Isolation and characterisation of a channel inhibitor from Bunodosoma capense
- Authors: Van Losenoord, Wynand
- Date: 2019
- Subjects: Bioactive compounds , Potassium channels Medical microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/44134 , vital:37114
- Description: Voltage gated ion channels have recently become a subject of investigation as possible pharmaceutical targets. Research has linked the activity of ion channels directly to antiinflammatory pathways, energy homeostasis, cancer proliferation and painful diabetic neuropathy. Sea anemones secrete a diverse array of bioactive compounds including potassium and sodium channel inhibitors. A novel sodium channel inhibitor (molecular mass of 4619.7 ± 0.6 Da) with a predicted sequence: CLCNSDGPSV RGNTLSGILW LAGCPSGWHN CKKHKPTIGW CCK was isolated from Bunodosoma capense using a modified stimulation technique to induce the secretion of the neurotoxin rich mucus confirmed by an Artemia nauplii swimming assay. The peptide purification combined size-exclusion and reverse-phase high performance liquid chromatography. A thallium-based ion flux assay confirmed the presence of a sodium channel inhibitor and purity was determined using a modified tricine SDS-PAGE system. The peptide isolated indicated a tight conformation with the presence of multiple disulfide bonds in a cystine knot conformation. An IC50 value of 26 nM was determined for sodium channel inhibition on MCF-7 cells, indicating increased toxicity in comparison to sodium channel inhibitors previously isolated from Bunodosoma species.
- Full Text:
- Date Issued: 2019
- Authors: Van Losenoord, Wynand
- Date: 2019
- Subjects: Bioactive compounds , Potassium channels Medical microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/44134 , vital:37114
- Description: Voltage gated ion channels have recently become a subject of investigation as possible pharmaceutical targets. Research has linked the activity of ion channels directly to antiinflammatory pathways, energy homeostasis, cancer proliferation and painful diabetic neuropathy. Sea anemones secrete a diverse array of bioactive compounds including potassium and sodium channel inhibitors. A novel sodium channel inhibitor (molecular mass of 4619.7 ± 0.6 Da) with a predicted sequence: CLCNSDGPSV RGNTLSGILW LAGCPSGWHN CKKHKPTIGW CCK was isolated from Bunodosoma capense using a modified stimulation technique to induce the secretion of the neurotoxin rich mucus confirmed by an Artemia nauplii swimming assay. The peptide purification combined size-exclusion and reverse-phase high performance liquid chromatography. A thallium-based ion flux assay confirmed the presence of a sodium channel inhibitor and purity was determined using a modified tricine SDS-PAGE system. The peptide isolated indicated a tight conformation with the presence of multiple disulfide bonds in a cystine knot conformation. An IC50 value of 26 nM was determined for sodium channel inhibition on MCF-7 cells, indicating increased toxicity in comparison to sodium channel inhibitors previously isolated from Bunodosoma species.
- Full Text:
- Date Issued: 2019
The extraction of bioactive compounds from spent coffee grounds and their application in a cosmetic formulation
- Authors: Mdubeki, Ntokozo
- Date: 2019
- Subjects: Bioactive compounds , Plants -- bioactive compounds Plant biotechnology Coffee -- Processing Chemistry, Technical Cosmetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/41460 , vital:36485
- Description: Residues obtained during coffee processing, known as spent coffee grounds, are an abundant, low-cost and versatile feedstock for a wide range of high-value biological compounds exhibiting a number of properties which include anti-cellulite and antioxidant properties for skin-care applications. The objective of the present work was to obtain a concentrated extract containing high-value biological methylxanthines and polyphenolic compounds with antioxidant activity from the spent coffee grounds. An investigation of the effect of extraction conditions on the concentration of the bioactive compounds in the extract was done. The recovery of both methylxanthines and the polyphenolic compounds with antioxidant activity was achieved through a one-step extraction method under reduced pressure using a non-toxic solvent blend. Various experimental designs were implemented with the aim of optimising the recovery of the selected bioactive compounds, namely, caffeine, theophylline and theobromine from spent coffee grounds by using solvent blends of water and ethanol. A number of factors considered were: (i) repeatability, (ii) extraction time, (iii) composition of solvent blends, (iv) extraction vacuum (pressure)/temperature settings and (v) operating variables. The results suggested that an optimal recovery of the bioactive compounds will be obtained at a two-hour extraction time with a solvent blend of 100% water to maximise caffeine, theophylline, total phenolic content and percentage antioxidant activity, while extraction of theobromine is optimal using aqueous ethanolic solvent mixtures. The optimal vacuum (pressure) setting