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.