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

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.