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
The anticancer activity of Cyathula prostrata on two malignant cell lines
- Authors: Schnablegger, Gerald
- Date: 2010
- Subjects: Medicinal plants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10309 , http://hdl.handle.net/10948/1563 , Medicinal plants
- Description: Plants have always been a source of medicine and are still being used by traditional healers in the rural part of Africa, Asia and India to treat a range of illnesses including cancer. The in vitro anticancer activity of an 80 percent ethanol extract of Cyathula prostrata, an annual branching shrub used by traditional healers in Nigeria to treat cancer was investigated. No previous studies have outlined the possible pathways and mechanisms used by cancer cells when treated with C. prostrata. Dose response analysis was performed to determine the effective cytotoxic concentrations of C. prostrata on HeLa (cervical cancer cell line) and U937 (myelo-monocytic cell line). The IC50 values were 100.8 μg/ml and 64.4 μg/ml for HeLa and U937 cells, respectively. All further experiments were performed using 125 μg/ml C. prostrata extract and 50 μM cisplatin as positive control. With the use of the fluorescent DNA binding dye propidium iodide, the induction of tumour cell death by C. prostrata extract has been linked to cell cycle arrest in the G1 phase at 24 and 48 h. In both cell lines, more than 80 percent of the C. prostrata treated cells were found in the G1 phase after 48 hours of treatment. The annexin V-FITC/PI assay revealed an increase in the percentage apoptotic cells from 4.9 percent to 53.1 percent at 24 h and 8.3 percent to 50.3 percent at 48 h. Since apoptosis induction can occur via a number of different pathways, distinct features were used as markers to investigate the mode of action of this C. prostrata extract. Markers such as activated caspase-8, p21 and cyt-c, were investigated with the aid of fluorescently labelled (FITC) antibodies with analysis using flow cytometry. No change in p21 levels was observed in response to treatment with the extract for up to 48 h. Cell cycle arrest in G1 was therefore not induced by this cyclin-CDK inhibitor. Increase in caspase-8 activation was observed in response to treatment with the extract with no cyt-c release from the mitochondria. The lack of cyt-c release was due to no change in mitochondrial membrane potential, which was investigated with the aid of fluorescent mitochondrial dyes and flow cytometric techniques. Caspase-8 activation is unique to the extrinsic apoptotic pathway. The results from this study therefore show that C. prostrata extract induces apoptosis via the extrinsic pathway and that this activation in independent of the mitochondria. The levels of hTERT, the catalytic subunit of telomerase, were investigated as an additional molecular target for C. prostrata. This was also investigated using FITC labelled antibodies and flow cytometry. A decrease in hTERT levels was observed following C. prostrata treatment. The findings from this study suggest that the extract acts through multiple targets, by inducing: cell cycle arrest in the G1 phase through an unknown mechanism; apoptosis through an extrinsic death receptor pathway and replicative senescence through inhibition of telomerase.
- Full Text:
- Date Issued: 2010
- Authors: Schnablegger, Gerald
- Date: 2010
- Subjects: Medicinal plants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10309 , http://hdl.handle.net/10948/1563 , Medicinal plants
- Description: Plants have always been a source of medicine and are still being used by traditional healers in the rural part of Africa, Asia and India to treat a range of illnesses including cancer. The in vitro anticancer activity of an 80 percent ethanol extract of Cyathula prostrata, an annual branching shrub used by traditional healers in Nigeria to treat cancer was investigated. No previous studies have outlined the possible pathways and mechanisms used by cancer cells when treated with C. prostrata. Dose response analysis was performed to determine the effective cytotoxic concentrations of C. prostrata on HeLa (cervical cancer cell line) and U937 (myelo-monocytic cell line). The IC50 values were 100.8 μg/ml and 64.4 μg/ml for HeLa and U937 cells, respectively. All further experiments were performed using 125 μg/ml C. prostrata extract and 50 μM cisplatin as positive control. With the use of the fluorescent DNA binding dye propidium iodide, the induction of tumour cell death by C. prostrata extract has been linked to cell cycle arrest in the G1 phase at 24 and 48 h. In both cell lines, more than 80 percent of the C. prostrata treated cells were found in the G1 phase after 48 hours of treatment. The annexin V-FITC/PI assay revealed an increase in the percentage apoptotic cells from 4.9 percent to 53.1 percent at 24 h and 8.3 percent to 50.3 percent at 48 h. Since apoptosis induction can occur via a number of different pathways, distinct features were used as markers to investigate the mode of action of this C. prostrata extract. Markers such as activated caspase-8, p21 and cyt-c, were investigated with the aid of fluorescently labelled (FITC) antibodies with analysis using flow cytometry. No change in p21 levels was observed in response to treatment with the extract for up to 48 h. Cell cycle arrest in G1 was therefore not induced by this cyclin-CDK inhibitor. Increase in caspase-8 activation was observed in response to treatment with the extract with no cyt-c release from the mitochondria. The lack of cyt-c release was due to no change in mitochondrial membrane potential, which was investigated with the aid of fluorescent mitochondrial dyes and flow cytometric techniques. Caspase-8 activation is unique to the extrinsic apoptotic pathway. The results from this study therefore show that C. prostrata extract induces apoptosis via the extrinsic pathway and that this activation in independent of the mitochondria. The levels of hTERT, the catalytic subunit of telomerase, were investigated as an additional molecular target for C. prostrata. This was also investigated using FITC labelled antibodies and flow cytometry. A decrease in hTERT levels was observed following C. prostrata treatment. The findings from this study suggest that the extract acts through multiple targets, by inducing: cell cycle arrest in the G1 phase through an unknown mechanism; apoptosis through an extrinsic death receptor pathway and replicative senescence through inhibition of telomerase.
