PAMELA ALVES CASTILHO

Título da Dissertação: EFFECTS OF A MYRCIARIA JABOTICABA PEEL EXTRACT ON STARCH AND TRIGLYCERIDE ABSORPTION AND THE ROLE OF

CYANIDIN-3-O-GLUCOSIDE

Orientadora: Profa. Dra. Anacharis Babeto de Sá-Nakanishi

Data da Defesa: 13/05/2021

GENERAL ABSTRACT

Introduction

Obesity is a metabolic disorder, characterized by excessive accumulation of body fat and related to several comorbidities, such as type 2 diabetes and dyslipidemia. In recent years, the incidence of obesity has become a public health problem worldwide, with incalculable social costs. A group of drugs widely used in the treatment and control of these pathologies are the inhibitors of the enzymes α-amylase and pancreatic lipase. However, these drugs have major side effects, such as abdominal pain, flatulence, diarrhea, and decreased vitamin absorption, which often leads to discontinuation of therapy. For this reason, natural compounds have been explored as potential alternatives to the treatment of obesity and diabetes.

Myrciaria jaboticaba belongs to the Myrtaceae family, is a fruit plant native to Brazil, widely cultivated mainly in the Southeast region. The fruits are globose, with juicy pulp, generally sweet, and called jabuticaba. The peel of jabuticaba has a high content of phenolics, anthocyanins, ellagic acid, and a high antioxidant and anti-inflammatory potential. Cyanidin-3-O-glycoside (C3G), the main anthocyanin present in the peel, is responsible for the color as well as for its significant antioxidant, anti-inflammatory, and anti-obesity properties. As much as some studies reported the inhibitory activity of the jabuticaba peel on the enzymes amylase and pancreatic lipase, these were carried out in vitro, that is, there are no reports that evaluate these effects in animals.

Objective

In this context, to take advantage of the residue obtained from the food industry, this work had the central objective of evaluating the effect of jabuticaba peel extract on the absorption of starch and triacylglycerols in mice. In addition, conduct a comparative study with its main constituent, C3G, on these phenomena.

Materials and methods

All experimental procedures were previously approved by the Ethics Committee on the use of Animals in Experimentation - CEUA-UEM under nº 9577260819. The peel of jabuticaba was purchased from the Belo farm (State of São Paulo) and was used for the preparation of the extract hydroethanolic 70%, and even was lyophilized. The extract was characterized and the profile of phenolic compounds was determined, and classified into anthocyanidin and non-anthocyanidin in high-performance liquid chromatography (HPLC) coupled to a mass spectrum. Evaluation of the effects of an extract and cyanidin-3-O-glycoside (C3G) concentration curve on the activity of pancreatic amylase and lipase were performed in vitro. Similarly, kinetic assays were performed to characterize the activity of these enzymes in the presence of at least 2 different concentrations of the extract. All experimental procedures were completely controlled and the activity of each of the enzymes, under the different conditions of evaluation, was calculated from the spectrophotometric readings.

For in vivo tests, male mice, Swiss lineage (28 to 33g) were used in an 18-hour fast. Four oral tolerance tests were performed on mice: 1) Starch tolerance test: animals received, via gavage, an overload of commercial corn starch (1g / Kg). 2) tolerance test to triacylglycerols (TG): animals received, via gavage, an overload of olive oil (5mL / Kg). 3) Fatty acid tolerance test: animals received an overload of oleic acid + glycerol via gavage. All tests were performed in the presence and absence of a curve of doses of the extract of the peel of the jabuticaba, as well as with at least two doses of C3G. Negative control animals received water, and positive control: received acarbose (50mg / kg) or orlistat (50mg / kg). Blood samples were collected before and after overloading starch or TG or glycerol plus oleic acid and the plasma levels of glucose or triacylglycerols were quantified depending on the purpose of the assay. Statistical analysis and numerical interpolation for the determination of the IC50 (effective concentration responsible for half of the maximum inhibition) were performed in the Scientist Software from MicroMath Scientific Software (Salt Lake City, UT).

Results and discussion

The chemical characterization of the extract revealed that the peel of the jaboticaba has 12 phenolic compounds, ten of which are non-anthocyanins and two anthocyanins. Cyanidin-3-O-glycoside is the main phytochemical found in the last group and makes up 1.2 g% of the extract. The extract of the bark of jabuticaba inhibited the activity of amylase and pancreatic lipase in a dose-dependent manner. The IC50 was 1963 μg / mL and 143.9 μg / mL, respectively. This means that the extract showed a much greater inhibitory action on lipase (+13.6 times) than on pancreatic α-amylase. These were parabolic inhibitions, and the extract was characterized as a mixed inhibitor. To evaluate the possible contribution of C3G to these enzymatic inhibitions, experiments were carried out with C3G with concentrations of up to 200 μg / mL. That is concentrations well above the concentrations of C3G present in the IC50 of the extract, of 23.55ug / mL for amylase and 1.77ug / mL for lipase. Even so, C3G contributed little to the inhibition of these enzymes by the extract. Thus, C3G can be considered a weak pancreatic lipase inhibitor. Since it is not primarily responsible for the inhibitory effect of the extract on amylase and pancreatic lipase, an effect probably attributed to other polyphenols present in the extract.

