http://hdl.handle.net/123456789/19 Pharmacy 2026-03-08T23:14:07Z http://hdl.handle.net/123456789/2123 BIOASSAY-GUIDED ISOLATION, CHARACTERISATION AND CYTOTOXICITY OF MOSQUITO REPELLENT COMPOUNDS FROM SELECTED MEDICINAL PLANTS ADENIYI-AKEE, MUKARAM AKINTUNDE Malaria, a tropical disease caused by Plasmodium parasites and spread by the bites of infected female Anopheles mosquitoes, remains a major public health issue. Mosquito repellents are used as a control measure.Existing synthetic repellents present challengesof high insecticide resistance, allergic reactions, and non-biodegradable residues. This has necessitated the search for alternative repellent compounds from natural sources.This study was designed to evaluate the repellent activity of selected ethnomedicinal plants against wild adult female Anopheles gambiae (s.l.), and characterise isolated bioactive compounds. Dry powdered plant parts of Citrus limon Linn. (seeds), Citrus paradisi Macf. (seeds), Citrus sinensisL.(seeds), Jatropha curcas L.(seeds), Dennettia tripetala G. (fruits), and Afromomum melegueta K. (fruits) were successively extracted with n-hexane (Hex), ethyl acetate (EA) and methanol by cold maceration. Phytochemical screening of the extracts was done using standard methods. The extracts were screened against A. gambiae for their repellent activity using human bait method, whileN,N-diethyl-meta-toluamide(DEET) and acetone were used as positive and negative controls, respectively. Extracts with the highest percentage repellency, calculated using standard formula, were subjected to bioassay-guided fractionation and separation using chromatographic techniques. Isolated compounds were evaluated for cytotoxicity using brine shrimp lethality assay (safety cutoff, LC50> 100µg/mL), and characterised by spectroscopic (IR, 1D and 2D NMR) and mass spectrometric analyses. Repellent activity data were analysed using one-way ANOVA at α0.05. Extraction of the plant materials yielded eighteen extracts. Phytochemical screening of the extracts revealed the presence of alkaloids, flavonoids, tannins, glycoside, saponins, and anthraquinones. The screened extracts, tested at 1.5, 2.5, and 5mg/mL, gave percentage repellency of ≤ 68.1, ≤ 74.9, and ≤ 97.5%, respectively (DEET, 100%). At 5 mg/mL, the percentage repellency of the three most active extracts (C. limon (Hex, 97.5%), D. tripetala (EA, 87.7%), and C. limon (methanol, 86.8%) were not significantly different (α˂0.05). Citrus limon (Hex) fractionation yielded 10 fractions with percentage repellency of ≤ 89.5 %, whileD. tripetala (EA) yielded eleven fractions which were pooled into four (DTH1, 55.7%; DTH2, 60.6%; DTEA, 77.2%, and DTM, 71.1%). n-Hexane-100% and Hex/EA (9:1) fractions of C. limon, DTEA and DTM fractions of D. tripetala showed the most repellency effects of 89.5%, 71.1%, 77.2% and 71.1%, respectively. Chromatographic purification of n-hexane-100%, Hex/EA (9:1) and DTEA gave seven compounds. They were identified as palmitic acid (a), 14-oxotricosanoic acid (b),n-octyl stearate(c), 15-(heptanoyloxy) pentadec-9-enoic acid (d), 6,8-dimethoxy-3-undecyl-1H- [2]benzopyran-1-one (e), 1,2,3-propanetriyl tris(5-eicosenoate) (f), and α-linoleic acid (g) with percentage repellency of 65.9, 63.6, 51.7, 77.9, 75.2, 37.0, and 57.3%, respectively. Cytotoxicity evaluation of compounds a-e gaveLC50ranging from 63.2 to 171.1 µg/mL, with compounds c (106.1 µg/mL) and e (171.1 µg/mL) being adjudged safe. Compoundd showed the highest repellent activity against A. gambiae mosquito. Extracts of Citrus limon and Dennettia tripetalacontain potentially useful mosquito repellent compounds. n-Octyl stearate and 6,8-dimethoxy-3-undecyl-1H-[2]benzopyran-1- one (both from C. limon) may serve as leads for the development of novel bio-degradable and safe mosquito repellent compounds. 