- Full Text:
- Date Issued: 2010
The in vitro biological activities of three Hypoxis species and their active compounds
- Authors: Boukes, Gerhardt Johannes
- Date: 2010
- Subjects: Potatoes -- Africa , Potatoes -- Therapeutic use , Medicinal plants
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10322 , http://hdl.handle.net/10948/1228 , Potatoes -- Africa , Potatoes -- Therapeutic use , Medicinal plants
- Description: The African potato is used as an African traditional medicine for its nutritional and medicinal properties. Most research has been carried out on H. hemerocallidea, with very little or nothing on other Hypoxis spp. The main aim of this project was to provide scientific data on the anticancer, anti-inflammatory and antioxidant properties of H. hemerocallidea, H. stellipilis and H. sobolifera chloroform extracts and their active compounds. The hypoxoside and phytosterol contents of the three Hypoxis spp. were determined using TLC, HPLC and GC. H. hemerocallidea and H. sobolifera chloroform extracts contained the highest amounts of hypoxoside and β-sitosterol, respectively. For the anticancer properties, cytotoxicity of the Hypoxis extracts and its purified compounds were determined against the HeLa, HT-29 and MCF-7 cancer cell lines (using MTT), and PBMCs (using CellTiter-Blue®). H. sobolifera had the best cytotoxicity against the three cancer cell lines, whereas H. stellipilis stimulated HeLa and HT-29 cancer cell growth. IC50 values of hypoxoside and rooperol were determined. DNA cell cycle arrest (using PI staining) occurred in the late G1/early S (confirmed by increased p21Waf1/Cip1 expression) and G2/M phases after 15 and 48 hrs, respectively, when treated with Hypoxis extracts and rooperol. H. sobolifera and rooperol activated caspase-3 and -7 (using fluorescently labelled antibodies) in HeLa and HT-29 cancer cells, and caspase-7 in MCF-7 cancer cells after 48 hrs. Annexin V binding to phosphatidylserines in rooperol treated U937 cells confirmed early apoptosis after 15 hrs. The TUNEL assay showed DNA fragmentation in the three cancer cell lines when treated with H. sobolifera and rooperol for 48 hrs. A shift pass the G2/M phase has led to the investigation of endoreduplication, which was confirmed by cell/nucleus size, and anti-apoptotic proteins (Akt, phospho-Akt, phospho-Bcl-2 and p21Waf1/Cip1). U937 cell differentiation to monocyte-macrophages was optimized using PMA and 1,25(OH)2D3, which was confirmed by morphological and biochemical changes. For the anti-inflammatory properties, Hypoxis extracts and rooperol significantly increased NO production in monocyte-macrophages (pre-loaded with DAF-2 DA) and phagocytosis of pHrodoTM E. coli BioParticles®. The treatments had no effect on COX-2 expression in monocyte-macrophages. The phytosterols significantly increased IL-1β and IL-6 secretion xv (using the FlowCytomix Multiplex human Th1/Th2 10plex Kit I) in the PBMCs of one donor. For the antioxidant properties, Hypoxis extracts and rooperol significantly increased ROS production in undifferentiated and differentiated U937 cells, which were pre-loaded with DCFH-DA. Hypoxis extracts and purified compounds had ferric reducing activities, but only rooperol had ferric reducing activities significantly greater than ascorbic acid. β-sitosterol, campesterol and cholesterol significantly increased SOD activity in Chang liver cells, while H. stellipilis, H. sobolifera and rooperol decreased SOD activity. Anticancer, anti-inflammatory and antioxidant properties of the Hypoxis extracts may be attributed to the β-sitosterol content, because Hypoxis chloroform extracts contained very little or no hypoxoside. Unidentified compounds, and synergistic and additive effects of the compounds may have contributed to the biological effects. This study confirms previous reports that rooperol is the active compound. Results provide scientific data on the medicinal properties of one of the most frequently used medicinal plants in South Africa.