Based on these results, it is expected at least a certain interference of the extract on the starch and triacylglycerol absorption process in mice. To evaluate this hypothesis, the starch tolerance test revealed that, the extract inhibited the absorption of starch only in high doses. Extract doses of 250 and 500 mg/kg reduced the glycemic increase induced by starch (area under the curves) by 51 and 84%, respectively. The dose of 500 mg/kg, had an effect very similar to acarbose (50 mg/kg), the classic inhibitor of starch absorption. C3G experiments, in doses far above that found in the extract (250 and 500 mg/kg), 10 and 20 mg/kg, did not interfere with the starch absorption process. In addition, the extract, at least in the doses evaluated, did not interfere with the transport of glucose through intestinal cells.

Similarly, jabuticaba bark extract reduced the absorption of triacylglycerols (TG) in a dose-dependent manner, after an oral overload of olive oil. However, this effect was observed at doses of extract well below the doses responsible for inhibiting the absorption of starch. Extract doses of 5 to 250 / mg kg reduced the response curve to TG overload progressively towards the curve obtained with orlistat (positive control). The dose of 1mg / Kg promoted a transient stimulus in the absorption of TG followed by inhibition. The 5 mg/kg dose reduced the area under the TG tolerance test curve by 67%. Numerical interpolation revealed that the concentration of the extract responsible for a 50% decrease in TG absorption (IC50) was 3.65 mg/kg. The effect of cyanidin-3-O-glycoside (0.2 and 2 mg/kg) on this process was also evaluated. The lowest dose (0.2 mg/kg), similarly to the lowest dose of the extract, had a double effect, as observed by the initial increase in serum TG concentration and subsequent

decline. On the other hand, the highest dose, 2.0 mg/kg, reduced the absorption of TG compared to the control. This complex phenomenon reveals that, at least in low doses, both the extract and C3G had a double effect, with a rapid and transient stimulus of TG absorption, but that this was overlaid by an inhibitory action that predominated over time. These results, however, revealed that the extract of the bark of the jabuticaba inhibited the absorption of TG in much lower doses than expected since the IC50 of the extract for lipase was 143.9ug / mL. Thus, it can be expected that the extract could interfere in steps after the hydrolysis of TG. In addition, this interpretation is reinforced by the effect of C3G, which is a weak inhibitor of lipase, but a strong inhibitor of the absorption of triacylglycerols in the concentrations present in the extract of the bark of the jabuticaba. In this sense, we evaluated the effect of the extract (5 and 25mg / Kg) and C3G (0.2 and 2mg / Kg) on the fatty acid absorption process in animals. Both the extract and C3G prevented an increase in TG plasma levels after an overload of oleate plus glycerol. The degree of inhibition was very similar for the extract and C3G doses used. Thus, it is concluded that the inhibitory effect of the extract on the absorption of fatty acids depends on a significant contribution, if not predominant, of C3G. The general process of fatty acid absorption (AG) and subsequent release of TG is highly complex. The exact mechanism by which C3G exerts this effect cannot be described using this work. However, it is possible to suggest an inhibitory action of C3G on one of the proteins involved in the transport of AG and or on the reactions that transform AG into TG via monoacyl-glycerol or 3P glycerol.

Conclusion

It can be concluded that the extract of jabuticaba bark presents favorable perspectives as an inhibitor of fat absorption and that cyanidin-3-O-glycoside, one of its main constituents, seems to play a decisive role in this effect. Thus, both the bark extract of jabuticaba and cyanidin-3-O-glycoside isolated present a real possibility of application as pharmacological agents or in diets for the treatment of obesity. An important characteristic of the extract of jabuticaba bark is the low doses necessary to reduce the absorption of fat. This also extends to C3G. Jaboticaba peels are usually discarded as waste by the industrial processing of the fruit, a fact that generates cheap raw material in large quantities for semi-purified pharmaceutical formulations and food additives. Of course, for this, more mechanistic and perhaps clinical studies are certainly desirable.

Keywords: Enzyme inhibition, pancreatic α-amylase, pancreatic lipase, obesity, diabetes.

Artigos Publicados Vinculados a Tese:

https://pubs.rsc.org/en/content/articlelanding/2021/fo/d0fo02927k