Experimental Animals:
Healthy Female Wistar rats weighing 150–200g were used. The rats were housed in groups, under controlled conditions of temperature (22 ± 1 °C) and relative humidity (~ 50 %) as well as 12-hour light / dark cycle. Animals were allowed food (standard laboratory rodent’s chow) and water ad libitum.
Plant material
Solenstemma argel was brought from Omdurman market, the plant then taxonomically authenticated at the herbarium of medical and aromatic plant research institute (MAPRI).
Preparation of the Solenstemma argel extract
Solenstemma argel plant sample was grounded using mortar and pestle and successively extracted by soaking 96 % ethanol for about seventy-two hours with daily filtration and evaporation. Solvents were evaporated under reduced pressure to dryness using rotary evaporator apparatus and the extracts were combined together. Extracts allowed to air till complete dryness(18).
Phytochemical screening tests:
Test for unsaturated Sterols and Triterpenes
Ten milliliters of Solenstemma argel extract were evaporated to dryness on a water bath and the cooled residue was stirred several times with petroleum ether to remove most of the coloring materials. The residue was then extracted with 20ml chloroform. The chloroform solution was dehydrated over Sodium Sulphate anhydrous. Portion of 5 ml of the chloroform solution was mixed with 0.5 ml of Acetic Anhydride followed by two drops of concentrated Sulphuric acid. The gradual appearance of green, blue pink to purple color was taken as an evidence of
the presence of sterols (green to blue) and or triterpenes (pink to purple) in the sample.
Test for Alkaloids
About 7.5ml of the Solenstemma argel extract were evaporated to dryness on a water bath. 5 ml of 2N HCl were added and stirred while heating on the water bath for 10 minutes, cooled, filtered and divided into two test tubes.
Few drops of Mayer’s reagent were added to one test tube while to the other tube few drops of Valser’s reagent were added. A slight turbidity or heavy precipitate in either of the two test tubes was taken as presumptive evidence for the presence of alkaloids.
Tests for Flavonoids
A volume of 17.5 ml of the Solenstemma argel extract was evaporated to dryness on water bath, cooled and the residue was defatted by several extractions with petroleum ether. The defatted residue was dissolved in 30 ml of 80% ethanol and filtered. The filtrate was then used for following tests:
To 3 ml of the filtrate, 1 ml of 1% Aluminum Chloride solution was in methanol was added. Formation of a yellow color indicated the presence of flavonoids, flavones or and chalcone.
To 3 ml of the filtrate, 1ml of 1% Potassium Hydroxide solution was added. A dark yellow color indicated the presence of flavonoids compounds (flavones or flavonenes), chalcone and or flavonols.
Tests for Tannins
Seven milliliters the Solenstemma argel extract were evaporated to dryness on water bath. The residue was extracted several times with N-hexane and filtered. The insoluble residue was stirred with 10 ml of hot saline solution. The mixture was cooled, filtered and the volume of the filtrate was adjusted to 10 ml with more saline solution. The solution was then used for following tests:
To 5 ml of the solution, few drops of gelatin salt reagent were added. Immediate formation of a precipitate was taken as an evidence for the presence of tannins in the plant sample.
To 5 ml of the solution, few drops of Ferric Chloride test reagent were added. The formation of blue, black or green was taken as an evidence for the presence of tannins.
Test for Saponins
One gram of the original dried powder Solenstemma argel leaves was placed in a clean test tube. 10 ml of distilled water were added and the tube was stoppered and vigorously shaken for about 30 seconds. The tube was then allowed to stand and observed for the formation of honeycomb. The appearance of honeycomb, which persisted at least for an hour, was taken as an evidence for the presence of Saponins.
Test for Anthraquinone glycosides
Ten grams of the powdered Solenstemma argel leaves were boiled with 10 ml of 0.5N KOH and 1 ml of 3% hydrogen peroxide solution. The mixture was extracted by shaking with 10 ml of benzene. 5 ml of the solution were shaken with 3ml of 10% Ammonium Hydroxide solution and the two layers were allowed to separate. The presence of Anthraquinone was indicated if the alkaline layer was found to have assumed pink or red color.
Test for Cumarins
Three grams of the original powdered plant sample were boiled with 20 ml distilled water in test tube. A filter paper spotted with 0.5 N KOH was attached to the test tube. The filter paper was allowed to be saturated with vapor and then inspected under UV light. Absorption of UV light indicate the presence of cumarins.
Induction of pregnancy
Adult female Wistar rats weighing 150–200g were allowed to mate with proven male rats in the ratio of 2 females to 1 male/cage. Vaginal smear for sperm detection was taken as evidence for copulation and that day was designated as day 1 of pregnancy. We adopted that method which described by Al-Harbi et al and (19) and ElTahir et al (20), with slight modifications.
Tests to investigate fetal abnormalities
Fetuses were removed on 21st day of pregnancy by caesarian section under light diethyl ether anesthesia. Litter size, alive and dead fetus number of resorption were recorded. Fetal and placental weights were also recorded.
The fetuses were examined for external gross malformation and histopathological examination of the liver.
Histopathological method
Slices of the liver were immediately fixed in 10% neutral buffered formalin, and then embedded in paraffin wax, sectioned at 5µm and stained routinely with hematoxylin and eosin (H&E).
Experimental design and treatment protocol:
The selected pregnant rats were divided into two groups of 7 animals each. The first group received normal saline and served as control. Rats in second group received Solenstemma argel extract intraperitoneal from day 7 to day 16 of pregnancy [ period of organogenesis in rats(20)].
Statistical work:
The results were analyzed using IBM SPSS Statistics version 24 and box plots using R-Statistics. Results summaries were presented as mean ± Standard Error of the Mean (S.E.M.). Differed inferential statistical tests (t-test, one-way ANOVA, Chi square test, etc.) were performed. Difference between groups was considered statistically significant at P-value < 0.05.