2023-01-01T00:00:00Z http://hdl.handle.net/123456789/1584 ISOLATION OF BIOACTIVE COMPOUNDS FROM SELECTED NIGERIAN MEDICINAL PLANTS FOR MANAGEMENT OF LETROZOLE-INDUCED POLYCYSTIC OVARIAN SYNDROME IN RATS OGUNLAKIN, AKINGBOLABO DANIEL Polycystic Ovary Syndrome (PCOS) is an endocrine disorder with a global prevalence of 5-10% among women of reproductive age. Women with PCOS have a 2.7-fold increased risk of developing ovarian and cervical cancers. Ethnobotanical survey revealed that plants including Kigelia africana (Lam.) Benth. (KA), Basella alba L. (BA), Tetracera potatoria G. Don (TP) and Mormodica charantia L. (MC) are used for treatment of PCOS in southwestern Nigeria. However, there is no reported scientific evidence to validate these claims. This study was, therefore, designed to investigate the effect of the four plants in alleviating polycystic ovary conditions in rats and the associated risk of ovarian and cervical cancers. Letrozole (1 mg/kg) was used for the induction of PCOS in thirty female albino rats (180-200 g, n=5). Methanol leaf extracts of KA, BA, TP and MC (100 mg/kg b.w.) and Clomiphene citrate (1 mg/kg b.w., standard drug) were administered for 15 days. Histopathological evaluation of the ovaries was done microscopically. Luteinizing hormone (LH), follicle stimulating hormone (FSH) and estradiol were measured using ELISA. Extracts of the most active plants, KA (FHI-111350) and TP (IFE-17794) were successively partitioned into n-hexane, dichloromethane and ethyl acetate fractions, and screened for PCOS inhibitory activity. Compounds were isolated from the active fractions using chromatographic techniques. Structures of isolated compounds were elucidated using spectroscopic techniques. Anti-proliferative effect of fractions, isolated compounds and derivatised cinnamic acid analogues were determined on cervix adenocarcinoma (HeLa) and Chinese Hamster ovarian (CHO 1) cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Doxorubicin was used as standard. Data were analysed using one-way ANOVA followed by Student’s t-test at α0.05. Animals treated with KA showed normal ovarian stroma with moderate fibroblastic tissues. The levels of FSH in KA, BA, TP and MC treated groups were 0.96±0.08, 1.10±0.23, 0.81±0.04 and 0.69±0.01 mIU/mL, respectively compared to control group (0.93±0.19 mIU/mL). The TP (0.19±0.05 mIU/mL) significantly reduced the level of LH compared with the control group (0.22±0.0l mIU/mL). Hexane and ethyl acetate fractions displayed selective moderate inhibitory effect (IC50 = 5.3±1.10 µg/mL) on CHO 1 cell line compared to doxorubicin (IC50 = 0.8±0.01 µg/mL). Compounds 3-(3, 4-dimethoxyphenyl) acrylic acid (1), sitosterol (2), methyl 3-(3,4- dihydroxyphenyl) acrylate (3), 2, 3-dihydro-5-(hydroxymethyl) furan-2, 3, 4-triol (4), p-coumaric acid (5) and caffeic acid (6) were isolated from KA, while apigenin (7) was isolated from TP. iii Compound 1 displayed moderate anti-proliferative activity against HeLa cell line (IC50 = 33.5±0.60 µg/mL). Compound 7 inhibited proliferation of HeLa cell line (IC50 = 6.2 µg/mL) and had moderate inhibitory effect on CHO l cell line (IC50 = 22.2 µg/mL). Derivatised compound N'-(2, 6- dimethoxybenzylidene)-3-(4-methoxyphenyl) acrylohydrazide (8) inhibited both CHO l (IC50 = l8.4±4.l µg/mL) and HeLa (IC50 = 22.4±0.4 µg/mL) cells. The extracts of Kigelia africana and Tetracera potatoria had inhibitory effects on polycystic ovary condition, irregular oestrual cycle and hormonal imbalance in female albino rats. The phenylpropanoids and derivatised analogues exhibited antiproliferative effect on ovarian and cervical cancer cell lines, which could contribute to their use for management of polycystic ovary syndrome. 