Biological activity report of Synergicfood by Andes Wisdom Chile (November 2nd, 2018) Introduction Diabetes and obesity are two of the most widespread diseases among the population of developed countries. It is estimated that over 13% of the adult population is obese (WHO 2015) and it continues to increase. The same tendency can be seen in incidence of diabetes. Currently there are about 350 million diabetic people around the world of which more than 90% (about 135 million people) suffer from diabetes type II. This type of diabetes is directly related to diet, body weight, and physical inactivity (WHO 2015) The prevalence of both diseases worldwide creates the need to find medicine and functional foods for their treatment or prevention. Being of special interest to those capable of inhibiting the enzymes responsible for the control of lipids in obesity (lipase enzyme) and carbohydrates (α-glucosidase enzyme) as well as the formation of glycated proteins in the case of diabetes. Today it is believed that the protein glycation plays a key role in the pathogenesis of chronic diseases related to aging and particularly to diabetes. Glucose can react with other molecules such as proteins and lipids without the intervention of enzymes and form toxic substances, called end products of advanced glycation (AGEs) through a process called glycation. The formation of endogenous AGEs depends mostly on blood glucose levels which explain the increasing production of glycosylated proteins and the accumulation of AGEs in patients with diabetes. The main complications associated to endogenous AGEs are nephropathy, retinopathy, neuropathy and cardiovascular diseases. Also, some authors suggest that industrial processes based on heat treatment application with low water content during food processing contributes exogenously to the production, exposure and accumulation of AGEs in the organism and the development of problems associated with chronic diseases. Consequently, products with anti-glycemic capacity appear to be a good alternative for delaying ageing and preventing age-associated diseases such as diabetes. Three possible therapeutic strategies have been described to regulate these processes. 1. Glycemic Index Control Therapy. 2. Antioxidant therapy. 3. Antiglycan therapy. Recent studies indicate that raw extracts from agro-industrial products are rich in phenols and polyphenols that have inhibitory activities of advanced glycosylation products (AGEs), which could be used in the production of functional foods to prevent complications of diabetes and other pathologies associated with advanced protein glycation (Glycation) such as aging, cancer, inflammation, neurodegenerative diseases, among others. The tendency on the current demand of consumers is to transform food into an integral system delivering nutrition, health and wellbeing. Functional foods and supplements were positioned as an alternative to maintain health at a time when health services are increasingly expensive for the consumer and chronic diseases are a global public health problem. Therefore, these foods have become a requirement for the food market. Index of contents INTRODUCTION 2 OBJECTIVES 5 SAMPLES 5 METHODS 5 QUANTIFICATION OF TOTAL PHENOLIC COMPOUNDS 5 IN VITRO EVALUATION OF THE CAPACITY INHIBITION OF DIGESTIVE ENZYMES 6 INHIBITORY ACTIVITY PANCREATIC LIPASE 6 ACTIVITY INHIBITORY OF ENZYME Α-GLUCOSIDASE 6 IN VITRO EVALUATION THE INHIBITION CAPACITY OF NON-ENZYMATIC PROTEIN GLYCATION. 7 INHIBITION CAPACITY OF PROTEIN GLYCATION 7 RESULTS AND DISCUSSION 8 ANTIOXIDANT CAPACITY 8 ACTIVITY INHIBITORY OF SAMPLES ON DIGESTIVE ENZYMES Α-GLUCOSIDASE AND PANCREATIC LIPASE 8 Inhibition of pancreatic lipase 9 Inhibition of the enzyme -glucosidase 11 ANTIGLING ACTIVITY 12 CONCLUSION 13 BIBLIOGRAPHY 14 Objectives To evaluate the functional properties of Synergicfood, the functional food, for its future use in reducing the risk of obesity and diabetes type II. Samples 1. Food: Synergicfood Methods Quantification of total phenolic compounds The total content of phenols has been determinate by the Folin-Ciocalteu method, for microplates that use gallic acid as reference material. The principle of the method is based on the measurement of the reducing capacity of the analyzed sample, i.e. the amount of sample required to produce the reduction of the Folin-Ciocalteu reagent is determined. This reagent usually appears in yellow, but when reduced it loses its colour and turns blue. The phenolic compounds inside a basic medium can reduce the Folin- Ciocalteu reagent, which produces its colour change. The concentration of these phenolic compounds can be determined based on the Lambert-Beer law through a spectrophotometric analysis at 735 nm after a reaction time of 2 hours. For this, 2 mg of the samples were used, put in a flask with 50 mL of distilled water and then shaken. 