Programme: Applied Biology

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MOLECULAR DOCKING, LIGAND QUALITY AND ANTIPLASMODIAL EVALUATION OF BENZAMIDE, COUMARIN AND BENZODIAZEPINE ANALOGS
(Covenant University Ota, 2025-04) ADEBAYO GLORY PIPELOLUWA; Covenant University Thesis
Malaria chemotherapy is an essential strategy for malaria elimination but resistance has challenged existing antimalarials, including frontline artemisinin combination therapy (ACT); hence, new antimalarial drugs must be discovered and developed. This study investigated the antiplasmodial efficacy and cytotoxicity through in vitro models while also testing the antiplasmodial efficacy, and the in vivo acute toxicity of benzamide, coumarin and benzodiazepine analogss. This study also evaluated the ligand quality of the molecules and their possible Plasmodium falciparum protein targets. Three molecules, 4- amino-N-hydroxybenzamide (AHB), ethyl 2-oxo-2H-chromene-3-carboxylate (ECC), and 2,2,4-trimethyl-2-3-dihyro-1H-benzo[b][1,4] diazepine (BDZ) were screened for their in vitro antiplasmodial activities tested against P. falciparum 3D7 standard strain using the SYBR Green Dye I measuring IC50 and their cytotoxicities against MCF-7 breast cancer cells using the [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Their antiplasmodial efficacies were determined using Peter’s 4-day suppressive test against Plasmodium berghei in M. musculus while acute toxicities were investigated in the Mus musculus (mice). Ligand qualities were determined using ligand efficiency metrics, and molecular docking was conducted to determine the ligand interactions between ECC and the following enzymatic proteins, P. falciparum dihydroorotate dehydrogenase (PfDHOH) and P. falciparum purine nucleoside phosphorylase (PfPNP); and the molecular interaction between BDZ and PfDXR - Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase, P. falciparum falcipain-2 and P. falciparum plasmepsin X (PfPMX). AHB showed no cytotoxicity against MCF-7 at (CC50) = 277.7 μM, while ECC showed inhibition with CC50= 3.930 μM, and BDZ showed no cytotoxicity CC50= 7373 μM. The in vitro antiplasmodial activity showed potency at (AHB)IC50 = 0.0020 ± 0.008 μM, (ECC) IC50= 0.0010 ± 0.002 μM, (BDZ) IC50= 0.0036 ± 0.003 μM respectively. BDZ showed the highest selectivity index at > 200,000, suggesting that it exhibited the best safety/efficacy among the three compounds. AHB displayed LD50 = >5000 mg/kg while ECC and BDZ displayed LD50 = 3162.28 mg/kg. Histopathological examinations showed non-toxicity by the three analogs on the liver and kidney of M. musculus. The percentage suppression of AHB (80.53 ± 3.26 %) at 400 mg/kg, was comparable to the standard chloroquine (81.71 ± 1.82 %) at 100 mg/kg where the mean survival time for both exceeded 30 days. ECC and BDZ showed excellent efficacies (70.98 ± 20.89 % and 83.66 ± 11.67 %) at 200 mg/kg, comparable to chloroquine 80.97 ± 5.82 %. The chemosuppression values for AHB and BDZ were significant at P value < 0.05. The ligand quality of ECC and BDZ displayed good Ligand Efficiency compared to chloroquine and artemisinin and higher enzyme affinities, and ligand efficiency dependent lipophilicity than the standard drugs. ECC and BDZ displayed good characteristics. The docking studies displayed strong hydrophobic interactions between ECC, PfDHODH, and PfPNP, suggesting good potency. BDZ’s binding with PfDXR, Pffalcipain-2, and PfPMX also displayed potency derived from hydrophobic and hydrogen interactions. Conclusively, this study showed AHB, ECC and BDZ were non-toxic to mammalian cells rodents’ liver and kidneys. These molecules exhibited good antiplasmodial inhibitory potential against both P. falciparum in vitro and P. berghei in vivo. ECC and BDZ displayed high ligand efficiency and strong molecular interactions with their protein targets. Therefore, all three analogs can be moved for further optimization in drug development.