2021-02-01T00:00:00Z http://hdl.handle.net/123456789/1582 MOLECULAR AND BIOLOGICAL STANDARDISATION OF Alstonia boonei DE WILD. AND Alstonia congensis ENGL. LEAVES AND STEM-BARKS OMITOLA, Opeyemi Josephine Alstonia boonei (AB) and Alstonia congensis (AC), belonging to the family Apocynaceae are different but closely related species, indigenous to Africa. The two plants possess varied biological activities including antimalarial, antihypertensive and antidiarrhoeal. However, the apparent similarities between the two species could lead to misidentification and inappropriate medicinal utilisation, necessitating the need to establish diagnostic characters for each species for proper identification. This study, was therefore, aimed at evaluating molecular, pharmacognostic, antispasmodic and antidiarrhoeal profiles of the two plants for their pharmacopoeial standardisation. Deoxyribonucleic acid was isolated from nine accessions of AB and AC leaves (ABL and ACL) collected from southwestern Nigeria and amplified, using Internal Transcribed Spacer (ITS) region. Micromorphological diagnostic characters of leaves and stem-barks were studied by light microscopy. Physico-chemical and elemental analysis of the powdered samples were determined using incineration method and Atomic Absorption Spectroscopy, respectively. Aqueous extraction of plant samples was done, extracts were concentrated in vacuo and freeze dried. The freeze-dried extracts were partitioned successively into dichloromethane (DCM), ethyl acetate and aqueous fractions. Antispasmodic activities of the extracts and fractions were evaluated on high-potassium induced and spontaneous contractions on isolated rat ileum. The AB and AC stem-barks (ABSb, ACSb) extracts and their dichloromethane fractions (DCM-ABSb, DCM ACSb) showing high antispasmodic activity were evaluated for in vivo antidiarrhoeal activities in mice (22- 25 g b.w). Loperamide (5mg/kg) was used as standard. Compounds were isolated from the most active fraction (DCM-ABSb) using chromatographic techniques (Column, TLC). Structures of the isolated compounds were elucidated using spectroscopic techniques (NMR and MS) and their antispasmodic activities evaluated. Data were analysed using descriptive statistics, unweighted pair group method with arithmetic mean and one-way ANOVA and Tukey’s multiple comparison at α0.05. The amplification of the ITS region discriminated between the two Alstonia species. The adaxial epidermal cells of both species were polygonal, straight anticlinal walls and leaves were hypostomatic. The vascular bundle of ABL mid-rib was arc-shaped, while that of ACL was V-shaped. Moisture content values of stem bark of AB (6.4±0.06%) was significantly different from that of AC (7.2±0.4%), while the values were not significantly different for the leaves. Elemental analysis revealed that calcium was the most abundant mineral in the leaves of AB (68.2±3.2 mg/g) and AC (65.7 ±1.0 mg/g). The stem-bark extracts of AB and AC were antispasmodic with IC50 of 0.03±0.2, 0.12±0.01 mg/mL and 1.15±0.1, 1.05±0.8 mg/mL on high-potassium induced and spontaneous contractions, respectively. The DCM-ABSb fraction exhibited significant antispasmodic activity (IC50: 0.02±0.05, 0.31±0.02 mg/mL) on high-potassium induced and spontaneous contractions, respectively. The DCM-ABSb (200 mg/kg b.w.) showed significant antidiarrhoeal activity (87.3%) comparable to Loperamide (87.5%). The isolated compounds from DCM-ABSb were β-amyrin and boonein with only boonein exhibiting antispasmodic activities on both high-potassium induced (IC50: 0.09±0.01 µg/mL) and spontaneous (0.29±0.05 µg/mL) contractions. Alstonia boonei and Alstonia congensis were identified as two distinct species. The diagnostic indices of the two plants provided pharmacopoeial standards for their identification. The isolated boonein could serve as a template for the development of antidiarrhoeal drugs 2021-02-01T00:00:00Z