Pharmacological activity of ethanolic extract of Alhagi maurorum roots

1.1 Instrumentation

The plant roots were powdered on Retsch GmbH mill model 5657 HAAN.

Filtrations were done using Spectrum quantitative laboratory filter papers from Curtin Scientific Company, Huston, USA.

Centrifugations of the aqueous solutions were done on ECCD centrifugation machine.

Freeze dryings were done on Freeze-dryer Mobile 12 SL (The Virtis Company Gardiner, NY, USA.

2.1 Plant material

The plant was collected from Riyadh region in Saudi Arabia in December 2008, and identified by Department of Botany and Microbiology, Faculty of Science; King Saud University, Riyadh. The roots were taken out, cleaned and dried in the shade for 2 weeks and then powdered.

2.2 Preparation of the extracts for biological activity tests

Of 100 g powdered roots were extracted with 500 ml of absolute ethanol under reflux for 2 h. The solution was filtered and the solvent was removed under vacuum distillation to give 3 g (3%) of reddish precipitate.

2.3 Standard procedures for biological activity tests of the extracts

The effects of the extract on mice rectal temperature were studied following the method described by Gray et al. (1987).

The effects of the extract on the rectus abdominis muscle were studied following the method described by Fleisher et al. (1960).

The effects of the extract on the heart using the ECG (electro-cardio gram) were studied following the method described by Bakheet et al. (1999). The effects of the extract on the guinea-pig ureter were studied using the method described by Washizn (1968).

2.4 Preparation of test samples

The extract was used in form of suspension in 0.25% aqueous sodium carboxy methyl cellulose. After suspension, the mixture was emulsified by vortexing (shaking). A glass rode is usually inserted inside a tube containing the suspension to facilitate emulsification during vortexing in the vortex mixer.

2.5 Biological activity tests

1. Effect on rectal temperature: Rectal temperature of mice was measured using an Aplex rectal thermometer (Haly) fitted with thermistor probes. The probe was inserted to a depth of 2.5 cm into the rectum of each mouse and temperature reading was allowed to stabilize, recorded and the probe then removed. The temperature was taken first after 15 min, then at intervals of 30 min.

2. Effect on the rectus abdominis muscle of the frog: Initially, each frog was decapitated and then pithed by inserting a needle into the spinal cord. The frog was then fixed to a wooden board with its abdomen facing upwards. The outer skin of the frog was cut longitudinally and horizontally to expose the abdominal wall. The two rectus abdominis muscles were located and the two muscles were cut and removed completely from the animal and placed in Krebs’ solution. Then each muscle was suspended vertically in the chamber of an organ bath containing Krebs’ solution and aerated using (95% O₂ + 5% CO₂). The temperature of the bathing fluid was adjusted to 37 °C. Each tissue was allowed to equilibrate with the bathing fluid for 30 min. Each dose of the drug was allowed to contact the tissue for a time to obtain the maximum response. The extract was allowed to contact the tissue for 5 min before addition of the nicotine agonist. The percentage inhibition induced by the extract on the agonist submaxional dose was then calculated.

3. Effect on the electro-cardio gram (ECG) of rats: Male Wister rats (250 g) were anesthetized with urethane and prepared for measurement of the ECG waves. The limbs of the animal were fastened to a dissection board with the animal lying on its back. The ECG lead II was recorded by the aid of subcutaneous needle electrodes and the record was displayed in chart of the instrument. The recording speed was adjusted to 25 mm/s. The extract was injected intraperitoneally and the effect was then followed for 20 min to observe any disturbances in the cardiac rhythm. The heart rate before and after the extract injection was recorded and the percentage changes were then calculated.

4. Effect on the ureter: Guinea-pigs were killed by blows on the neck, and the abdominal cavity of each animal was opened. The kidney and the ureters were located and the whole length of the ureter (from the kidney to the urinary bladder) was cut and placed in Krebs’ solution. The ureter was then prepared for the study of the effect of the extract. Of 2 cm length of each ureter was suspended in an organ bath containing oxygenated (95% O₂ + 5% CO₂) Krebs’ solution. Each tissue was connected to an isometric transducer connected to a physiograph (Narco Biosystems, USA). Each tissue was allowed to equilibrate with the bathing fluid for 30 min. Initially the effects of different doses of the extract were determined. The effect of the extract on the standard agonists (histamine, acetyl choline (ACh) were then determined. The extract was allowed to contact the tissue for 5 min before the addition of the agonist. The percentage inhibition induced by the extract on the selected dose of the agonist was then calculated.

3.1 General screening of the extract in conscious mice

Intraperitoneal administration of the extract into conscious mice in doses of 1.6 g/kg produced mild sedation. The extract also decreased the locomotion activity of the animals and skeletal muscle relaxation suggesting an action at the skeletal muscles neuromuscular junctions. The extract also decreased the rectal temperature of the animals 3.2 °C. These results directed the attention to study the effects of the extract on the rectal body temperature of the mice and on the skeletal muscle of the frog. The extract produced noticeable changes in the heart rate and this directed the attention to study its effects of the heart rate via registering the electro-cardio gram (ECG).

3.2 Effect on mice rectal temperature

Administration of the extract in doses of 0.25 and 0.5 g/kg (I.P.) into mice did not induce any changes in the rectal temperature. However, administration of the extract in doses of 1 g/kg (I.P.) decreased the body temperature with a maximum of 3.3 °C 60 min after administration of the extract. Thereafter the temperature started to rise again. The cumulative results are shown in Table 1.

3.3 Effect on the frogs rectus abdominis muscle (skeletal muscle)

Exposure of the muscle to the extract in concentration of 1, 2, 4, 10 or 25 mg/ml of bathing fluid did not induce any contraction.

3.4 Effect on ACh-induced contraction of the frogs rectus abdominis muscle

1. Exposure of the frog’s rectus abdominis muscle to the nicotinic receptor stimulant acetyl choline (ACh) in concentration of 1–4 μg/ml induced concentration-dependent contractions.

2. Exposure of the tissue to the extract in a concentration of 4 μg/ml bathing fluid for 5 min antagonise ACh (3 μg/ml)-induced contraction by 70 ± 2.1% (N = 4). When the dose of ACh was increased up to 8 μg/ml in presence of the extract blockade, it did not reverse completely the blockade. The maximum reversal of antagonism was 27.7, suggesting that the extract blocked the action of ACh in a non-competitive manner.

3.5 Effect on the rat heart

When rats were anaesthetized with urethane and the ECG was monitored to investigate the effects of the extract on the heart rate and force of contraction, the results revealed that the extract at a dose of 1 g/kg induced bradycardia only and not myocardial depressant. The results are shown in Table 2.

3.6 Effect on calcium oxalate solubility

When 1 g calcium oxalate was added to 5 ml of 20% solution of the extract and the mixture was mixed and left to stand for 3 days, there was no solubilization of the calcium oxalate crystal. The weight of undissolved calcium oxalate was not changed compared with the initial weight.

3.7 Effect on the guinea-pig ureter

Addition of histamine in doses of 3 μg/ml bathing fluid to the isolated guinea-pig ureter induced continuous contractions. Addition of the extract in doses of 5 mg/ml bathing fluid completely suppressed histamine induced contractions. Addition of another dose of histamine did not reverse the inhibition.