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. The potential of these natural substances is that they are safer for the human body, since they are found in various foods, and therefore could be more accepted for the treatment of conditions such as obesity and diabetes compared with other synthetic origin compounds. The results on the study of total content of phenols associated to the functional food Synergicfood produced based on grape juice extract and rich in fibers showed a high content of total phenols (Table 1). It is estimated that the amount of total phenols in the sample is related to the antioxidant capacity of an extract. Table 1. Results obtained for the quantification of total phenols of the samples analyzed.

Sample Total Phenols mgEAG/100g SynergicFood 1559 ± 37,5*

*Values correspond to the average of three repetitions ± Standard deviation

Inhibitory activity of samples on the digestive enzymes α-glucosidase and pancreatic lipase

Recent studies suggest that CF can reduce energy intake by regulating the digestion and absorption of carbohydrates and lipids (Buchholz and Melzig, 2015, Hanhineva et al., 2010). The condition for these nutrients to be absorbed in the small intestine is that they must first be subjected to the action of the enzymes that carry out their digestion process (Sanders, 2016). The enzymes involved in the complex process of digestion of carbohydrates and lipids are α-amylases, α-glucosidases and lipases, respectively. Therefore, the inhibition of these enzymes represents a mechanism by which CFs could act as an anti- obesogenic agent (Buchholz and Melzig, 2015; Xiao et al., 2013a; Xiao et al., 2013b).

The digestion of dietary carbohydrates is mainly regulated by the intestinal α-glucosidase (E.C. 3.2.1.20) and the pancreatic α-amylase (E.C. 3.2.1.1). Therefore, the control of glucose absorption in the small intestine by inhibiting these enzymes is considered as a way to prevent the development or exacerbation of obesity (Samaha et al., 2003). Several studies have reported the ability of CF to inhibit the activity of these enzymes. Compounds such as kaempferol 3-(6-methylglucuronide) have been reported to inhibit low concentrations (65.22 μM), like 50% of the activity of α-glucosidase in the intestine (Yang et al., 2016). Likewise, it has been reported that tannic acid is able to inhibit the activity of this enzyme with an IC50 of 0.44 μg / mL, showing to be more effective than acarbose (IC50> 0.60 μg / mL), which is a synthetic inhibitor of the intestinal α- glucosidase and pancreatic α-amylase (Xiao et al., 2015). It is also well known that dietary lipids represent the main source of unwanted calories and that the metabolism of these nutrients is closely related to the development of obesity. To date, many enzymes involved in the different stages of lipid metabolism are known, which are therapeutic targets for the prevention and treatment of obesity (Loli et al., 2015, Shi and Burn, 2004a b). Among these enzymes is lipase (LP), which plays a fundamental role in the digestion of dietary lipids for subsequent absorption in the small intestine (Whitcomb and Lowe, 2007). Currently, the LP is the target of orlistat®, a specific inhibitor of this enzyme, which has reported to reduce the absorption of dietary lipids up to ~ 30% (Sumithran and Proietto, 2014). It has been described that this drug is the most used to treat obesity given its ease acquisition without a prescription, however, the adverse effects after its use should not be ignored (Beyea et al., 2012). This way, as it has been seen with other digestive enzymes, FC could represent a promising alternative to act as natural anti- obesogenic agents by inhibiting LP activity (Birari and Bhutani, 2007; Buchholz and Melzig, 2015). Clearly the best way to approach this disease is through a lifestyle modification, however, in the recent years various therapeutic strategies have emerged to defeat it, such as the use of drugs (Sergent et al., 2010, 2012). Drugs currently used include orlistat, lorcaserin, and phentermine/topyramate, which have been approved by the FDA (Food and Drug Administration). Although the effectiveness of such drugs has been demonstrated, adverse health effects reported after use should not be ignored (Fidler et al., 2011; Kim et al., 2013a; O'Neil et al., 2012; Smith et al., 2010). Because of this, there is a need to explore new compounds that are effective and safe (Chakrabarti, 2009, Robinson and Niswender, 2009).