FUNGAL-MEDIATED VANILLIC ACID PRODUCTION THROUGH BIOCONVERSION OF GALLIC ACID OBTAINED FROM RICE BRAN
(Covenant University Ota, 2025-08) OGUNBAJO, Oluwafemi Omolade; Covenant University Dissertation
Gallic and vanillic acids are naturally occurring phenolic compounds widely used in the pharmaceutical, food, and cosmetic industries due to their antioxidant properties. Rice bran is a nutrient-rich agro-industrial by-product. However, there are challenges of improper disposal and underutilization as a substrate for the production of valuable bioactive compounds. This study investigated the fungal-mediated bioconversion of rice bran into gallic acid under solid-state fermentation (SSF) and its subsequent transformation into vanillic acid through submerged fermentation (SMF) using Aspergillus niger. It was inoculated into a fermentation medium containing pre-treated rice bran and mineral salts and incubated for 5 days at 30 °C. Fermentation was monitored for pH, temperature, UV-Vis absorbance (276nm), and titration. The produced gallic acid was characterized using HPLC, GC-MS, and FTIR. Thereafter, 1% Methanol was introduced into a mineral salt medium containing the produced gallic acid as the substrate, and inoculated 1ml of the inoculum and incubated at 30 °C for 5 days. This was monitored and characterized as in the gallic acid production. During fermentation, the temperature ranged 28-33 °C, pH decreased from 6.5 to 5.2, the absorbance rose from 0.205 to 0.681 nm, and titration increased from 0.016 to 0.075 mol (p<0.05). HPLC quantified 6552.2 mg/L total phenolics, with gallic acid at 2569.8 mg/L. FTIR revealed gallic acid functional groups such as O–H, C=O, and C=C, while GC-MS identified volatile compounds including O-toluic acid, 2(1H)-naphthalene derivatives, and 3H-Cyclodeca[b]furan-2-one. For the vanillic acid production, fermentation filtrate temperature stayed at 29-30 oC, with the pH increasing in acidity from 6.5 to 4.3 through the 5-day period. This corresponded with the results of the titration, showing 0.016 to 0.079 mol results.The GC-MS showed volatile organic compounds present in vanillic acid, including protocatechuic, catechol, and syringic acids. HPLC quantified a total of 15,31203 mg/L of vanillic acid. FTIR revealed vanillic acid functional groups such as OCH₃, OH, and C=O. The results of this study provides strategic insights for sustainable bioprocess approaches in support of SDGs 3 and 12.
ANALYSIS OF PHYTATE CONTENT VARIABILITY AND MOLECULAR CHARACTERISATION OF GENES IN COWPEA LANDRACES
(Covenant University Ota, 2025-08) OMODIAGBE, David Eromosele; Covenant University Dissertation
Phytate is a naturally occurring phosphorus-storage compound in seeds, but it reduces the bioavailability of iron, zinc, and calcium in human diets. Cowpea (Vigna unguiculata (L.) Walp), an essential dietary protein source widely consumed in Nigeria, contains notable phytate levels, which may hinder micronutrient absorption with regular consumption. This study investigated phytate content in Nigerian cowpea accessions and explored candidate genes involved in its biosynthesis to guide future efforts for nutritional improvement. Thirty (30) cowpea landraces were obtained from the National Centre for Genetic Resources and Biotechnology (NACGRAB). Phytate concentration was quantified using acid digestion followed by filtration and titration. To explore the genetic basis of phytate accumulation, protein sequences of known phytate biosynthetic genes from Arabidopsis thaliana, Glycine max, and Phaseolus vulgaris were retrieved from Phytozome 14.0. These sequences were used for BLASTp searches to identify putative homologues in cowpea. Homologous cowpea sequences were aligned and subjected to phylogenetic analysis using the Neighbour-Joining method in MEGA v12.0.11 with 10,000 bootstrap replicates to assess evolutionary relationships. The results showed significant variation in phytate content, 3.96‒30.94mg/g (p < 0.001) across cowpea accessions. The cowpea sequences displayed strong homology to known phytate biosynthetic enzymes in seed, MIPS (Myo-inositol-3-phosphate synthase), ITPK (Inositol 1,3,4-triphosphate 5/6-kinase), IPK2 (Inositol 1,4,5-tris-phosphate kinase), MRP (Multidrug-resistance-associated protein ATP-binding cassette), IMP (Inositol monophosphate phosphatase), MIK (Myo-inositol kinase), and IPK1 (Inositol 1,3,4,5,6 pentakisphosphate 2-kinase), with low E-values and high identity percentages. Phylogenetic clustering of cowpea sequences alongside those from related legumes suggests potential functional conservation, although expression-level validation is necessary. Quantitative gene expression profiling to further clarify the roles of these genes in phytate biosynthesis and accumulation is needed. These identified genes could be prioritised for reverse genetics or transcriptomic studies, provided that tissue-specific expression and off-target impacts are carefully evaluated
