Experimental Diabetes Model
The study was conducted on an experimental model of male Wistar albino rats (Institut Pasteur, Algiers), weighing between 200–250 g of body weight at the start of the experiment. After an adaptation period of one week, the animals are divided into 5 groups (n = 10), in a controlled environment, with a constant temperature (22 ± 2°C), a humidity of 50 ± 5% and light-dark cycles of 12 hours each. The rats had food and water ad libitum.
After overnight fasting, diabetes is induced in these rats by intraperitoneal injection of a single dose (200 mg/kg) of a 98% alloxan monohydrate solution (ALX) (SIGMA-ALDRICH, St Louis, USA). After injection, the rats are put back in the cages and systematically receive free access to food and a 5% glucose solution to drink overnight to avoid hypoglycemic shock.
48 hours after injection of alloxan, fasting and post-prandial blood glucose (1h30 after the meal) were measured. Animals with a fasting blood sugar level greater than 2 g/l are considered diabetic and randomly divided into 5 groups of 10 rats each.
Biological analyzes
For blood glucose determination, blood samples are taken from the tail of the rats. After cleaning the tail with alcohol, the rats are pricked with a fine needle; a drop of blood is collected and then placed on a strip for blood glucose reading (Accu Chek Performa Reader).
For the exploration of the other parameters, the sample is taken at the level of the retro-orbital venous plexus, using a hematocrit capillary previously immersed in an EDTA solution (1%). The blood was collected in heparinized tubes, then immediately centrifuged at 4000 rpm for 10 minutes at a temperature of 10°C using a thermostatically controlled centrifuge (Sigma 3–16 KL, Ref: 10360), the sera were stored at -20°C. These samples were taken from fasting rats.
Vegetable material
The young leaf shoots of Ranunculus repens L. were collected between March and April 2019 in the Wilaya of Jijel (Algeria) during the flowering period. After their identification by the Laboratory of Phytopharmacology and Toxicology (University of Batna 1) the leaf samples are cleaned and then dried at room temperature in a ventilated place in the shade. The plant material was pulverized in powder form.
Preparation of the aqueous extract of flavonoids
The extraction of flavonoids is based on the degree of solubility of flavonoids in organic solvents. This method consists of two main steps: The first phase of extraction is done with methanol to solubilize the flavonoids and the second is carried out with diethyl ether (extraction of free genins) and ethyl acetate (extraction of monoglycosides) and n-butanol (to solubilize di and triglycosides).
The extraction of flavonoids is carried out from the dry matter finely ground by 85% methanol. The aqueous phase thus obtained is kept for 48 hours at 40°C to accelerate the diffusion of the molecules in the solvents then filtered through Büchner under reduced pressure and subjected to low pressure evaporation at 35°C (Rota Vapor, Büchi 461, Germany). The filtrate is freed from waxes, lipids and chlorophyll by three successive washes with petroleum ether to give an aqueous phase.
In order to separate the flavonoids into aglycone, monoglycoside and di and triglycoside fractions, the aqueous phase is mixed with diethyl ether to obtain an organic phase containing the aglycone flavonoids and the methoxylated aglycones. The remaining aqueous phase in turn undergoes three extractions with ethyl acetate in order to recover in the organic phase certain aglycone flavonoids but especially the monoglycosides. The remaining aqueous phase is mixed with n-butanol to recover in particular di flavonoids and triglycosides.
The final aqueous phase contains mostly the more polar glycosylated flavonoids. The collected fractions are concentrated by evaporation at low pressure at 35°C then freeze-dried for 24 hours (ALPHA 1–2 LD, Fisher Bioblock). Lyophilization makes it possible to obtain a product that is easily soluble in water and which, after adding water, has the same characteristics as the original product. Each lyophilisate is weighed to calculate the extraction yield, expressed in grams of lyophilisate per 100 g of dry matter.
Dosage of flavonoids
The quantitative evaluation of flavonoids in the different fractions is carried out according to the aluminum trichloride method: 4 ml of diluted extract solution was mixed with 4 ml of aluminum trichloride solution (2% in methanol). After 15 min, the absorbance was measured at 415 nm. Quercetin (Q) was used as a reference compound to produce the calibration curve.