0.5 mL of each of these solutions were taken and mixed with 0.75 mL of Folin-Ciocalteu reagent, leaving them at room temperature for 3 min. Subsequently, 0.75 mL of 20% sodium carbonate was added. They were shaken vigorously and continue to stand for 90 min at room temperature. After this time, 50ul of the mixture were taken and transferred to a well of the microplate to measure the absorbance at 765 nm with the BioTek equipment. Obtained readings were interpolated in a calibration curve of the gallic acid standard (R2 = 0.99). The results were expressed in milligrams of equivalent gallic acid per gram of product (mg AGE / g). The calculation was made using the following equation: Total Phenols = [(AS – AB) - b]/m Where, AS stands for the absorbance of the sample with the Folin-Ciocalteu reagent; AB for the absorbance without the reagent; b & m are the intercept and slope of the calibration curve, respectively. In vitro evaluation of the capacity to inhibit digestive enzymes Inhibitory activity pancreatic lipase The determination of the inhibition from lipase enzyme, which divides triglycerides into fatty acids and monoacylglycerol, is based on a series of consecutive reactions that triggers the action of the lipase enzyme, forming a quinone, coloured compound at the end of the process and stoichiometrically whose concentration can be determined by spectrophotometry. The reaction was carried out in 96-well polypropylene microtiter plates. A volume of 25 μL of the sample was incubated with 150 μL of the substrate, composed of a mixture of 0.2 mM p-nitrophenyl-palmitate (p-NPP), 5 mM deoxypuracolic acid dissolved in 50 mM monobasic phosphate buffer (pH 8,0). After pre-incubation, 25 μL of the enzyme pancreatic lipase previously dissolved in reaction buffer at the concentration of 10 mg / ml was added. After an incubation at 37 ° C for 5 min, the absorption of each reaction mixture is shown at the wavelength of 420 nm in a Synergy ™ HT plate reader from BioTek®. As a positive control of the assay, tetrahydrolipistatin (Orlistat MK¾) was used. The results were expressed as a percentage of inhibition of pancreatic lipase, using the equation: % I = (1-AS/AC) x 100. Where, AS stands for the absorbance of the Sample and AC for Control Absorbance. The control will represent 100% of the activity of the enzyme and corresponds to the reaction mixture without the sample (AC), which is replaced by the diluent. Inhibitory activity of the α-glucosidase enzyme Inhibitors of alpha-glucosidase (such as acarbose) decrease the absorption of carbohydrates from the digestive tract, thus reducing blood glucose levels after meals. These medications help the body reduce the blood glucose level in patients with and without diabetes. To evaluate the inhibitory effect of the food Synergicfood, the activity of the enzyme alpha-glucosidase was evaluated indirectly by means of the p-nitrophenyl released (its absorption is at 450nm). Therefore, a unit of α-glucosidase released by 1.0 μmol of D-glucose and 1.0 μmol of p-nitrophenol, in response to p- nitrophenyl α-D-glucoside which was at a pH of 6.8 and a temperature of 38. ° C. Finally obtaining as results that, the greater the inhibition of the enzyme, the lower the amount of p-nitropheny. Acarbose was used as a positive control of the reaction. The calculation of the percentage of inhibition was found by the formula that appears below described in the section of inhibition of pancreatic amylase. In vitro evaluation of the inhibition capacity from non-enzymatic protein glycation. Capacity to inhibit protein glycation. The glycation model system was composed of bovine serum albumin (BSA) at a final assay concentration of 1mg / ml in 0.01M PBS buffer (pH 7.4) with the addition of 80.05% sodium azide) and methylglyoxal (MGO) (5mM). The model was prepared in the absence and presence of the extracts. The test concentrations evaluated were: Pure Synergicfood and ½ diluted. The reactions were incubated at 37 ° C for 72 hours under agitation. All reactions were carried out through triplicate, a BSA reaction was included only as a negative control and aminoguanidine (AMG) (10 mM equivalent to 0.6 mg / ml) as the inhibition standard. Inhibition of glycation reaction was determined by fluorescence analysis with 96-well fluorescence plates using the Biotek® system through an excitation of 370 nm an emission of 460 nm and a gain of 68 nm. AGEs formation inhibition was calculated as: inhibition % = (F control − F control blank) × 100 / (F extract – F extract blank) Here (F control – F control blank), corresponds to the difference of fluorescence intensity between BSA incubated with MGO and BSA without MGO and (F extract - F extract blank) corresponds to the difference between the fluorescence intensity of BSA + MGO incubated with Synergicfood and BSA + MGO incubated without Synergicfood. Results and Discussion Antioxidant Capacity The consumption of food rich in phenolic compounds (PC) have been associated with health benefits due to its antioxidant, anti-inflammatory and antimicrobial capacities. Besides, it has been reported that PC own the ability to inhibit digestive enzymes such as pancreatic lipase (PL) and the glycation of proteins which suggests their potential as natural anti-obesogenic and antidiabetics agents.