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Identification of bioactive ingredients from Cleome gynandra and the physicochemical study of their microencapsulation in beta-cyclodextrin
Thesis (PhD (Chemistry))--University of Pretoria, 2022.
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University of Pretoria
2022
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| _version_ | 1867613504893091840 |
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| access_status_str | Open Access |
| author2 | Maharaj, Vinesh J. |
| author_browse | Maharaj, Vinesh J. |
| author_facet | Maharaj, Vinesh J. |
| collection | Thesis |
| dc_rights_str_mv | © 2022 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Thesis (PhD (Chemistry))--University of Pretoria, 2022. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/86075 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:37:12.475Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/86075 Identification of bioactive ingredients from Cleome gynandra and the physicochemical study of their microencapsulation in beta-cyclodextrin Maharaj, Vinesh J. buntubonke@gmail.com Dlamini, Nomusa R. Labuschagne, Philip Gom, Buntubonke Cleome gynandra Magnetic resonance imaging (MRI) Bioelectrochemistry Chemical extraction methods Enzymes UCTD Thesis (PhD (Chemistry))--University of Pretoria, 2022. In this study single solvent system extraction, sequential extraction and supercritical fluid extraction were used to prepare several extracts which were initially selectively investigated for their inhibition of selected enzymes (α-glucosidase, α-amylase, renin, HMG-CoA reductase and xanthene oxidase), inhibition of selected bacteria strains (Staphylococcus aureus and Escherichia coli) and cancer cytotoxicity of lung cancer A549 cell line. The greatest enzyme based activity was seen against α-glucosidase where the ethyl acetate, acetone, n-hexane and supercritical fluid (CO2-ethanol) extracts exhibited activities with 91.76 ± 1.16, 89.97 ± 1.77, 92.67 ± 0.17 and 96.55 ± 0.15 %inhibition, respectively, at 200 μg/mL. In bacterial assays, the ethanol extract exhibited the greatest inhibition activity against Escherichia coli with the MIC value 0.052 ± 0.016 mg/mL while in lung cancer cytotoxicity assays the n-hexane extract was the most active (>84% inhibition in the concentration range of 0.125 – 1.00 mg/mL, over 48-hour incubation and >75 % over 24-hour incubation). The solvent-solvent partition product of the ethyl acetate extract, pEtOAc fraction, displayed IC50 value 8.75 µg/mL, better than the 25.40 µg/mL of the crude extract and 37.10 µg/mL of the positive control. This fraction was further fractionated using column chromatography via a bioassay-guided fractionation approach resulting in seven semi-pure fractions displaying activities in the range 82.0 – 98.4 %inhibition at 25 µg/mL. Chemical profiling using UPLC-QTOF-MS led to several unidentified high molecular mass compounds and a novel compound cleogynone A. Fractionation of the ethanol extract was followed by bioautographic antibacterial analysis of 22 fractions against both Escherichia coli and Staphylococcus aureus where the brightest spot of largest diameter indicative of the greatest activity was located against Escherichia coli and the most active fraction was further fractionated via semi-preparative HPLC yielding eight semi-pure sub-fractions with MIC values in the range 0.012 – 0.094 mg/mL comparable to gentamicin the positive control. Chemical profiling of the active fraction and sub-fractions using UPLC-QTOF-MS and MassLynx data processing led to the tentative identification of rutin, kaempferol-3-glucoside-3''-rhamnoside and isorhamnetin 3-O-robinoside as possible biomarkers, while processing using the Waters® UNIFI® Scientific Information System led to the identification of rutin (quercetin-3-O-rutinoside) and nictoflorin (kaempferol 3-O-rutinoside) as the major compounds and kaempferol, quercetin and nepitrin were represented by minor peaks. Purchased standards for nictoflorin and rutin displayed no inhibition of Escherichia coli. Bioassay-guided fractionation of the n-hexane extract initially using column chromatography for lung cancer cytotoxicity led to several active fractions. Three semi-pure and most abundant, reasonably active fractions of mid-polarity were further