http://hdl.handle.net/123456789/1580 ISOLATION AND CHARACTERISATION OF ACETYLCHOLINESTERASE AND PROLYL ENDOPEPTIDASE INHIBITORS FROM SELECTED MEDICINAL PLANTS ONOJA, OJOGBANE JOEL Acetylcholinesterase (AChE) and Prolyl endopeptidases (PEP) are involved in the catalytic hydrolysis of acetylcholine and cleavage of neuropeptides influencing the activity of the brain, leading to some symptoms of Alzheimer’s disease (AD).Inhibition of AChE and PEP is considered a promising strategy for AD management.There are no effective drugs for the management of AD which necessitated the search for new potent neurotherapeutic agents.This study, was therefore, aimed at isolating and identifying inhibitors of AChE and PEP from selected medicinal plants identified from Nigerian ethnomedicine. Ten plants selected were macerated in methanol and their extracts evaluated for AChE inhibitory activity using Ellman colorimetric in vitro assay. The most active plant extracts: Phyllanthus muellerianus (PM) leaves (FHI 111339), Tinospora cordifolia(TC) stem (FHI 112287) and Cola hispida(CH) seed (FHI 111321) were successively partitioned into n-hexane, dichloromethane, ethyl acetate and aqueous fractions. The fractionswere assessed for AChE inhibitory activity using eserine as standard. In vitro analyses were used to evaluate the antioxidant potential of the plant extracts. Active fractions were subjected to chromatographic separations to isolate and purify bioactive compounds. Structures of isolated compounds were determined by spectroscopic analyses. Compounds were screened for AChE and PEP inhibition using in vitro colorimetric assays and metal chelating potential, respectively. Bacitracin was used as standard for PEP. Molecular docking was done using software (MOE 2015.010) to anticipate the binding affinity between drug candidates with protein targetsusing PDB ID: 3IVM (Prolyl endopeptidase) and 10CE (Acetylcholinesterase). Standard curves were generated and calculation of IC50 values was done using Microsoft Excel. Data were analysed using One way ANOVA followed by Dunnet’s Multiple Comparison testat α0.05 Methanol extracts of PM (IC50 of 1.29±0.70 mg/mL) and CH (IC50 of0.87±0.40 mg/mL) gave promisingAChE inhibitory activity at 5 mg/mL. Ethyl acetate fraction of PM (0.74±0.12 mg/mL) and CH(0.66±0.24 mg/mL) gave the highestAChE inhibitory activity compared to eserine. The dichloromethane fraction of TC exhibited good metal chelating activity (IC50 of 0.20±0.08 mg/mL). Beta-sitosterol, daucosterol, 1-octacosanol, rel-(2s,3s,4r,16e)-2-[(2'r)-2'-hydroxynonadecanoylamino]-heneicosadec-16-ene-1,3,4-triol, four clerodane diterpenoids and five alkaloids were isolated from TC.Two steroids, 5-hydroxymethylfurfural, and 2-hydroxyquinoline-4-carboxylic acidwere isolated from CH. Stigmasterol and β-sitosterol glucoside were isolated from PM. Oxoglaucine (Oxoaporphinoid alkaloid) from TC exhibited the highest AChE inhibitory activity (IC50 of 0.80±0.09 mg/mL) compared to eserine (IC50=0.53±0.34 mg/mL) at 1 mg/mL, while stigmasterol from PM demonstrated the highest PEP inhibitory activity (IC50= 0.77±2.9 mM) compared to bacitracin (IC50= 0.13±1.5 Mm) at 1 mM.Oxoglaucine also gave the highest metal chelating potential (IC50= 0.22±0.00 mg/mL) compared to EDTA (IC50 of 0.05±0.11 mg/mL). Molecular docking revealed hydrophobic, hydrogen bonding and π -stacking interactions among tested compounds and the active site of AChE andPEPresidue, which indicates their ability to mitigate the symptoms associated with AD. Oxoglaucine and stigmasterol isolated from Tinospora cordifoliaand Phyllanthus muellerianus, respectivelyshowed high acetylcholinesterase and prolyl endopeptidase inhibitory activities. The two biomolecules could serve as potential leads for novel drug development for the management of Alzheimer’s disease. 2021-01-01T00:00:00Z