- Full Text:
- Date Issued: 2010
- Authors: Boukes, Gerhardt Johannes
- Date: 2010
- Subjects: Potatoes -- Africa , Potatoes -- Therapeutic use , Medicinal plants
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10322 , http://hdl.handle.net/10948/1228 , Potatoes -- Africa , Potatoes -- Therapeutic use , Medicinal plants
- Description: The African potato is used as an African traditional medicine for its nutritional and medicinal properties. Most research has been carried out on H. hemerocallidea, with very little or nothing on other Hypoxis spp. The main aim of this project was to provide scientific data on the anticancer, anti-inflammatory and antioxidant properties of H. hemerocallidea, H. stellipilis and H. sobolifera chloroform extracts and their active compounds. The hypoxoside and phytosterol contents of the three Hypoxis spp. were determined using TLC, HPLC and GC. H. hemerocallidea and H. sobolifera chloroform extracts contained the highest amounts of hypoxoside and β-sitosterol, respectively. For the anticancer properties, cytotoxicity of the Hypoxis extracts and its purified compounds were determined against the HeLa, HT-29 and MCF-7 cancer cell lines (using MTT), and PBMCs (using CellTiter-Blue®). H. sobolifera had the best cytotoxicity against the three cancer cell lines, whereas H. stellipilis stimulated HeLa and HT-29 cancer cell growth. IC50 values of hypoxoside and rooperol were determined. DNA cell cycle arrest (using PI staining) occurred in the late G1/early S (confirmed by increased p21Waf1/Cip1 expression) and G2/M phases after 15 and 48 hrs, respectively, when treated with Hypoxis extracts and rooperol. H. sobolifera and rooperol activated caspase-3 and -7 (using fluorescently labelled antibodies) in HeLa and HT-29 cancer cells, and caspase-7 in MCF-7 cancer cells after 48 hrs. Annexin V binding to phosphatidylserines in rooperol treated U937 cells confirmed early apoptosis after 15 hrs. The TUNEL assay showed DNA fragmentation in the three cancer cell lines when treated with H. sobolifera and rooperol for 48 hrs. A shift pass the G2/M phase has led to the investigation of endoreduplication, which was confirmed by cell/nucleus size, and anti-apoptotic proteins (Akt, phospho-Akt, phospho-Bcl-2 and p21Waf1/Cip1). U937 cell differentiation to monocyte-macrophages was optimized using PMA and 1,25(OH)2D3, which was confirmed by morphological and biochemical changes. For the anti-inflammatory properties, Hypoxis extracts and rooperol significantly increased NO production in monocyte-macrophages (pre-loaded with DAF-2 DA) and phagocytosis of pHrodoTM E. coli BioParticles®. The treatments had no effect on COX-2 expression in monocyte-macrophages. The phytosterols significantly increased IL-1β and IL-6 secretion xv (using the FlowCytomix Multiplex human Th1/Th2 10plex Kit I) in the PBMCs of one donor. For the antioxidant properties, Hypoxis extracts and rooperol significantly increased ROS production in undifferentiated and differentiated U937 cells, which were pre-loaded with DCFH-DA. Hypoxis extracts and purified compounds had ferric reducing activities, but only rooperol had ferric reducing activities significantly greater than ascorbic acid. β-sitosterol, campesterol and cholesterol significantly increased SOD activity in Chang liver cells, while H. stellipilis, H. sobolifera and rooperol decreased SOD activity. Anticancer, anti-inflammatory and antioxidant properties of the Hypoxis extracts may be attributed to the β-sitosterol content, because Hypoxis chloroform extracts contained very little or no hypoxoside. Unidentified compounds, and synergistic and additive effects of the compounds may have contributed to the biological effects. This study confirms previous reports that rooperol is the active compound. Results provide scientific data on the medicinal properties of one of the most frequently used medicinal plants in South Africa.
- Full Text:
- Date Issued: 2010
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