fractionated using a combination of flash column chromatography and preparative thin-layer chromatography to yield two novel compounds, cleogynone A and cleogynone B whose molecular ions were identified using UPLC-QTOF-MS and chemical structures were elucidated using NMR with their crystal structures subsequently determined by single crystal X-ray diffraction. A known compound, named here, cleogynone C was confirmed by comparison of the NMR data to those in literature. Further characterisation of the pure compounds involve melting point analysis, IR, and optical rotation analysis. These three compounds were evaluated for their invitro anticancer activity against lung cancer (A549), breast cancer (MDA-MB-468) and colorectal cancer (HCT116 and HCT15) in the concentration range 0.39 – 25.0 µg/mL. Against the colorectal cancer (HCT15), cleogynone A, B and C showed the highest activities 82.97±0.56, 81.74±0.34 and 83.67±3.16 % inhibition, respectively, at 25 µg/mL concentration over 48 hr treatment. Cleogynones B and C showed the greatest activity of 89.34±5.46 and 87.76±1.22 % inhibition activity, respectively, against HCT116 over 24 hr treatment at the highest test concentration (25 µg/mL). The compounds displayed moderate activity with the highest activity 66.58±1.30 %inhibition displayed by cleogynone C against breast cancer (MDA-MB-468) while all three compounds displayed poor activity against lung cancer (A549) ≤ 51.38±0.39 at the highest concentration (25 µg/mL). Several β-CD n-hexane formulations were prepared via supercritical CO2 technique at varying temperature, pressure and exposure time to study the effect of varying such operating parameters on the feasibility of the formulation, while one β-CD-n-hexane complex formulation was obtained via the spray-drying technique for comparison. The solubility assessments were achieved by area integrating the peaks of chromatographic profiles generated using UPLC-QTOF-MS. Formulation was successful in improving the aqueous solubility for most of the supercritical CO2 formulations as well as the spray-dried formulation. High pressure formulation conditions enhance the formulation but negatively impacts the aqueous solubility. While formulations via the supercritical CO2 technique did not show improvement of anticancer activity the spray drying technique resulted in the greatest anticancer activity improvement. The aqueous solubility of the novel compounds cleogynone A and cleogynone B was not improved in the presence of β-CD in water, no improvement of anticancer activity against the lung-, breast- and colorectal cancer cell lines. Council for Scientific and Industrial Research (CSIR) The Department of Science and Innovation (DSI) Chemistry PhD (Chemistry) Unrestricted 2022-07-08T12:16:10Z 2022-07-08T12:16:10Z 2022-09 2022-05 Thesis * S2022 https://repository.up.ac.za/handle/2263/86075 10.25403/UPresearchdata.20110799 en © 2022 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | Cleome gynandra Magnetic resonance imaging (MRI) Bioelectrochemistry Chemical extraction methods Enzymes UCTD Identification of bioactive ingredients from Cleome gynandra and the physicochemical study of their microencapsulation in beta-cyclodextrin |
| title | Identification of bioactive ingredients from Cleome gynandra and the physicochemical study of their microencapsulation in beta-cyclodextrin |
| title_full | Identification of bioactive ingredients from Cleome gynandra and the physicochemical study of their microencapsulation in beta-cyclodextrin |
| title_fullStr | Identification of bioactive ingredients from Cleome gynandra and the physicochemical study of their microencapsulation in beta-cyclodextrin |
| title_full_unstemmed | Identification of bioactive ingredients from Cleome gynandra and the physicochemical study of their microencapsulation in beta-cyclodextrin |
| title_short | Identification of bioactive ingredients from Cleome gynandra and the physicochemical study of their microencapsulation in beta-cyclodextrin |
| title_sort | identification of bioactive ingredients from cleome gynandra and the physicochemical study of their microencapsulation in beta cyclodextrin |
| topic | Cleome gynandra Magnetic resonance imaging (MRI) Bioelectrochemistry Chemical extraction methods Enzymes UCTD |
| url | https://repository.up.ac.za/handle/2263/86075 |