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
Chemical transformations and phytochemical studies of bioactive components from extracts of Rosmarinus officinalis L
- Authors: Okoh, Omobola Oluranti
- Date: 2010
- Subjects: Essences and essential oils , Rosmarinus , Lamiaceae , Solution (Chemistry) , Extractive distillation , Medicinal plants , Bioactive compounds
- Language: English
- Type: Thesis , Doctoral , PhD (Chemistry)
- Identifier: vital:11331 , http://hdl.handle.net/10353/354 , Essences and essential oils , Rosmarinus , Lamiaceae , Solution (Chemistry) , Extractive distillation , Medicinal plants , Bioactive compounds
- Description: Variations in the yield, chemical composition, antibacterial, and antioxidant properties of the essential oils of Rosmarinus officinalis L. cultivated in Alice, Eastern Cape of South Africa over a period of 12 months using the solvent-free microwave extraction and traditional hydrodistillation methods were evaluated. The GC-MS analyses of the essential oils revealed the presence of 33 compounds with 1,8-cineole, a-pinene, camphor, verbenone, bornyl acetate and camphene constituting about 80 percent of the oils throughout the period of investigation, with the solvent-free microwave extraction method generally yielding more of the major components than the hydrodistillation method. Each of the major components of the oils varied in quantity and quality of yield at different periods of the year. The method of extraction and time of harvest are of importance to the quantity and quality of essential oil of Rosmarinus officinalis. Higher amounts of oxygenated monoterpenes such as borneol, camphor, terpene- 4-ol, linalool, a-terpeneol were present in the oil of SFME in comparison with HD. However, HD oil contained more monoterpene hydrocarbons such as a-pinene, camphene, β-pinene, myrcene, a-phellanderene, 1,8-cineole, trans- β-ocimene, γ-teprinene, and cis-sabinene hydrate than SFME extracted oil. Accumulation of monoterpene alcohols and ketones was observed during maturation process of Rosmarinus leaves. Quantitative evaluation of antibacterial activity, minimum inhibitory concentration values were determined using a serial microplate dilution method. The essential oils obtained using both methods of extraction were active against all the bacteria tested at a concentration of 10 mg mL-1. The minimum inhibitory concentrations for the SFME extracted oils ranged between 0.23 and 1.88 mg mL-1, while those of the HD extracted oils varied between 0.94 and 7.5 mg mL-1, thus suggesting that the oil obtained by solvent free microwave extraction was more active against bacteria than the oil obtained through hydrodistillation. The antioxidant and free radical scavenging activity of the obtained oils were tested by means of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH+) assay and β- carotene bleaching test. In the DPPH+ assay, while the free radical scavenging activity of the oil obtained by SFME method showed percentage inhibitions of between 48.8 percent and 67 percent, the HD derived oil showed inhibitions of between 52.2 percent and 65.30 percent at concentrations of 0.33, 0.50 and 1.0 mg mL-1, respectively. In the β-carotene bleaching assay, the percentage inhibition increased with increasing concentration of both oils with a higher antioxidant activity of the oil obtained through the SFME than the HD method. Thin layer chromatography (TLC) was used to analyze the chemical composition of the extracts using three eluent solvent systems of varying polarities i. e. CEF, BEA and EMW and sprayed with vanillin-sulfuric acid. The chemical composition of the different extracts was similar with the exception of methanol and water extracts which had only one or two visible compounds after treating with vanillin-spray reagent. To evaluate the number of antibacterial compounds present in the fractions, bioautography was used against two most important nosocomial microorganisms. S. aureus (Gram positive) and E. coli (Gram negative). Nearly all the crude serial extraction fractions contained compounds that inhibited the growth of E. coli. The hexane extract had the most lines of inhibition followed by ethyl acetate. Bioassay-guided fractionation against E. coli was used to isolate antibacterial compounds. The largest number of antibacterial compounds occurred in the hexane fraction. Furthermore we tried to complete the characterization by extracting and studying other biologically important plant metabolites such as phenolic compounds to evaluate the antioxidant capacity of Rosmarinus extracts.
