ABSTRACT
Diabetes mellitus is among the chronic diseases considered emergencies. Products obtained from medicinal plants have been used for thousands of years by the population in the treatment of various diseases. The objective of this work was to evaluate the possible effects of inputs obtained from the artichoke (Cynara scolumus L.) on biochemical markers in diabetic rats. The dry extract was obtained by hydroethanolic (70% v / v) maceration. Male Wistar rats were treated with alloxan (150 mg / kg of mass dissolved in 0.9% saline) intraperitoneally. The extract (0.2g extract / kg) was given in normal and diabetic rats for 30 days. Animals from the Control and Diabetes groups received only water. Biochemical markers were determined in the serum by enzymatic colorimetric method. The chemical characterization was performed by quantifying the total polyphenol content by the Folin-Ciocalteau method and quantification of the total flavonoid in dry extract (1 mg / mL), by the complexation method with aluminum chloride. The evaluation of fasting blood glucose was positive, when considered that the glycemic levels from “diabetic group” was partially higher when compared with “water group”, “extract group” and “diabetic extract group”. The treatment with extract for the diabetic animals also prevented significantly the serum levels of fructosamine and presented serum levels of total cholesterol and triglycerides significantly lower than non-treated animals. However, the treatment with artichoke extract was able to control glycemic levels at short and medium term, showing an improvement in lipid profile.
Key words: Artichoke. Oxidative stress. Phytochemical screening. Flavonoids. Total polyphenols.
1 INTRODUCTION
Chronic noncommunicable diseases (NCDs) cause about 16 millions of premature deaths per annum. According to the annual report of the World Health Organization’s (WHO), published in 2016, heart diseases and lung diseases, cerebrovascular accidents, cancer and diabetes are largely responsible for high rates of premature deaths. In Brazil, studies point to three major groups responsible for the “NCDs epidemic”, diabetes, cardiovascular disease and brain stroke [Malta & Silva, 2013].
The diabetes mellitus is featured by a chronic disorder that affects the metabolism of carbohydrates, fats and proteins. The diabetes mellitus (DM) is considered a public health problem due to its association to several risk complications, such as microvascular changes, in which retinopathy, neuropathy and macrovascular complications can be cited, including coronary heart disease and stroke.
Of chronic complications inherent to diabetes mellitus, diabetic neuropathy and cardiopathy according to [Moreira et al., 2008], affects about 30% to 40% of individuals with type 1 DM and about 10% to 40% those with type 2 DM, representing the main macrovascular complication of diabetes and the biggest cause of end-stage renal failure worldwide.
Therefore, the search for compounds with capacity to reduce or prevent chronic complications of diabetes mellitus, have been the target of several studies since the costs for the treatment of NCDS have a great impact on the country’s health system. Otherwise, use of natural products has been a good source for discovery of new drugs. Only for the treatment of type 1 and 2 diabetes mellitus, from 1981 to 2014, 29 new drugs from natural sources have been approved [Newman & Cragg, 2016 ].
2 METHODOLOGY
As a natural source, leaves from Artichoke (Cynara scolumus L .) were used to extract production and purchased from a company which produces plant extracts, Biotae©.
2.1 Crude extraction preparation
Dried and sprayed artichoke leaves (Cynara scolumus L.) were submitted to exhaustive maceration, in proportion of 1 part of powder to 10 parts of hydroethanolic solution at 70% (v/v). The mixture (powder + extractor liquid) was filtered through a paper filter and the extractive solution (extractor liquid + secondary liquid) was dried under reduced pressure at 45°C, eliminating the organic solvent. The water was removed by the lyophilization process. After the organic and aqueous solvent elimination, crude extract was stored at -20°C.
2.2 Determination of the total phenol content
The content of polyphenols was determined in crude extract aliquots by the method of Folin & Ciocalteau, using gallic acid as standard [Singleton, Orthofer & Lamulela, 1999]. The results were expressed in grams equivalent of gallic acid (g GAE/100g extract). All determinations were performed in triplicate.
