Sodium Salicylate

Plant material The fruits of Astragalus hamosus were purchased from a local medicinal plant shop in Tehran and identified by M.Kamalinejed (School of Pharmacy, Shahid Beheshti University of medical sciences, Tehran, Iran), the voucher specimen (8005) was deposited in School of Pharmacy.
Preparation of extract and fractionsFor preparation of the hydro-alcoholic extract, 300 g of dried and grinded pods of Astragalus hamosus were macerated in ethanol 70% for three times (each time 24 h). The extract was then filtered and concentrated with vacuum evaporator and the percentage yield was 13%.
To yield different fractions, dried hydro alcoholic extract was suspended in water and partitioned by hexane, chloroform and ethyl acetate (each solvent in duplicate). Each fraction was evaporated to obtain hexane fraction (42 g), chloroform fraction (10 g), ethyl acetate fraction (4 g) and water fraction (25 g) which were used for bioassay.
Phytochemical screeningPhytochemical investigations of the HAAH were carried out using standard methods and tests (11 (link)-13 ). The test for tannins was carried out by subjecting 1 g of extract in 2 mL of distilled water, filtered and ferric chloride reagents were added to the filtrate. The extract was subjected to frothing test for the identification of saponins and to Fehling's test for glycosides. Alkaloids were detected in the alkaloid fraction obtained by a classical acid: base extraction procedure for alkaloids and analyzed by TLC in chloroform: methanol: ammonia solution 25% 8:2:0.5 as solvent system, spots were detected after spraying with Dragendorff’s reagent. The presence of flavonoids was determined using 1% aluminum chloride solution to the extract and yellow coloration. Another test for flavonoid, dilute ammonia (5 mL) was added to the extract and then concentrated sulphuric acid (1 mL) was added. Steroids were detected by adding 1 mL of acetic anhydride to 0.25 g methanolic extract of each sample with 1 mL H₂SO₄. The color changed from violet to blue or green indicating the presence of steroids. The test for anthraquinones was performed with 0.5 g of extract boiled with 10 mL sulphuric acid and filtered. Then filtrate was shaken with 5 mL CHCl₃ and CHCl₃ layer was removed to another tube and 1 mL of ammonia was added and colour change was observed. Detection of terpenoids (triterpenoids) was carried out by adding 2 mL of CHCl₃ to 0.5 g of extract and then adding carefully concentrated H₂SO₄ (3 mL) to form a layer and reddish to brown color in interface.
Animals42 adult male wistar rats (150-200 g) and 77 adult male albino mice (25-35 g) were housed in animal unit of Iran University of Medical Sciences under standard laboratory conditions (temperature 23 ± 2 ˚C) with 12 h dark and 12 h light cycle. The animals had free access to standard dry pellet diet and tap water ad libitum.
Anti-inflammatory activity Formalin induced rat paw edemaThe test was carried out using the method described by Hunskaar and Hole (14 (link)).Initially, 42 adult wistar rats were divided into 6 groups. The animals in each group were treated with HAAH at doses of 100, 300, 700 and 1000 mg/Kg i.p. and 300 mg/Kg sodium salicylate as positive control group and 1 mL distilled water as negative control group according to our previous studies. Then the rat paw edema induced by injection of 50 µL of 2.5% formalin (in normal saline 0.9%) into sub-planar tissue of the paws of rats and paw volumes were measured by plethysmometer during 8 days after formalin injection. Animals were treated with plant extract and drugs every day. Then the percentage inhibition of edema was calculated by the following formula:
Percentage Inhibition= ((Vt-V0)/V0) ×100
Vt= volume of animals’ paw after injection
V0=volume of animals’ paw before injection
Analgesic activityAcetic acid-induced writhing testThe acetic-acid writhing test was performed using the reported procedure (15 ).
Groups of rats (n=7), were administered with100, 300, 700, 1000 mg/Kg of HAAH i.p., 300 mg/Kg sodium salicylate as positive control group and 1 mL distilled water as negative control group. After 30 minutes the animals were administered with i.p. injection of 0.1 mL acetic acid (0.6%). Then the count of abdominal contractions of animals during 30 minutes after acetic acid injection was reported and the Percentage Analgesic Activity (PAA) was calculated by using the following formula:
PAA = ((C- C_D)/C_D) ×100
C = Mean of contractions’ count in animals treated with different doses of Astragalushamosus extract and sodium salicylate
C_D = Mean of contractions’ count in animals served as negative control
Hot plate testThe Hot plate test was performed using the reported procedure (1 (link)).
Anti-nociceptive effect of HAAH and its fractions was investigated using hot plate test in seventy-seven adult male albino mice. The same procedure was applied to the animals of each group and the latency time was measured and compared to control group. The Percentage Analgesic Activity (PAA) was calculated by using the following formula:
PAA= ((L_a-L_b)/L_b) ×100
L_a =Latency time after treatment with drug or extract
L_b=Latency time before treatment with drug or extract
The analgesic effects of different fractions of HAAH were also evaluated with the same procedure. Morphine used as positive control.
Statistical analysisThe results are reported as mean ± S.E.M. The statistical analyses were performed using one way analysis of variance (ANOVA). Group differences were calculated by post hoc analysis using Tukey’s test. For all tests, differences with values of P<0.05 were considered significant.

For the determination of ammonia formation, 100 μL of the complex dissolved in acetonitrile (2.8 mM) and 900 μL of urea solution (60 mM) were mixed in a 5 mL flask with stirring. The reaction was kept at 20 °C. Aliquots of 100 μL of the reaction were taken at different reaction times (10, 30, 60, 120, 120, 480, and 600 s) and were mixed with 250 μL of hypochlorite solution (2.5%) and 250 μL of sodium citrate/NaOH solution (0.38 M and 0.46 M). After mixing this solution, 300μL of sodium salicylate (2.75 M)/sodium nitroprussiate (9.4 μM) solution was added. After incubation of the solution at 20 °C for 15 minutes, the solution was analyzed by UV-Vis spectroscopy, monitoring the band absorption at 650 nm. Quantification was performed by means of a calibration curve with seven points (R² = 0.99). Control reactions without the catalysts were also performed. All analyses were performed in triplicate.

Table 6 lists the chemicals used in this work: sodium salicylate (Panreac, mass fraction purity > 0.99; molar mass = 160.11 g mol⁻¹), β-cyclodextrin (mass fraction purity > 0.97; molar mass = 1134.98 g mol⁻¹), and tetrasodium 5,11,17,23-tetrakissulfonatemethylene-2,8,14,20-tetra(ethyl)resorcin[4]arene (Na₄EtRA). This last compound was synthesized according to what was reported in the literature [24 (link)]. All these chemicals were used without further purification, but they were stored under low pressure in a desiccator over silica gel.
All solutions were freshly prepared by using Millipore Milli-Q water (specific resistance = 18.2 MΩ cm, at 298.15 K). For diffusion measurements, these were prepared (at 25 °C) in calibrated volumetric flasks. For the other measurements, they were prepared by the direct weighing of both solute and solvent. To determine its concentration, the water content of the corresponding chemicals was taken into account. Before each experiment, solutions were de-aerated for 30 min, approximately.