Plant material
The leaves of A. hirtum were collected in November 2012 from El-Zohria botanical garden, Cairo, Egypt and identified by Prof. Dr. Mahmoud Abdelhady Hassan Professor of Horticulture, Faculty of Agriculture, Minia University. A voucher sample (Mn-ph-Cog-016) was kept in the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Minia University, Minia, Egypt.
Preparation of the extract and fractions
The air dried powdered leaves (5 Kg) of A. hirtum were extracted with 95% ethanol and concentrated under reduced pressure. The concentrated ethanolic extract (550 g) was suspended in the least amount of distilled water, transferred to a separating funnel and partitioned successively with petroleum ether, chloroform and finally with ethyl acetate. The fractions were concentrated under reduced pressure to afford petroleum ether (150 g), chloroform (8 g) and ethyl acetate fractions (18 g). The remaining mother liquor was concentrated to give the aqueous fraction (280 g).
Preparation of the crude polysaccharides
The concentrated aqueous fraction (280 g) was dissolved in the least amount of distilled water, transferred to a conical flask and polysaccharides allowed to settle by drop wise addition to 1 l of methanol, followed by vigorous shaking, then filtrated using glass Büchner funnel and vacuum pump. The residue (crude polysaccharides) was collected and dried using vacuum drying oven and then kept for further investigation.
Animals
The animals used in this study include female and male albino rats weighing 200 ± 50 g and mice weighing 30 ± 5 g, obtained from animal house of Faculty of Medicine, Assiut University. They were housed under standardized environmental conditions, and fed with standard diet and water. The study was conducted following approval by the Institutional Animal Ethical Committee of Faculty of Pharmacy, Minia University, Minia, Egypt.
Acute toxicity
The acute toxicity of the total ethanolic extract of Abutilon hirtum leaves was determined by measuring the lethal dose for 50% of the laboratory animals (LD50) [14]. Different dose levels (1, 2, 2.5, 3 up to 3.5 g/ kg, p.o) of the total ethanolic extract (suspended in 0.5% CMC) were orally administrated to different groups of mice (30 ± 5 g, each containing six mice). The control group received an equivalent dose of the vehicle (0.5% CMC). Both the test and control groups were observed for 24 h under normal environmental conditions, with free access to food and water.
Anti-inflammatory activity
The total ethanolic extract and different fractions of A. hirtum leaves were evaluated for their anti-inflammatory activity using the carrageenan-induced paw edema method [15]. Female albino rats (200 ± 50 g) were randomly divided into seven groups (six animals per group). The specified dose of extract, fractions, and standard drug were suspended in 0.5% CMC solution. The control group administered the vehicle (0.5% CMC solution), while the standard group was given indomethacin orally at a dose level of 8 mg/kg. The total ethanolic extract and different fractions were administrated orally at a dose level of 300 mg/kg through 2 h after carrageenan injection (0.1 ml, 1% w/v in normal saline, s.c.) into the sub-plantar tissue of the right hind paw. The paw thickness (mm) was measured using a vernier caliper at 0, 0.5, 1, 2, 3, 4 and 5 h after administration of the tested extract, fractions and standard drug. The percentage of inhibition of the rat paw edema was calculated as follows [16]:
$$ \%\mathrm{Inhibition}=\frac{\left({\mathrm{ET}}_{\mathrm{c}}-{\mathrm{ET}}_{\mathrm{t}}\right)}{{\mathrm{ET}}_{\mathrm{c}}}\kern0.5em \times \kern0.5em 100 $$
ETc is Paw edema thickness of control group and ETt is Paw edema thickness of treated group.
Analgesic activity
The total ethanolic extract and different fractions of A. hirtum leaves were evaluated for their analgesic activity using hot plate method [17]. Mice (30 ± 5 g) were grouped into seven groups (six animals each). The control group administered the vehicle (0.5% CMC solution) and the standard group administered 100 mg/kg, p.o. of acetylsalicylic acid. The tested extract and different fractions were suspended in 0.5% CMC solution and were administrated orally at a dose level of 300 mg/kg. The animals were placed on a hot plate and the temperature of the metal surface was maintained at 54 °C. The time (s) of the response produced by the animal as tail withdrawn, licking paws or jumping due to radiant heat is noted and recorded at 0, 0.5, 1, 2, 3, 4 and 5 h after the administration of the tested extract, fractions and the standard drug. The percentage of thermal pain stimulus protection was calculated according to the following formula [18]:
$$ \%\mathrm{Protection}\ \mathrm{against}\ \mathrm{thermal}\ \mathrm{stimulus}=\frac{\left({\mathrm{T}}_{\mathrm{t}}-{\mathrm{T}}_{\mathrm{c}}\right)}{{\mathrm{T}}_{\mathrm{c}}}\times 100 $$
Where Tt is the reaction time of treated group and Tc is the reaction time of control group.
Antipyretic activity
The total ethanolic extract and different fractions of A. hirtum leaves were tested for their antipyretic activity using yeast-induced pyrexia method [19, 20]. The test was performed on female albino rats (200 ± 50 g) by subcutaneous injection (in the back, below the nape of the neck) of 20% aqueous suspension of yeast in a dose of 10 ml/kg to induce pyrexia. The pyretic animals were grouped into seven groups (six animals each). The control group orally administered the vehicle (0.5% CMC solution), while the reference group was given acetylsalicylic acid at a dose level of 100 mg/kg, p.o. The tested extract and different fractions were suspended in 0.5% CMC solution and were administrated orally at a dose level of 300 mg/kg through 2 h after yeast injection. The rectal temperature of each animal was recorded by inserting a thermometer 2 cm into the rectum at 0, 0.5, 1, 2, 3, 4 and 5 h after administration of the tested extract, fractions and the reference drug.
Anti-diabetic activity
The antidiabetic activity of the total ethanolic extract and different fractions of A. hirtum leaves was evaluated using streptozotocin-induced hyperglycemia method [21, 22]. The test was performed on adult male albino rats (200 ± 50 g) by intraperitoneal injection of streptozotocin (80 mg/kg). Blood glucose level was measured after 3 days up to one week for assessment of hyperglycemia. Rats with blood glucose level above (200 mg/dl) were considered to be diabetic and were used in this study. The diabetic rats were divided into eight groups (six rats each). The control group was administered the vehicle (0.5% CMC solution), while the standard group was given of 150 mg/kg p.o metformin. The tested extract and different fractions were suspended in 0.5% CMC solution and were orally administrated at a dose level of 300 mg/kg. Blood glucose levels were measured at intervals of 0 (fasting), 0.5, 1, 2, 3, 4 and 5 h by collecting blood samples from the tail vein (caudal vein). The percentage of change in blood glucose level was calculated by the following formula [23, 24]:
$$ \%\mathrm{lowering}\ \mathrm{blood}\ \mathrm{glucose}\ \mathrm{level}=\frac{\left({\mathrm{W}}_{\mathrm{c}}-{\mathrm{W}}_{\mathrm{t}}\right)}{{\mathrm{W}}_{\mathrm{c}}} $$
Where Wt is the blood glucose concentrations of treated group and Wc is the blood glucose concentrations of control group.
Statistical analysis
Results of all biological studies were expressed as means ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett’s test was used to determine significance when compared to the control group. p values less than 0.05, 0.01, and 0.001 were considered significant (*p < 0.05, **p < 0.01,*** p < 0.001). Graph Pad Prism 5 was used for statistical calculations (Graph pad Software, San Diego California, USA).