2.3 Determination of flavonoids content
The content of total flavonoid content in crude extract was performed in solutions containing the crude extract (1 mg/mL), Fr-BuOH (0,2 mg/mL) and Fr-EtOAc (0,2 mg/mL). Stable complexation between aluminum and flavonoids in a solution containing methanol or ethanol was quantified by spectrophotometry at 425 nm, since at this wavelength it is possible to quantify the flavonoid content without the interference of phenolic compounds. [Souza, 2005] Quercetin in ethanolic solution was used as standard at concentrations of 10, 20, 40, 60, 80, 100 μg/mL, obtaining the analytical curve. The flavonoid content was determined by interpolating the absorbance of the samples against the calibration curve and the values were expressed as quercetin equivalent (g of quercetin/100g of sample). The analyzes were performed in triplicate.
2.4 Experimental Groups
The animals used in this experiment were Wistar lineage rats, male and aging 8 weeks and body mass of (326, 64g, ± 28,5). [King, 2012]. Animals were obtained from the Central vivarium of José do Rosário Vellano university. The entire experimental part of this work was approved by the ethics committee on animal experimentation under approval number 09A/2018. The animals were housed at vivarium the laboratory of pharmacology and experimental biochemistry, with a maximum of 5 animals per polyethylene box, type kaefiq, autoclavable, acid resistant, in the measures of 49X34X16 cm. Initially, the animals were submitted to a vermifugation protocol with albendazol 10% in cycles of 24 to 48 hours ad libitum during 1 week. Water and feed were replenished daily ad libitum to the animals.
Animals groups were divided into four groups:
a) Non-diabetic group: was composed of non-diabetics animals and were not subjected to treatment with the artichoke extract (n = 5).
b) Diabetic group: was composed of animals subjected to treatment with the alloxan and not treated with artichoke extract (n = 6).
c) Extract group: was composed for non-diabetic animals and treated with artichoke extract (n = 10).
d) Diabetic extract group: was composed for animals subjected to treatment with alloxan and treated with artichoke extract (n = 14).
2.5 Induction of diabetes mellitus
To induce diabetes, the diabetogenic drug, alloxan, was administered in rats, intraperitoneally, at a concentration of 150m mg/Kg of body weight, dissolved in saline solution 0,9% (pH 4,5). The animals which showed casual blood glucose over 200 mg/dL were considered diabetics and evenly distributed between diabetic groups so that all groups had a statistically equal glycemic [Jaouhari, Lazrek & Jana, 2000].
2.6 Crude extract administration
Crude extract was reconstituted in water and administered for gavage technique for 30 days. The amount of crude extract administered to the animals was 200 mg of extract per kilogram of animal mass [Gary, 2003].
2.7 Biological parameters
The Biological parameters were evaluated during the treatment of the animals, by monitoring food consumption and liquid intake. At the end of the treatment, the animals were anesthetized to get the blood collected and conducted biochemical tests for these samples.
2.8 Obtaining biological samples
At day 31, the animals were anesthetized with thiopental (60 mg/kg of mass) intraperitoneally and the blood was collected by cardiac puncture and distributed in different tubes without additives and with a clot activator gel to obtain the serum.
2.9 Evaluation of glycemic control
To evaluate the glycemic control, fasting blood glucose and fructosamine parameters were measured. The glucose concentration was measured in the serum by an endpoint colorimetric enzymatic method. The determination of serum fructosamine concentration was made by a colorimetric kinetic method, based on the reduction of nitrobluetetrazole, in semi-automated equipment from the Bio2000® brand com kits da marca labtest®.
2.10 Evaluation of the lipid profile
The concentration of total cholesterol and triglycerides were determined in the serum by an endpoint colorimetric enzymatic method da marca labtest® [Burtis & Ashwood, 1999].
2.11 Determinação de creatinina
Os níveis séricos de creatinina serão determinados pelo método de Jaffé modificado, utilizando kit adquirido comercialmente, cujo procedimento de medição foi calibrado com o material de referência SRM 914 doNational Institute of Standards and Technology (NIST), tornando os resultados rastreáveis ao método definitivo (espectrometria de massas com diluição isotópica) [Burtis & Ashwood, 1999].
2.12 Statistical analysis
The observed values of each variable were subjected to analysis of variance. Multiple comparisons between the means of the different treatments were performed using Tukey test at 5% of probability in the Sisvar version 5.3 program.