was found to be 250 mbar to maximise percentage antioxidant activity. The methylxanthines and total phenolic content were not affected by the vacuum (pressure) and temperature settings. Freezedrying the spent coffee grounds before extraction ensured long-term stability of a reproducible sample and achieved better recovery of the important bioactive compounds. Extraction was performed under reduced pressure in order to reduce solvent reflux temperature and to improve extraction efficiency. The quantitative and qualitative determinations of the above-mentioned high-value compounds were performed by high-performance liquid chromatography (HPLC), which confirmed the presence of the methylxanthines in the extracts. In the extraction of methylxanthines, it was found that caffeine was more abundant in the extract as compared to theophylline and theobromine. The extracts were incorporated into three cosmetic gel formulations. Release studies of caffeine, theobromine and theophylline were performed using the three formulated cosmetic gels with the use of a Franz diffusion cell. It was found that theobromine and theophylline were not detected from the gel samples as they were present in too small amounts in the original extracts and there was no significant difference in the release rates of caffeine when comparing the three different gels. Caffeine was released within 15 minutes. All three gels displayed a pseudoplastic viscosity profile and the different viscosity values of the three gels did not influence the release of caffeine significantly.
- Full Text:
- Date Issued: 2019
- Authors: Mdubeki, Ntokozo
- Date: 2019
- Subjects: Bioactive compounds , Plants -- bioactive compounds Plant biotechnology Coffee -- Processing Chemistry, Technical Cosmetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/41460 , vital:36485
- Description: Residues obtained during coffee processing, known as spent coffee grounds, are an abundant, low-cost and versatile feedstock for a wide range of high-value biological compounds exhibiting a number of properties which include anti-cellulite and antioxidant properties for skin-care applications. The objective of the present work was to obtain a concentrated extract containing high-value biological methylxanthines and polyphenolic compounds with antioxidant activity from the spent coffee grounds. An investigation of the effect of extraction conditions on the concentration of the bioactive compounds in the extract was done. The recovery of both methylxanthines and the polyphenolic compounds with antioxidant activity was achieved through a one-step extraction method under reduced pressure using a non-toxic solvent blend. Various experimental designs were implemented with the aim of optimising the recovery of the selected bioactive compounds, namely, caffeine, theophylline and theobromine from spent coffee grounds by using solvent blends of water and ethanol. A number of factors considered were: (i) repeatability, (ii) extraction time, (iii) composition of solvent blends, (iv) extraction vacuum (pressure)/temperature settings and (v) operating variables. The results suggested that an optimal recovery of the bioactive compounds will be obtained at a two-hour extraction time with a solvent blend of 100% water to maximise caffeine, theophylline, total phenolic content and percentage antioxidant activity, while extraction of theobromine is optimal using aqueous ethanolic solvent mixtures. The optimal vacuum (pressure) setting was found to be 250 mbar to maximise percentage antioxidant activity. The methylxanthines and total phenolic content were not affected by the vacuum (pressure) and temperature settings. Freezedrying the spent coffee grounds before extraction ensured long-term stability of a reproducible sample and achieved better recovery of the important bioactive compounds. Extraction was performed under reduced pressure in order to reduce solvent reflux temperature and to improve extraction efficiency. The quantitative and qualitative determinations of the above-mentioned high-value compounds were performed by high-performance liquid chromatography (HPLC), which confirmed the presence of the methylxanthines in the extracts. In the extraction of methylxanthines, it was found that caffeine was more abundant in the extract as compared to theophylline and theobromine. The extracts were incorporated into three cosmetic gel formulations. Release studies of caffeine, theobromine and theophylline were performed using the three formulated cosmetic gels with the use of a Franz diffusion cell. It was found that theobromine and theophylline were not detected from the gel samples as they were present in too small amounts in the original extracts and there was no significant difference in the release rates of caffeine when comparing the three different gels. Caffeine was released within 15 minutes. All three gels displayed a pseudoplastic viscosity profile and the different viscosity values of the three gels did not influence the release of caffeine significantly.
- Full Text:
- Date Issued: 2019
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