoxidative reaction with BSA and methylglyoxal by ESI Q-TOF-MS/MS analysis. The addition of CGA or CSE to the glycoxidation system decreased the presence of arginine adducts. The novel structure formed by interaction of proteins and phenol caused a significant increase (p < 0.05) of antioxidant character of the parental protein. The results are of great interest because this change in protein function may provide protection against oxidation reactions and diseases associated to this of damage. The experiments in INS-1E beta cell showed that CSE, pure CGA and caffeine did not affect pancreatic beta cells viability and oxidative status, when assayed under physiological conditions. All concentrations of CSE and CGA ≥ 5 µM significantly increased (p < 0.05) the enzymatic activity of GPx. CSE (1-10 µg/mL) and the dose of 10 µM of CGA, significantly increased (p < 0.05) beta cell insulin secretion in the presence of 4 and 10 mM of glucose. On the other hand, CSE (1 µg/mL) and CGA (10 µM) reinforced the antioxidant defence and increased insulin secretion in response to glucose in beta cells stressed with STZ. Bioaccesibility of CSE compounds was affected during in vitro gastrointestinal digestion decreasing concentrations of caffeine (25%), phenolic content (40%) and CGA (82%). The overall antioxidant capacity of CSE was reduce at 15% and 50% as measured by ABTS and ORAC, respectively. Although a significant reduction of the bioaccesibility of CGA and caffeine occurred during digestion, results suggest that physiological active concentrations of both compounds remain available to act in the body. The study of the bioavailability of CSE bioactive components in the organism shows that 24 h after the intake of a single dose, intact CGA was not found in urine of rats fed with CSE (containing 0.254 mg of CGA/day) or CGA (0.321 mg of CGA/day), while a significant excretion (p < 0.05) of hippuric acid was observed only after the ingestion of CGA alone. In addition, non-metabolized caffeine and paraxanthine was higher in urine after consumption of pure caffeine than after the treatment with CSE. Altogether results on bioccesibility and excretion indicated that caffeine and CGA in CSE were metabolised. The supplementation of rats with pure CGA and caffeine tended to reduce (p < 0.1) STZ-NA-induced oxidation of pancreas proteins. Pre-treatment of animals with CSE and CGA significantly reduced (p < 0.05) STZ-induced pancreas GSH depletion. Results confirm the bioavailability of caffeine and CGA in CSE and support their biological implications in diabetes. Conclusion In conclusion, the findings derived from the present investigations demonstrate that one of the mechanisms by which CSE bioactive compounds inhibit AGEs formation is the generation of novel structures with antioxidant properties. The antiglycative effect of CSE may provide protection against complications in diabetics. Further investigations should be carried out to demonstrate the hypothesis. CSE protects pancreatic beta cells from oxidative stress and modulates insulin secretion. Caffeine and CGA in CSE are bioavailable and exert antidiabetic effects following different mechanism of actions. The effects observed on diabetes biomarkers can be associated to the synergic effect of CGA, caffeine, their metabolites and others coffee components. Although further studies should be conducted to identify all the CSE components able to affect the biomarkers of diabetes and its effects in humans, the present study suggests that an effect of CSE consumption in diabetes is biologically plausible, and that effect should be ascribed to the particular chemical and complex composition in bioactive compounds of the extract. The valorisation of CS into a sustainable product for diabetes is feasible.