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Production of Astaxanthin from Cultured Green Alga Haematococcus pluvialis and its Biological Activities

Ranga Rao, A. (2011) Production of Astaxanthin from Cultured Green Alga Haematococcus pluvialis and its Biological Activities. PhD thesis, University of Mysore.

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Abstract

Micro algal biotechnology has gained importance due to its potential to produce bioactive molecules. Among the micro algae, Haematococcus pluvialis is a green alga belonging to chlorophyceae, which produces astaxanthin & its esters in large quantities under stress conditions. Carotenoids are being intensively investigated regarding their potential to prevent diseases and vitamin A deficiency. The present study is focused on the production of astaxanthin from Haematococcus pluvialis and its biological activities. This thesis consists of effect of various stress conditions on the production of astaxanthin in Haemtococcus pluvialis; isolation and characterization of astaxanthin & its esters; evaluation of stability of astaxanthin in various edible oils; safety evaluation of biomass; evaluation of its biological activity; bioavailability and vitamin A conversion; anticancer properties in rat experimental models. Culture of Haematococcus was done using appropriate media & incubation conditions in laboratory. In sodium nitrate (25 mM) treated culture, the maximum biomass yield was 3.3 g/l, total carotenoid content was 2.9% and astaxanthin content was 2.5%, whereas in a potassium chloride 16 mM supplemented culture the biomass yield was 2.5 g/l, total carotenoid content was 2% and astaxanthin was 1.87%. Among various carbon source studied, ammonium carbonate (3 mM) treated culture showed the biomass yield of 2.9 g/l, total carotenoid was 2.6% and astaxanthin was 2.2%. Among the various solvents used, ethylacetate, isopropyl alcohol: hexane (1:1) and acetone were found to be efficient for extraction of the carotenoids from the Haematococcus cells. Carotenoids, astaxanthin & its esters were quantified and identified by following techniques such as thin layer chromatography, high performance liquid chromatography and liquid chromatography mass spectrum using APCI mode. The major carotenoids were quantified in Haematococcus biomass and identified as astaxanthin & its esters followed by neoxanthin, violaxanthin, astaxanthin, lutein, zeaxanthin, β-cryptoxanthin and β-carotene. These were confirmed by their retention times and the absorption spectra of the respective reference standards. Based on the mass spectral data obtained from an astaxanthin mono-di esters such as ME C16:0, ME C17:2, ME C17:1, ME C17:0, ME C18:4, ME C18:3, ME C18:2, ME C18:1, DE C16:0/C16:0, DE C16:0/C18:2, DE C18:1/C 18:3, DE C18:1/C18:2 and DE C18:1/C18:1 were identified in H. pluvialis. Edible oils were also used as a vehicle of carotenoid delivery. To achieve maximum extractability various oils were tried. The maximum extractability of carotenoids was found in palm oil and olive oil. Astaxanthin & its esters were further confirmed by recording unambiguous 13C NMR, 1H NMR, HSQC 2D, NOESY 2D and COSY 2D NMR. Further, COSY and NOSEY spectrum confirmed the existence of both cis and trans forms of astaxanthin & its esters in H. pluvialis. The Haematococcus extracts exhibited 80% antioxidant activity in -carotene linoleate model system (-CLAMS), 1, 1-diphenyl -2-picrylhydrazyl (DPPH) method, and hydroxyl radical scavenging model systems. Antibacterial properties of H. v pluvialis extracts were evaluated against selected bacteria. Among the different solvent extracts of H. pluvialis, chloroform extract exhibited highest antibacterial effect followed by ethyl acetate extract. Astaxanthin & its esters also showed significant antioxidant activity and hepatoprotective ability in carbon tetrachloride induced albino rats. Among the groups of experimental rats, the one which was treated with astaxanthin esters at 250 μg/kg b.w. showed maximum hepatoprotective activity i.e protection when compared to control treated group. However, pretreatment of rats with 250 μg/kg b.w of astaxanthin esters preserved catalase, peroxidase and SOD activities, when compared with control values in untreated animals. Feeding trials, for experimental rats were carried out to assess the safety and efficacy of Haematococcus biomass as a source of astaxanthin. The study revealed that Haematococcus biomass was safe in both single and repeated dose in experimental rats. In vivo experiments demonstrated that astaxanthin from Haematococcus was effective in retinol formation and its accumulation in serum and liver. Time course study of carotenoids in rats after administration of Haemtococcus biomass showed peak levels in plasma, liver and eyes at 2, 4 & 6 h respectively. In the repeated dose study, the astaxanthin levels in plasma, liver and eyes of rat over 15 days after intubation of H. pluvialis biomass was recorded. In liver, astaxanthin levels were 1.7 and 1.8 fold higher than in plasma and eyes. In plasma and liver, antioxidant enzymes catalase, SOD, peroxidase activity was higher in astaxanthin treated rats when compared to untreated control rats. The anticancer properties of astaxanthin & its esters was also studied in human glioma cell lines (LN-229, HNGC2) and liver hepatocellular carcinoma cell line (HepG2). The apoptosis cells were observed under phase-contrast and confocal microscope. Anticancer properties of astaxanthin & its esters were evaluated against skin carcinogenesis, which is reported for the first time. Tyrosinase enzyme activity was inhibited by using astaxanthin & its esters using in vitro models. Pre feeding of rats with astaxanthin esters 250 μg/kg b.w. prior to treatment with carcinogen showed 3-4 fold reduction in tumor index. Hematological and histopathological studies were examined which substantiate the protective role of astaxanthin esters. The plasma astaxanthin and retinol content were estimated in serum and liver homogenate. Biochemical changes like lipid peroxidation, catalase, superoxide dismutase, glutathione reductase activities were estimated in control, cancer induced animal groups. Astaxanthin metabolites isolated from plasma, liver from in vivo studies were characterized by HPLC and LC-MS (APCI) techniques to elucidate their structure. Epoxy carotenoids were tentatively identified in liver and plasma. This study emphasizes the influence of astaxanthin and its beneficial effects on the metabolism in experimental animals. Based on animal experiments and bioavailability studies, it was evident that astaxanthin & its esters could possibly be safely used as nutritional, antioxidant, anticancer agent.

Item Type: Thesis (PhD)
Uncontrolled Keywords: micro algae, Haematococcus pluvialis, Micro algal biotechnology, astaxanthin
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 14 Carotenoid Chemistry
500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology > 01 Algae
Divisions: Plant Cell Biotechnology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 15 May 2012 06:40
Last Modified: 15 May 2012 06:40
URI: http://ir.cftri.com/id/eprint/10754

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