Treatment with the butanol extract of Ranunculus repens L.
The diabetic rats are divided into 5 groups of 10 rats each: an untreated control group receiving a physiological solution (NaCl 0.9%), 3 other groups treated for 28 days with the butanol extract of Ranunculus repens L, at a daily dose of 200, 400 and 600 mg/kg of body weight by gavage. The 5th group is a control group receiving glibenclamide which is a hypoglycemic drug, at a dose of 2.5 mg/kg.
The evolution of glycaemia is measured before the treatment and 2 hours after the treatment.
Effect of flavonoids on glucose complexation in vitro: Glycosylation test
The purpose of this test is to estimate the ability of flavonoids to complex free glucose in vitro and therefore to demonstrate their role as glucophage. For this, 10 µl of a glucose solution is mixed with 10 µl of each flavonoid extract at different concentrations: 0.1, 0.2 and 0.4 mg/ml. The mixture thus obtained is incubated at 37°C for 15 min. The dosage of glucose not bound to the flavonoid extracts is measured according to the enzymatic reference method with hexokinase:
Glucose is phosphorylated to glucose-6-phosphate by the action of ATP and hexokinase (HK). Then, a second enzyme, glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the oxidation of glucose-6-phosphate by NADP+ to form NADH + H+ and the 6-phosphoglucono-δ-lactone.
Since the product has no absorbance, this involves measuring the absorbance of the NADPH, a coenzyme involved in the enzymatic reaction.
Thus, the concentration of NADH formed is directly proportional to the concentration of glucose. It is measured by the increase in absorbance at 340 nm by spectrophotometry.
Evaluation of the antioxidant power of flavonoids
The antiradical capacity of di and triglycosyd flavonoids is evaluated in vitro by the DPPH° method (2, 2 - Diphenyl-1-picrylhydrazyl): Briefly, 15 µl of different concentrations of flavonoids 0.1, 0.2, 0.4 mg/ml and a solution prepared with 0.158 mg/ml of trolox are added to 1.5 ml of DPPH°. Absorbance is read every 30 s for 5 min at 515 nm. The dust removal effect of the flavonoids is expressed as a percentage of reduced DPPH° starting from 100% of the control (DPPH° alone) according to the following relationship:
Radical scavenging activity (%) = [(Abscontrol−Abssample)/Abscontol]×100
Evaluation of the effect of flavonoids on hepatic glycogen storage
The evaluation of the level of hepatic glycogen content in hyperglycemic rats is carried out according to the following protocol: the control group as well as the four other groups receive a solution of glucose at a dose of 4 g/kg in a physiological solution orally.
After 3 hours of effect of the flavonoids, the glycaemia of the animals is measured, and then the rats are sacrificed by chloroform anesthesia and undergo abdominal dissection. The livers are removed and fixed in Bouin (formaldehyde/picric acid/acetic acid).
5 grams of fresh liver is cut into small pieces and then boiled in 50 ml of distilled water for 2 minutes. The liver fragments are drained, and then crushed with the mortar. 25 ml of distilled water are added to the ground material and the suspension obtained is boiled for 5 minutes. The broth is filtered under vacuum on Büchner then the filtrate is recovered with 3 drops of HCl (to precipitate the proteins) and is filtered again. The filtrate is treated with 4 times its volume of 95% alcohol then filtered under vacuum and the final filtrate is taken up with 2 ml of distilled water.
To measure the glycogen, 3 ml of distilled water and a drop of Lugol are added to 1 ml of glycogen extract obtained and the optical density of the mahogany brown color is read at 470 nm. The glycogen concentration is deduced from a standard range established with pure glycogen as standard.
Statistical analyzes
Results were expressed as mean ± STD error (M ± SE) of 10 rats per 5 group (N = 10). Statistical evaluation is performed using Student's t test. The differences in level ∗𝑝 < 0,05 were considered statistically significant.