Pancreatic lipase inhibition. The inhibition of this enzyme promotes a lower fat absorption and a possible way for treat metabolic alterations such as hypertriglyceridemia (abnormal concentration of triglycerides in the blood). In Table 2, the results obtained can be observed against the activity of the pancreatic lipase enzyme of Synergicfood. This food showed a 67% inhibition of the enzymatic activity at concentrations of 10 mg / ml and an IC50 of 6.5 mg / ml. This IC50 value indicates the in vitro potency of any compound when compared to known patterns such as the drug orlistat®, which is a potent, specific and long-acting inhibitor of gastrointestinal lipases. This agent exerts its therapeutic activity in the lumen of the stomach and small intestine by forming a covalent bond with the active site of the serine in gastric and pancreatic lipases. The inactivated enzyme is not available to hydrolyze dietary fat, in the form of triglycerides, to absorbable free fatty acids and monoglycerides. Inhibition values have been reported for different polyphenols of natural origin against this enzyme fundamentally associated with the content of flavonoids in samples, however, the inhibitory effect of the flavonoids will be directly proportional to the number and position of the phenolic hydroxyl groups that determine them as potential inhibitors. Several studies demonstrate the presence of activity of the lipolytic enzyme with IC50 of 7 mg / mL for extracts rich in catechin, quercitrin and rutin (Buchholz & Melzig, 2015), values that are close to that of orlistat®. According to the chemical analysis carried out in our laboratories, the extract of grape juice, from which the food is made, has a high content of these phenolic compounds, so the activity found may respond to these presences.

Sample Concentration SynergicFood (mg/mL) (% of inhibition)

10 67,1 +/- 6,3

5 38,4 +/- 9,0

2,5 20,3 +/-3,3

1,25 11,5 +/-3,7

0,62 5,6 +/- 0,3

0,31 2,1 +/-0,6

Table 2 Percentages of inhibition and IC 50 values of the samples against lipase

0,15 0

0,078 0

CI50 6,8 mg/mL +/- 0,14

CI50tetrahydrolipstatin (Orlistat MK®) 4,43 +/- 1,12

CI50: Concentration of the product that inhibits 50 percent of the reaction. The lower the IC50 value, the greater the efficiency of inhibition. Orlistat (tetrahydrolipistatin): positive control.

α-glucosidase enzyme inhibition. The enzyme α-glucosidase has an important function in the hydrolysis of carbohydrates necessary to accomplish an adequate digestion in the organism. Therefore, the inhibition of this enzyme contributes to the delay in time of the digestion of carbohydrates at the intestinal level and hereby hyperglycemia is avoided. In Table 3 we can see the results obtained against the inhibition of alpha glycosylase. The food Synergicfood can inhibit 31% at a concentration of 10 mg / mL. All the samples analyzed show IC50 higher than the positive control (Acarbose: 0.86 mg / mL)

Table 3 Percentages of inhibition and IC50 values of the samples against the alpha glucosidase enzyme Sample Concentration Synergicfood (mg/mL) (% of inhibition)

10 31.0 +/- 6.3

5 18.4 +/- 9.0

2.5 6.3 +/-3.3

1.25 1.5 +/-3.7

0.62 0

0.31 0

0.15 0

0.078 0

CI50 Greater than 10mg/mL

CI50: Concentration of the product that inhibits 50 percent of the reaction. The lower the IC50 value, the greater the efficiency of inhibition. Acarbose: positive control. Antiglycan Activity The results referring to an antiglycan activity in different samples expressed by % inhibition is shown in Table 4. The tests were performed using glucose to measure the formation or no inhibition of the amadori products formed at the beginning of glycation reaction. We use methylglyoxal to detect inhibition or no inhibition in the last steps of the reaction where the AGEs are formed. Synergicfood shows a greater inhibitory activity in the BSA plus MGO reaction, which could indicate that it acts in the last steps of the reaction of the formation in these products. Therefore, it directly inhibits the formation of AGEs. Table 4: Antiglycan activity (expressed in% inhibition of AGE formation) of the tested products.

BSA +GLU BSA + GLU BSA +MGO BSA+MGO

(Inhibition (IC50 mg/ml) (Inhibition IC50 mg/ml %) %) SynergicFo od (0,1 18.4 ± 4.12 1. 75.0 ± 0.20 mg/mL) 75 AMG 1.38 ± 0.82 nd 92.02 ± 0.01 (0,1 mg/ml) a 3.00 AMG (Aminoguanidine), BSA (Bovine Albumin Glu (Glucose), MGO (Methylglyoxal). Nd: not determined.

Conclusion

The functional food based on grape juice and fiber has an inhibitory effect on intestinal enzymes, such as lipase and alpha glucosidase in experiments carried out in vitro. In the case of the inhibitory activity of alpha glucosidase it is necessary to test concentrations greater than 10 mg / ml of the juice to determine the IC50 values and compare them with known pharmacological patterns. It is necessary to carry out studies that allow to define the type of inhibition that the food and its components have on the enzymes analyzed in order to better predict their biological effect. The beneficial effects of natural extracts rich in polyphenols depend on the absorption and distribution through the organism. Several studies show the bioavailability and absorption of some grape polyphenols in animals and humans. In this case, it is necessary to carry out these studies associated with the formulation of juice proposed by the producers. On the other hand, the food has the capacity to inhibit the formation of AGEs in vitro. Just as for the rest of the analyzes, it is necessary to carry out deepening studies and in vivo studies that allow to corroborate the biological activity found in vitro.

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