EXPRESSION PROFILES OF CYTOCHROME P450 GENES ASSOCIATED WITH PERMETHRIN RESISTANCE IN Anopheles gambiae s.l. IN ADO-ODO OTA, OGUN STATE
(Covenant University Ota, 2025-10) AINA, Motunrayo Oluwabunmi; Covenant University Dissertation
Malaria remains a significant tropical public health threat, where resistance to insecticides constitutes a severe hindrance to the efficacy of its primary vector control methods. Routinely applied pyrethroid insecticides are increasingly facing resistance associated with the overexpression of cytochrome P450 genes in the Anopheles gambiae sensu lato, underscoring the urgent search into these associated genes. This study assessed the expression profile of cytochrome P450 genes associated with permethrin resistance in Anopheles gambiae sensu lato collected from three localities in Ado-Odo, Ota. Ethical approval was obtained from the Covenant Health Research Ethics Committee (CHREC). Based on the WHO standard, female adult An. gambiae larvae (n=300) were collected using the dipping method and reared into adults in the Insectary Laboratory. These laboratory-reared mosquitoes were phenotypically identified using microscopy and genotypically characterised using polymerase chain reaction (PCR) based on species-specific primers. Thereafter, a WHO susceptibility bioassay was conducted in vivo for mosquitocidal activity against these adult mosquitoes in four replicates at a 0.75% permethrin concentration each on day 3 post-adult emergence. The relative expression of the cytochrome P450 genes (CYP6M2 and CYP6P3) was carried out using the quantitative real-time PCR (qRT-PCR). Higher occurrence rate of An. gambiae sensu lato. was recorded in Nestle (80%)), Chelsea (78%)) compared Gasline (30%) localities of Ado-Odo. In vivo insecticide susceptibility testing revealed consistently low mortality rates across all the replicates, ranging from 20% to 32% indicating increased resistance to permethrin. Results of relative expression of cytochrome P450 genes showed higher fold changes in CYP6M2 ranging from 0.63 to 122.4 than in CYP6P3 0.63 to 34.39 across the tested mosquito replicates. Thus, this study has further emphasized the prevalence of An. gambiae sensu lato members in Ado-Odo, Ota. Additionally, the results of higher permethrin resistant and upregulation of CYP6M2 and CYP6P3 genes inform the imminent need for integrated resistance surveillance with newer vector management for improved malaria control.
TARGET-SITE MUTATION GENOTYPING OF INSECTICIDE-RESISTANT FEMALE Anopheles MOSQUITOES IN OTA
(Covenant University Ota, 2025-10) TAIWO, Damilare Isaiah; Covenant University Dissertation
Malaria control techniques in sub-Saharan Africa significantly depend on insecticide-based measures such as insecticide-treated nets (ITNs) and indoor residual spraying (IRS). However, the resistance emergence in Anopheles mosquitoes, driven by target-site mutations, undermines their effectiveness and threatens elimination efforts. Key resistance markers include knockdown resistance mutations (Kdr) in the voltage-gated sodium channel (VGSC) gene and the G119S mutation in the Ace-1 gene, both of which reduce insecticide efficacy. This study investigated the morphological and molecular composition of Anopheles populations, their insecticide resistance status, prevalence of Kdr and Ace-1 mutations, and assessed the genetic diversity, gene flow, and population structure across breeding sites in Ota, Ogun State, Nigeria. Using Coetzee’s key, a total of 478 adult female mosquitoes were obtained and morphologically identified, with molecular assays confirming a uniform species composition of Anopheles gambiae (390 bp) across all sites. Susceptibility assays revealed strong pyrethroid resistance, with permethrin mortality rates of 15–25%, far below the WHO threshold of 90%. However, bendiocarb produced 98–100% mortality, indicating high susceptibility. Genotypic analysis showed predominance of the Kdr-W (L1014F) allele, especially in Nestle and Iju, where homozygous resistant individuals were frequent. The Kdr-E (L1014S) allele was rare and largely confined to homozygous susceptible genotypes, suggesting it is not yet established. Deviations from the Hardy-Weinberg equilibrium were detected in Nestle populations, while sequence alignment confirmed L1014F mutations in Atan, Iju, and Chelsea. Population genetic analyses showed no strong subdivision (χ² = 0.7885, Fst = -1.3586, Gst = -0.0395, Kst = -0.13760), but high haplotype diversity (Hd = 0.94–1.00), moderate nucleotide diversity (π = 0.27–0.31), and substantial gene flow (Nm ≈ 8.24), supported by PCA and phylogenetic clustering. This study reveals widespread pyrethroid resistance but sustained carbamate susceptibility, providing the first genetic dataset from Ota to guide surveillance and resistance management strategies.