- Full Text:
- Date Issued: 2010
- Authors: Okoh, Omobola Oluranti
- Date: 2010
- Subjects: Essences and essential oils , Rosmarinus , Lamiaceae , Solution (Chemistry) , Extractive distillation , Medicinal plants , Bioactive compounds
- Language: English
- Type: Thesis , Doctoral , PhD (Chemistry)
- Identifier: vital:11331 , http://hdl.handle.net/10353/354 , Essences and essential oils , Rosmarinus , Lamiaceae , Solution (Chemistry) , Extractive distillation , Medicinal plants , Bioactive compounds
- Description: Variations in the yield, chemical composition, antibacterial, and antioxidant properties of the essential oils of Rosmarinus officinalis L. cultivated in Alice, Eastern Cape of South Africa over a period of 12 months using the solvent-free microwave extraction and traditional hydrodistillation methods were evaluated. The GC-MS analyses of the essential oils revealed the presence of 33 compounds with 1,8-cineole, a-pinene, camphor, verbenone, bornyl acetate and camphene constituting about 80 percent of the oils throughout the period of investigation, with the solvent-free microwave extraction method generally yielding more of the major components than the hydrodistillation method. Each of the major components of the oils varied in quantity and quality of yield at different periods of the year. The method of extraction and time of harvest are of importance to the quantity and quality of essential oil of Rosmarinus officinalis. Higher amounts of oxygenated monoterpenes such as borneol, camphor, terpene- 4-ol, linalool, a-terpeneol were present in the oil of SFME in comparison with HD. However, HD oil contained more monoterpene hydrocarbons such as a-pinene, camphene, β-pinene, myrcene, a-phellanderene, 1,8-cineole, trans- β-ocimene, γ-teprinene, and cis-sabinene hydrate than SFME extracted oil. Accumulation of monoterpene alcohols and ketones was observed during maturation process of Rosmarinus leaves. Quantitative evaluation of antibacterial activity, minimum inhibitory concentration values were determined using a serial microplate dilution method. The essential oils obtained using both methods of extraction were active against all the bacteria tested at a concentration of 10 mg mL-1. The minimum inhibitory concentrations for the SFME extracted oils ranged between 0.23 and 1.88 mg mL-1, while those of the HD extracted oils varied between 0.94 and 7.5 mg mL-1, thus suggesting that the oil obtained by solvent free microwave extraction was more active against bacteria than the oil obtained through hydrodistillation. The antioxidant and free radical scavenging activity of the obtained oils were tested by means of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH+) assay and β- carotene bleaching test. In the DPPH+ assay, while the free radical scavenging activity of the oil obtained by SFME method showed percentage inhibitions of between 48.8 percent and 67 percent, the HD derived oil showed inhibitions of between 52.2 percent and 65.30 percent at concentrations of 0.33, 0.50 and 1.0 mg mL-1, respectively. In the β-carotene bleaching assay, the percentage inhibition increased with increasing concentration of both oils with a higher antioxidant activity of the oil obtained through the SFME than the HD method. Thin layer chromatography (TLC) was used to analyze the chemical composition of the extracts using three eluent solvent systems of varying polarities i. e. CEF, BEA and EMW and sprayed with vanillin-sulfuric acid. The chemical composition of the different extracts was similar with the exception of methanol and water extracts which had only one or two visible compounds after treating with vanillin-spray reagent. To evaluate the number of antibacterial compounds present in the fractions, bioautography was used against two most important nosocomial microorganisms. S. aureus (Gram positive) and E. coli (Gram negative). Nearly all the crude serial extraction fractions contained compounds that inhibited the growth of E. coli. The hexane extract had the most lines of inhibition followed by ethyl acetate. Bioassay-guided fractionation against E. coli was used to isolate antibacterial compounds. The largest number of antibacterial compounds occurred in the hexane fraction. Furthermore we tried to complete the characterization by extracting and studying other biologically important plant metabolites such as phenolic compounds to evaluate the antioxidant capacity of Rosmarinus extracts.
- Full Text:
- Date Issued: 2010
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