The bioactive materials were extracted from the powdered G. tournefortii using two extraction methods.

The Soxhlet apparatus was used to extract the oil fraction from the plant. Twenty grams of the powdered plant were extracted with 200 mL of 80% ethanol for 12 hours at 80°C. The solvent was removed using a rotary evaporator. The dry extract was resuspended in distal water and transported to a separatory funnel [14]. Then, an equal amount of petroleum ether was added and mixed well. The mixture was left at room temperature to stand until two distinct layers (aqueous and organic) were formed. The organic layer (petroleum ether) was collected, and the petroleum ether technique was repeated three times. The collected petroleum ether fraction was dried and stored in a glass container at 4°C.

The defatting procedure was conducted with petroleum ether. The marc was subjected to extraction with 250 cc of 80% methanol for 12 hours at 65°C using Soxhlet apparatus. A rotary evaporator removed the solvent. The resulting extract was preserved in a dark glass jar at 4°C [15]'

The phytochemical tests thoroughly examined the presence of triterpene, sterol, phenol, and flavonoids according to [16].

1. Tests for Sterol and Triterpene

a. Liberman Test

A few drops of acetic anhydride were added to the alcoholic extract and mixed thoroughly. Then, 1 ml of concentrated sulphuric acid was added slowly from the side of the test tube. The appearance of a reddish-brown ring indicated the presence of sterol.

b. Salkowski Test

A few drops of concentrated sulphuric acid were added to the alcoholic extract, shaken well, and allowed to stand until the appearance of a golden yellow indicated the presence of triterpenes.

2. Tests for Phenol and Flavonoids

Lead acetate, ferric chloride, and Shinoda tests: The Lead acetate test methodology included the addition of a few drops of aqueous basic lead acetate solution to the alcoholic extract; the emergence of a yellow precipitate signified the presence of flavonoids. A few drops of neutral ferric chloride solution were introduced to a tiny volume of alcoholic extract during the ferric chloride test. The emergence of a blackish-green hue indicated the existence of flavonoids. In the Shinoda test, a segment of magnesium ribbon and strong hydrochloric acid were introduced to the alcoholic solution extract. The emergence of red to pink hues after a few minutes indicated the presence of flavonoids.

3. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

The phytochemical screening by GC-MS was performed at Basra Oil Company Laboratory using an Agilent Technologies 7890B GC instrument paired with an Agilent Technologies 5977A MSD equipped with an EI signal detector. They utilized an HP-5ms 5% phenyl, 95% methyl siloxane (30m*250um*0.25) and precisely set the oven temperature at 40°C for 5 minutes. Then, ramped it up to 300°C at a consistent rate of 8°C/min for 20 minutes. The helium carrier gas flow rate was maintained at 1 ml/min, with a 3 ml/min purge flow. The injection mode was pulsed splitless with an injection temperature of 290°C. The injection sample volume was 0.5 microliter. The mass spectrometer used an Ion Source Temperature of 230°C, a scan speed of 1562(N2), and a mass range of 44-750 m/z. The data was run through the NIST 2014, 2020 Library database as an additional tool to confirm the identity of compounds [17].

4. Isolation of Beta-Sitosterol by HPLC

Using preparative high-performance liquid chromatography (HPLC) to separate the beta-sitosterol with mode SYKAM (Germany), C18 (250 mm x 4.6 mm, five μm particle size) column, using gradient mode with mobile phase including A = acetonitrile, B= 5 % acetic acid = (0 – 5 min) A: 30 %, (6 – 15 min) A = 50 %. Gradient elution was carried out for 20 minutes at a flow rate of 1 mL/min. The injection volume was 1mL, the detection was recorded with a UV detector at λ 220 nm, and the isolated constituent was recrystallized using hot methanol for purification[18].

5. Antileishmanial Activity

a. Preparation of Inoculum

Leishmania donovani and Leishmania tropica were obtained from the Center of Biological Technology Research at the College of Sciences at Al-Nahrain University. These were isolated from the bone marrow of an infected child and several confirmed cases, respectively, in Baghdad. The evaluation of the antileishmanial activity of the plant extract started by first propagating the organism. This was done by incubating them in Roswell Park Memorial Institute media (RPMI) enriched with 12% fetal calf serum at 25°C for 5 days to reach an average of 10^5 parasites/ml in a hemocytometer[19], [20].

b. Preparation of Plant Extract Concentrations

Several dilutions of the oil sample and methanolic sample (defatted) were made in dimethyl sulfoxide (DMSO) at the following concentrations: 1000, 900, 800, 700,600,500,400,300, and 200μg/ml to identify the antileishmanial activity. Furthermore, pentavalent antimonial (antileishmanial medicine) was used as a positive control [21].

c. Evaluation of the Samples’ Activity Against Leishmania Tropica and Leishmania Donovani

The antileishmanial activity was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [21]. 200µL of fresh promastigotes culture (10^5 cell/ml) was transformed to each well of a flat-bottom plate containing 96 wells, except four blank wells containing culture medium only. Subsequently, 200ul of each plant extract concentration (prepared previously) was added to all plate wells except the blanks. Whereas four wells contained culture only as a negative control, four wells contained sodium stibogluconate as a positive control, and the last four wells were blank. The plate was incubated at 25°C ± 1°C for 24 hours. Afterward, 10µL of MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well. The plate was incubated for an additional 4 hours at 25°C ± 1°C to assess metabolic activity. DMSO was added to each well as a solubilizing agent to dissolve the MTT purple dye inside the living matter, enabling the scanning process. An enzyme-linked immunosorbent assay (ELISA) reader at 490 nm was used to detect the optical density of each well. Finally, findings were compared with both positive and negative control. All tests were repeated in a tetraplicate. Calculating the IC50 The IC50 of each analytical sample was calculated using the following procedure: At each of the two locations, inhibition ratios (y) were plotted against sample concentrations (x). The associated regression line (y=ax+b) was generated. The IC50 value can be calculated using interpolation by connecting the two points surrounding the 50% inhibition point with a straight line. After selecting two points surrounding a 50% inhibition ratio, a regression line (y=ax+b) was created. The x (sample concentration) was determined by substituting 50 for y in the regression equation y=ax+b [22] [23]

d. Statistical Analysis

The % inhibition rate for each concentration gradient was computed, and a mean comparison was conducted to validate the significance of the results. A one-way ANOVA test was conducted using IBM software to compute the p-value and Least Significant Differences (LSD) to evaluate if the inhibition rate substantially varies from sodium stibogluconate.

1. Phytochemical Screening by Chemical Tests

The phytochemical contents were analyzed to assess the compound groups present in each extraction process, potentially linked to the observed bioactivity. All produced extracts, including oil and methanol extracts, exhibited the presence of numerous bioactive components, as detailed in Table 1. The Liberman test confirmed the presence of sterols by producing a reddish-brown ring upon the addition of a few drops of acetic anhydride and 1 ml of concentrated sulfuric acid. The Salkowski test indicated the presence of triterpenes by yielding a golden-yellow color after the addition of a few drops of concentrated sulfuric acid. The presence of phenols and flavonoids was established through Lead acetate, Ferric chloride, and Shinoda's tests.