Malaria is a deadly disease that continues to pose a threat to children and maternal well-being. This study was designed to identify the chemical constituents in the ethanolic fruit extract of Azadirachta indica, elucidate the pharmacological potentials of identified phytochemicals through the density functional theory method and carry out the antimalarial activity of extract using chemosuppression and curative models. The liquid chromatography-mass spectrometry (LC-MS) analysis of the ethanolic extract was carried out, followed by the density functional theory studies of the identified phytochemicals using B3LYP and 6-31G (d, p) basis set. The antimalarial assays were performed using the chemosuppression (4 days) and curative models. The LC-MS fingerprint of the extract led to the identification of desacetylnimbinolide, nimbidiol, O-methylazadironolide, nimbidic acid, and desfurano-6α-hydroxyazadiradione. Also, the frontier molecular orbital properties, molecular electrostatic potential, and dipole moment studies revealed the identified phytochemicals as possible antimalarial agents. The ethanolic extract of A indica fruit gave 83% suppression at 800 mg/kg, while 84% parasitaemia clearance was obtained in the curative study. The study provided information about the phytochemicals and background pharmacological evidences of the antimalarial ethnomedicinal claim of A indica fruit. Thus, isolation and structure elucidation of the identified phytochemicals from the active ethanolic extract and extensive antimalarial studies towards the discovery of new therapeutic agents is recommended for further studies.
The fruit of A indica (Meliaceae) was collected within Obafemi Awolowo University (OAU) campus. The fruit was identified, authenticated, and deposited at the Faculty of Pharmacy Herbarium, Ife by Mr. I. I. Ogunlowo of the Pharmacognosy Department, OAU, Ile-Ife, with voucher specimen number FPI 2423. The fruits were air-dried, powdered, and 500 g of the dried powder was macerated in 1500 mL ethanol for 48 hours with intermittent shaking. The resultant extract (17 g) was filtered, evaporated in vacuo, freeze-dried, weighed, and stored. A linear trap quadrupole (LTQ) Orbitrap spectrometer (Thermo Scientific, USA) was used to carry out liquid chromatography-mass spectrometry (LC-MS) analysis. The instrument is equipped with an Agilent 1200 HPLC system (Santa Clara, CA, USA) and connected to a photodiode array (PDA) detector. Sample preparation was done by making the fruit extract into a final concentration of 2 mg/mL in methanol and was centrifuged for 5 min at 6600 r/min and loaded for analysis. A reverse phase Luna C18 column (60 × 3 mm, 3 μm) (Phenomenex, Torrance CA, USA), was used to carry out high-performance liquid chromatography (HPLC) analysis of the sample. The mobile phase consists of water (+0.1% formic acid) A and methanol (+0.1% formic acid) B at a flow rate of 360 μL/min. The gradient was configured to be a linear gradient from 96% A to 100% B over 14 minutes, followed by 100% B for 4 minutes, then a return to the initial concentration of 96% A in 0.6 minutes, and allowed to equilibrate for 4.6 minutes. The column oven condition was kept at 30°C, and the injection volume was 6 μL. Spectrometry analysis was carried out in positive mode with a nominal mass resolving power of 60 000 at 400 m/z, spray voltage of 6 kV, and a scan rate of 1 Hz. The spectrometer was run with a capillary temperature of (300°C), a tube lens of 100 V, collision gases were argon and nitrogen as sheath gas (66 arbitrary units) and auxiliary gas (8 arbitrary units), respectively.7,23 Xcalibur software 2.2.48 was used for data analysis. Compounds were proposed by comparison of acquired MS data with literature. Density functional theory analysis of phytochemicals identified from the ethanolic extract of A indica fruit was performed using the Spartan 14 programme containing functional B3LYP (Lee-Yang Parr exchange-correlation functional method). Also, a 6-31G basis set was chosen for the DFT study.24 During the calculations, the values of the frontier orbital energies were computed from the most established conformation of the compounds using the following formulas: Seven-week-old Swiss albino mice of either sex weighing between 18 and 24 g (male and female, not pregnant) were obtained from the Animal House, Faculty of Basic Medical Sciences, College of Health Sciences, OAU, Ile-Ife, Nigeria, where they were housed in aluminium cages with wood shavings used as beddings and allowed free access to water and food (Growers’ mash) under 12-hour day/night cycle. The animal experimental methodology was approved by the Health Research and Ethics Committee of the Institute of Public Health, OAU, Ile-Ife, Nigeria. They were also handled in accordance with the National Institutes of Health (NIH) Guide for the care and use of laboratory animals (NIH Publication, No. 83-123 (revised), 1985). They were acclimatized for at least 7 days before use and randomly divided into groups of 5 mice each for the experiments. Plasmodium berghei strain NK65 sensitive to chloroquine (CQ), obtained from Professor O G Ademowo of the Institute of Advanced Medical Research and Training (IMRAT), University College Hospital, Ibadan, was used to assess the in vivo chemo-suppressive and curative antimalarial activity. The parasite strain was preserved via serial passage of blood taken from an infected mouse into an uninfected mouse. The donor mouse was sacrificed, and blood was withdrawn through cardiac puncture into a heparinized bottle to prepare the inoculum. It was diluted with normal saline solution so that 0.2 mL of the inoculum will contain 1.0 × 107 parasitized red blood cells. The chemosuppressive and the curative activities were performed by oral administration of the extract (100, 200, 400, and 800 mg/kg), CQ (10 mg/kg), and normal saline to groups of 5 mice each, 2 hours after infection and thereafter daily for 3 days in the chemosuppressive model, while in the curative model, the administration was done daily for 5 days starting from the third day after infection.25,26 The temperature of each mouse was taken using a digital clinical thermometer inserted into the rectum before the administration of the extracts or drugs. The level of parasitaemia was determined for each mouse on Day 4 (D4) and daily after infection for the chemosuppressive and curative models, respectively, by cell counting of 5 fields in a view of the microscope of a thin blood smear, fixed with methanol and stained with Giemsa, obtained from the tail of each mouse.25,26 The average parasitaemia in each group was calculated to determine the percentage chemo-suppressive and curative activities of the extract using the following formula: where A and B are the mean parasitaemia in the negative control and the test groups, respectively.27 The extract’s antimalarial chemo-suppressive activity was determined by the percentage reduction of parasitaemia in treated groups compared with the untreated infected group. The animals were further observed for 28 days for mortality while survival times and percentage of survivors were estimated.25,28 The percentage of survival time was calculated for each group by using the following formula: Values were expressed as mean ± standard error of the mean (SEM) and analysed statistically using 1-way analysis of variance (ANOVA) followed by Student-Newmann-Keuls’ post hoc for comparison to determine the source of significant difference for all values. Values of P < .05 were of statistical significance.
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