Fucoidans from South African brown seaweeds: establishing the link between their structure and biological properties (anti-diabetic and anti-cancer activities)
- Authors: Mabate, Blessing
- Date: 2022-10-14
- Subjects: Fucoidan , Diabetes Treatment , Cancer Treatment , Brown algae
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365677 , vital:65775 , DOI https://doi.org/10.21504/10962/365677
- Description: Type 2 diabetes mellitus (T2DM) and cancer are major non-communicable diseases causing a heavy morbidity-mortality and economic burden globally. The therapeutic efforts in managing these diseases are primarily chemotherapeutic and are associated with demerits, including side effects and toxicity, limiting the prescribed amounts. These dosage limits may cause drug resistance, another major challenge in maintaining quality global health. The pursuit of novel natural bioproducts is a reasonable strategy to add to the arsenal against T2DM and cancer. Fucoidans, sulphated fucose polysaccharides abundant in brown seaweeds, have recently become popular for their biological activities, including anti-diabetic and anti-cancer properties. However, endemic South African brown seaweeds have not been adequately explored. Therefore, this study sought to characterise fucoidans extracted from South African brown seaweeds and elucidate their structure to their biological activities. Also, this study highlighted carbohydrate and glucose metabolism as major target processes in the control efforts of T2DM and cancer using fucoidans. Harvested brown seaweeds were identified as Ecklonia radiata and Sargassum elegans. E. maxima was kindly donated by KelpX. The fucoidans were then extracted using hot water, EDTA assisted, and acid extraction protocols. The integrity of the extracted fucoidan was confirmed through structural analysis using FTIR, NMR and TGA. The fucoidan extracts were then chemically characterised to determine their carbohydrate and monosaccharide composition and sulphate content. The characterised fucoidans were profiled for inhibiting the major amylolytic enzymes, namely α-amylase and α-glucosidase. The mode of inhibition by fucoidans and synergy experiments with the commercial anti-diabetic drug acarbose were also investigated. Furthermore, the fucoidans were screened for potential anti-cancer activities on the human colorectal HCT116 cancer cell line. The cytotoxicity of fucoidans was quantified using the resazurin assay. The effect of fucoidan on HCT116 cell adhesion on the tissue culture plastic was also investigated using the crystal violet-based cell adhesion assay. In addition, cancer antimigration properties of fucoidans were also investigated using 2D wound healing and 3D spheroid-based assays. Furthermore, the long-term survival of HCT116 cells was investigated through the clonogenic assay after treatment with fucoidans. Lastly, glucose uptake and lactate export assays revealed the influence of fucoidan on glucose uptake and the glycolytic flux of HCT116 cells. Fucoidans were successfully extracted with a yield between 2.2% and 14.2% on a dry weight basis. EDTA extracts produced the highest yields than the water and the acid extracts. Ecklonia spp. fucoidans displayed the highest total carbohydrate content, with glucose and galactose being the major monosaccharides. S. elegans and commercial Fucus vesiculosus had lower carbohydrate contents but contained more sulphates than the Ecklonia spp. fucoidans. Furthermore, the extracted fucoidan contained little to no contaminants, including proteins, phenolics and uronic acids. In addition, the extracted fucoidans were determined to be >100 kDa through ultracentrifugation. Mass spectrometry also detected the most abundant peak for all fucoidans to be around 700 Da (m/z). Extracted fucoidans inhibited the activity of α-glucosidase more strongly than the commercial anti-diabetic agent acarbose but were inactive on α-amylase. Fucoidans were also shown to be mixed inhibitors of α-glucosidase. Compellingly, fucoidans synergistically inhibited α-glucosidase in combination with the anti-diabetic agent acarbose, highlighting prospects for combination therapy. Finally, fucoidans demonstrated some anti-proliferative characteristics on HCT116 cancer cells by inhibiting their ability to adhere to the tissue culture plate matrix. Furthermore, some fucoidan extracts inhibited the migration of HCT116 cancer cells from 3D spheroids. Some of our fucoidan extracts also inhibited HCT116 colony formation, demonstrating inhibition of long-term cell survival. The E. maxima water extract also inhibited glucose uptake by HCT116 cells, thereby influencing the glycolytic flux. In conclusion, biologically active fucoidans were successfully extracted from South African brown seaweeds. These fucoidans demonstrated anti-diabetic and anti-cancer properties, revealing their relevance as potential drugs for these diseases. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mabate, Blessing
- Date: 2022-10-14
- Subjects: Fucoidan , Diabetes Treatment , Cancer Treatment , Brown algae
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365677 , vital:65775 , DOI https://doi.org/10.21504/10962/365677
