This experimental study was done in Khartoum state from September 2019 to January 2020. The study population were ten sickle cell anemia samples obtained from known diagnosed sickle cell disease patients (HBSS) admitted to Khartoum teaching hospital.
Inclusion and exclusion criteria
Samples were collected from SCD patient, including both genders with different age. Patients with other hereditary disorders and cancer were excluded.
Study design and plant materials
This experimental study was evaluated the anti sickling activity of Moringa Oleifera leaves and seeds extracts both aqueous and methanol fractions at a concentration of 20 mg/ml depending on previous study by Mpiana et al., [3] who worked at a concentration range between 0 and 10 mg/ml for the antisickling activity of anthocyanins from Ocimum basilicum.
Fresh leaves, seeds and flowers of Moringa Lam. were harvested from Moringa Oleifera tree in Khartoum state. The tree is identified by botanist, the leaves and seeds were dried at room temperature, milled and weighed. The powder was extracted by cold maceration in absolute methanol for 72 hrs after which it was filtered and evaporated to dryness in vacuo at 40 ºC. The crude extract obtained was further fractionated into methanol and water. All extracts were subjected to antisickling assays.
Preparation of Methanolic Extract
Extraction was carried out according to the method described by previously. Briefly, 100 gm of the plant sample was coarsely powdered using mortar and pestle. Coarsely a sample was soaking with absolute methanol. Extraction carried out for three days with daily filtration and evaporation the solvent under reduced pressure using rotary evaporator apparatus. Sample extract was allowed to air in evaporating dish till complete dryness and the yield percentages were calculated as follows:
About 4 ml EDTA blood sample was obtained from patients and centrifuged at 3000 rpm for 10 minutes to remove the plasma. The resulting packed erythrocytes was washed 3 times with 1 ml sterile normal saline per 5 ml of blood. The sample then was centrifuged each time to remove the supernatant. Washed RBC then re-suspended in remaining suspension and was used for the analysis.
Procedure for anti-sickling activity evaluation
Washed erythrocyte was mixed with an equivalent volume of 2% sodium metabisulfite (Na2O5S2). 10 µl from the above mixture was spotted on a microscope slide then 10 µl from each plant extracts (aqueous leaves extract, methanol leaves extract, aqueous seeds extract and methanol seeds extract), each one of them was added and mixed with the blood mixture. 10 µl normal saline was added to one of the slides instead of the plant extract which served as internal negative control; all the slides was covered with a cover slip. Paraffin was applied to seal the edges of the cover completely to exclude air (hypoxia), and then, slides were incubated at 37 °C for 2-period interval (immediately and 60 minutes). Each slide was examined under the oil immersion light microscope, and RBCs was counted in five different fields of view across the slide. The numbers of both sickled and unsickled blood cells was determined, and the percentage of unsickled cells was calculated.
Ethical considerations
Approval for this study was obtained from Ethical committee of Alzaeim Alazhari University and permission from the administrators of Khartoum teaching hospital. Research purpose and objectives were explained to participant in a clear simple words. Participant has right to voluntary, verbal informed consent. Data were obtained with privacy.
Statistical analysis
Data were analysed using Statistical package for the social sciences (SPSS) version 21.0 software (SPSS for Windows). Independent T test at 5% level of significant was performed to determine the means (Standard Deviation) of percentage of unsickled cells during two incubation period interval. The percentage of unsickle cells was calculated using the formula:
Percentage of unsickling cells = Number of unsickling cells × 100 / Total cells