The whole plant A. flava was collected from the Biskra District in southern Algeria. The plant was identified by its vernacular name and later confirmed at Agronomic Institute, University of Banta, by Professor Bachir Oudjehih and Voucher specimen with the corresponding number (660/LCCE) was deposited at the Griffin Herbarium of the mentioned Institute.
Preparation of extracts
Dried and powdered plant material (1000 g) was macerated with the solvent mixture methanol/water 80:20 (v/v). The recovered quantity was filtrated and concentrated under vacuum at ambient temperature. The hydroalcoholic extract was submitted to liquid–liquid fractioning using (petroleum ether, dichloromethane, ethyl acetate, and n-butanol). The n-butanol fraction was used to evaluate the α-Glucosidase and α-Amylase inhibitory and the antihyperglycemic activity.
Only male Wistar albino rats weighted from 150 to 180 g, aged 2–3 months were handled to evaluate the antidiabetic activity. Rats were obtained from the Pasteur Institute, Algiers, Algeria, and were fed a standard laboratory diet and allowed water ad libitum. This study was approved by the ethics committee of Pasteur Institute, Algiers, Algeria, ethics with approval voucher number VB09268.
In vitro antidiabetic activities
α-Amylase inhibitory activity
The α-Amylase inhibition assay was determined by using the dinitrosalicylic acid (DNS) method . Different concentrations (62.5–1000 µg/mL) of BEAF were prepared from a 1 mg/mL stock solution of phosphate buffer. The samples (100 µL) were incubated with 100 µL of α -Amylase solution (0.02 mol /L for 30 min at room temperature. Then 100 µL of starch solution (1%) was added and incubated for another 10 min. The mixture was heated 5 min in a boiling water bath after adding 1 mL of DNS color reagent that stops the reaction. Then it was allowed to cool and was diluted by adding distilled water (5 mL). The blank was prepared by replacing the enzyme with a buffer for each set of concentrations of the test sample. The control was maintained without the addition of a sample that represented 100% enzyme activity. The absorbance of the color solution was measured at 540 nm using ultraviolet–visible spectrophotometer (Jenway™ 6305). Acarbose serves the experiment as a positive control.
α -Glucosidase inhibitory activity
The α -Glucosidase inhibitory activity of the BEAF was tested by measuring the 4-nitrophenol released from ρ-nitrophenyl α-D glucopyranoside (ρNPG). The different concentrations (62.5–1000 µg/mL) of a sample (200 µL) were added to the assay mixtures containing 700 µL of 10 mM ρ-nitrophenyl α -D glucopyranoside, 1.0 mL of potassium phosphate (0.1 M, pH: 6.8), 200 µL of enzyme solution and incubated for 6 min at 37 C. The addition of 100 mM sodium carbonate (1 mL) terminated the reaction. The liberated ρ-nitrophenol was estimated by determining the absorbance at 405 nm using a spectrophotometer (Jenway™ 6305). A positive control (Acarbose) was used to compare the inhibitory activity of BEAF. The percentage of inhibition was calculated to express the α -Glucosidase inhibitory activity .
In vivo antidiabetic activities
Oral Glucose Tolerance Test (OGTT) in rats
Rats were fasted for 12 h, except for water ad libitum, and haphazardly divided into three groups (n = 5). Group I (normal control) rats were treated with vehicle (NaCl 0.9%); groups II, III were treated with 250 mg/kg and 500 mg/kg n-butanol extract, respectively. Animals were orally administered in a unique dose, the n-butanol extract that was dissolved in 0.9% NaCl. Thirty minutes after treatment all the animals received 4 g/kg of glucose solution. Blood was collected from the tail tip of each rat and BGL was measured immediately before treatment using an Accu-chek Active™ test glucometer at 0 (as a baseline), 60, 90, and 120 min and of glucose administration .
Induction of experimental diabetes
A dose of 125 mg/kg of freshly prepared alloxan (Sigma-Aldrich (St. Louis, MO) (0.9% NaCl) was sufficient to induce diabetes by a single intraperitoneal injection of overnight fasted male rats . Animals were given free access to water and pellet diet after 30 min of alloxan administration. Animals were kept under rigorous surveillance, the fasting BGL was recorded after seven days alloxan injection using a glucometer. The selection of rats that will be included in the study was based on the criteria of having a fasting BGL greater than 200 mg/dL .
Acute and subchronic antidiabetic activity in alloxan-induced diabetes model
The chosen diabetic animals were scattered into 5 groups (n = 5). Group 1 was defined as a negative control, received a single dose of 1 mL/100 g of the vehicle, group 2 was considered as positive control, treated with glibenclamide (5 mg/ kg) as a reference drug. Groups 3 to 5 were treated with BEAF at three dose levels (100,250 and 500 mg/kg). Blood was collected from the tail tip of each mouse and BGL was measured immediately before treatment at 0 (as a baseline), 30, 60, 90, and 120 min of BEAF administration. Treatment was extended for 7 consecutive days (p.o.). At the end of day 7, the rats have fasted for 16 h and blood parameters were determined, the rats were thereafter sacrificed by cervical dislocation and the blood samples were collected through cardiac puncture from the posterior vena cava.
Estimation of biochemical parameters
The BGL was measured using an Accu-chek Active™ test glucometer by collecting the blood from the rat tail vein. For other plasma parameters, at the end of treatment, i.e., 7 days, blood was collected from rats retro-orbital plexus applying a light ether anesthesia method. The plasma was restricted by centrifuging at 3000 rpm for 5 min and was checked out for lipid profiles (total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL)), creatinine, and urea that were analyzed by standard enzymatic assays with an automatic analyzer (COBAS INTEGRA® 400 plus).
All the data were expressed as mean ± SEM, and one-way analysis of variance (ANOVA) followed by Dunnett’s test was used for statistical analysis by using GraphPad Prism software (version 7.0). Significance was assumed if P < 0.05.