- Description: Type 2 diabetes mellitus (T2DM) and cancer are major non-communicable diseases causing a heavy morbidity-mortality and economic burden globally. The therapeutic efforts in managing these diseases are primarily chemotherapeutic and are associated with demerits, including side effects and toxicity, limiting the prescribed amounts. These dosage limits may cause drug resistance, another major challenge in maintaining quality global health. The pursuit of novel natural bioproducts is a reasonable strategy to add to the arsenal against T2DM and cancer. Fucoidans, sulphated fucose polysaccharides abundant in brown seaweeds, have recently become popular for their biological activities, including anti-diabetic and anti-cancer properties. However, endemic South African brown seaweeds have not been adequately explored. Therefore, this study sought to characterise fucoidans extracted from South African brown seaweeds and elucidate their structure to their biological activities. Also, this study highlighted carbohydrate and glucose metabolism as major target processes in the control efforts of T2DM and cancer using fucoidans. Harvested brown seaweeds were identified as Ecklonia radiata and Sargassum elegans. E. maxima was kindly donated by KelpX. The fucoidans were then extracted using hot water, EDTA assisted, and acid extraction protocols. The integrity of the extracted fucoidan was confirmed through structural analysis using FTIR, NMR and TGA. The fucoidan extracts were then chemically characterised to determine their carbohydrate and monosaccharide composition and sulphate content. The characterised fucoidans were profiled for inhibiting the major amylolytic enzymes, namely α-amylase and α-glucosidase. The mode of inhibition by fucoidans and synergy experiments with the commercial anti-diabetic drug acarbose were also investigated. Furthermore, the fucoidans were screened for potential anti-cancer activities on the human colorectal HCT116 cancer cell line. The cytotoxicity of fucoidans was quantified using the resazurin assay. The effect of fucoidan on HCT116 cell adhesion on the tissue culture plastic was also investigated using the crystal violet-based cell adhesion assay. In addition, cancer antimigration properties of fucoidans were also investigated using 2D wound healing and 3D spheroid-based assays. Furthermore, the long-term survival of HCT116 cells was investigated through the clonogenic assay after treatment with fucoidans. Lastly, glucose uptake and lactate export assays revealed the influence of fucoidan on glucose uptake and the glycolytic flux of HCT116 cells. Fucoidans were successfully extracted with a yield between 2.2% and 14.2% on a dry weight basis. EDTA extracts produced the highest yields than the water and the acid extracts. Ecklonia spp. fucoidans displayed the highest total carbohydrate content, with glucose and galactose being the major monosaccharides. S. elegans and commercial Fucus vesiculosus had lower carbohydrate contents but contained more sulphates than the Ecklonia spp. fucoidans. Furthermore, the extracted fucoidan contained little to no contaminants, including proteins, phenolics and uronic acids. In addition, the extracted fucoidans were determined to be >100 kDa through ultracentrifugation. Mass spectrometry also detected the most abundant peak for all fucoidans to be around 700 Da (m/z). Extracted fucoidans inhibited the activity of α-glucosidase more strongly than the commercial anti-diabetic agent acarbose but were inactive on α-amylase. Fucoidans were also shown to be mixed inhibitors of α-glucosidase. Compellingly, fucoidans synergistically inhibited α-glucosidase in combination with the anti-diabetic agent acarbose, highlighting prospects for combination therapy. Finally, fucoidans demonstrated some anti-proliferative characteristics on HCT116 cancer cells by inhibiting their ability to adhere to the tissue culture plate matrix. Furthermore, some fucoidan extracts inhibited the migration of HCT116 cancer cells from 3D spheroids. Some of our fucoidan extracts also inhibited HCT116 colony formation, demonstrating inhibition of long-term cell survival. The E. maxima water extract also inhibited glucose uptake by HCT116 cells, thereby influencing the glycolytic flux. In conclusion, biologically active fucoidans were successfully extracted from South African brown seaweeds. These fucoidans demonstrated anti-diabetic and anti-cancer properties, revealing their relevance as potential drugs for these diseases. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
The quantification of fucoxanthin from selected South African marine brown algae (Phaeophyta) using HPLC-UV/Vis
- Authors: Mubaiwa, Byron Tawanda
- Date: 2015
- Subjects: Marine algae , Brown algae , High performance liquid chromatography , Functional foods , Xanthophylls , Carotenoids , Extraction (Chemistry)
- Language: English
- Type: Thesis , Masters , MPharm
- Identifier: vital:3868 , http://hdl.handle.net/10962/d1017879
- Description: Marine brown algae (seaweeds) are a rich source of fucoxanthin, a xanthophyll carotenoid that is naturally, an accessory pigment in the process of photosynthesis of sea vegetation such as Sargassum incisifolium. Fucoxanthin has been exploited by nutraceutical companies for its anti-obesity effects that has resulted in an increase of seaweed slimming preparations such as FucoThin™. The field is getting widespread consumer attention as interest in fucoxanthin has also transcended to its widespread biological potential which include cytotoxicity, anti-diabetic, anti-oxidant, anti-inflammatory and anti-plasmodium effects. We therefore wanted to identify a reliable source(s) of fucoxanthin from diverse samples of South African marine brown algae in order to explore our medicinal chemistry interests around the cytotoxicity and anti-malarial potential of fucoxanthin. A known source, Sargassum incisifolium, was used to isolate (maceration in CH₂Cl₂/MeOH at 35 °C followed by a hexane/EtOAc step gradient silica column of the crude extract and reversed phase semi-prep HPLC) and characterize (1D and 2D NMR) fucoxanthin (reference standard) in order to develop an analytical method for its determination in selected diverse brown algae commonly found in South Africa. The HPLC [Column: Phenomenex® Synergi™ (250 x 3.0 mm i.d); Mobile phase: ACN/H2O (95:5)] method developed for this analysis was validated according the guidelines set by the International Conference on Harmonization (ICH). Fifteen species were then assessed for fucoxanthin content (μg/g of dried weight) using the developed method. Stability studies on fucoxanthin were also carried out to assess photo- and pH degradation of fucoxanthin. Zonaria subarticulata (KOS130226-18) from Kenton-On-Sea beach and Sargassum incisifolium (PA130427-1) from Port Alfred beach were found to be the highest producers of fucoxanthin with 0.50 mg/g and 0.45 mg/g dried weight respectively. Fucoxanthin was found to be both photo-labile and sensitive to both acidic and basic pH environments. However, the pigment was more photostable in pure as opposed to extract form and also showed to be more stable at pH 10.0. Our findings show that Z. subarticulata and S. incisifolium could be reliable sources of fucoxanthin and can be considered as the algae to use in optimized extraction procedures in further studies. Also, when working with fucoxanthin, it is important to protect it from light. Any consideration of taking fucoxanthin preparation orally (as a nutraceutical) should consider protecting the active from the harsh conditions of the gastrointestinal tract. Any upscale production of fucoxanthin from seaweed should consider variations such as geographical, seasonal, lifecycle stage, etc. of identified algae as these may be important factors in obtaining effective concentrations of fucoxanthin.
- Full Text:
- Date Issued: 2015
- Authors: Mubaiwa, Byron Tawanda
- Date: 2015
- Subjects: Marine algae , Brown algae , High performance liquid chromatography , Functional foods , Xanthophylls , Carotenoids , Extraction (Chemistry)
- Language: English
- Type: Thesis , Masters , MPharm
- Identifier: vital:3868 , http://hdl.handle.net/10962/d1017879
- Description: Marine brown algae (seaweeds) are a rich source of fucoxanthin, a xanthophyll carotenoid that is naturally, an accessory pigment in the process of photosynthesis of sea vegetation such as Sargassum incisifolium. Fucoxanthin has been exploited by nutraceutical companies for its anti-obesity effects that has resulted in an increase of seaweed slimming preparations such as FucoThin™. The field is getting widespread consumer attention as interest in fucoxanthin has also transcended to its widespread biological potential which include cytotoxicity, anti-diabetic, anti-oxidant, anti-inflammatory and anti-plasmodium effects. We therefore wanted to identify a reliable source(s) of fucoxanthin from diverse samples of South African marine brown algae in order to explore our medicinal chemistry interests around the cytotoxicity and anti-malarial potential of fucoxanthin. A known source, Sargassum incisifolium, was used to isolate (maceration in CH₂Cl₂/MeOH at 35 °C followed by a hexane/EtOAc step gradient silica column of the crude extract and reversed phase semi-prep HPLC) and characterize (1D and 2D NMR) fucoxanthin (reference standard) in order to develop an analytical method for its determination in selected diverse brown algae commonly found in South Africa. The HPLC [Column: Phenomenex® Synergi™ (250 x 3.0 mm i.d); Mobile phase: ACN/H2O (95:5)] method developed for this analysis was validated according the guidelines set by the International Conference on Harmonization (ICH). Fifteen species were then assessed for fucoxanthin content (μg/g of dried weight) using the developed method. Stability studies on fucoxanthin were also carried out to assess photo- and pH degradation of fucoxanthin. Zonaria subarticulata (KOS130226-18) from Kenton-On-Sea beach and Sargassum incisifolium (PA130427-1) from Port Alfred beach were found to be the highest producers of fucoxanthin with 0.50 mg/g and 0.45 mg/g dried weight respectively. Fucoxanthin was found to be both photo-labile and sensitive to both acidic and basic pH environments. However, the pigment was more photostable in pure as opposed to extract form and also showed to be more stable at pH 10.0. Our findings show that Z. subarticulata and S. incisifolium could be reliable sources of fucoxanthin and can be considered as the algae to use in optimized extraction procedures in further studies. Also, when working with fucoxanthin, it is important to protect it from light. Any consideration of taking fucoxanthin preparation orally (as a nutraceutical) should consider protecting the active from the harsh conditions of the gastrointestinal tract. Any upscale production of fucoxanthin from seaweed should consider variations such as geographical, seasonal, lifecycle stage, etc. of identified algae as these may be important factors in obtaining effective concentrations of fucoxanthin.
- Full Text:
- Date Issued: 2015
Analysis of the anti-cancer activity of novel indigenous algal compounds in breast cancer: towards the development of a model for screening anti-cancer stem cell activity
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
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