Improvement of vitamin B12 status with Spirulina supplementation in Wistar rats validated through functional and circulatory markers.

Spirulina evaluated as a source of vitamin B12 through the modulation of vitamin B12 deficiency mediated physiological and biochemical changes in experimental animals. The B12 deficient male weanling Wistar rats were fed with Spirulina-supplemented diet for 10 weeks. An increase in urinary methylmalonic acid (22.70 ± 4.08 µmol/moles of creatinine) and plasma homocysteine (16.55 ± 0.48 µmol/L) levels in the B12 deficient group was observed, while these were equal to control in the Spirulina fed group (8.71 ± 0.48 µmol/mol of creatinine and 6.88 ± 1.18 µmol/L, respectively). The vitamin B12 levels in serum (874.27 ± 89.69), plasma (615.53 ± 26.5 pg/ml), kidney (10.19 ± 1.066 ng/g), and liver tissues (6.37 ± 0.62 ng/g) in the Spirulina fed group were similar to control. Severe atrophic changes in the testes and altered tissue architecture in lung and spleen as seen in the B12 deficient group were normalized in the Spirulina fed group. The study validates that Spirulina can improve the vitamin B12 status. PRACTICAL APPLICATIONS: The present study showed that the supplementation of Spirulina in the diet of vitamin B12 deficient rats leads to the normalization of vitamin B12 deficiency-induced circulatory and functional biomarkers along with biochemical and histological changes. Vegetarian sources for vitamin B12 are limited and the results presented here provide scientific validation for the use of Spirulina as a potential vegetarian source of bioavailable vitamin B12 .

Atheroprotective effect of novel peptides from Porphyridium purpureum in RAW 264.7 macrophage cell line and its molecular docking study.

OBJECTIVE: To explore the atherogenic foam cell prevention efficiency of two dipeptides purified from Porphyridium purpureum on RAW 264.7 cell line and to study its molecular interaction through molecular docking. RESULT: P. purpureum consists of 29.9% protein and 2.98% phycoerythrin on a dry weight basis. The two dipeptides namely of Histidine-Glutamic acid (HE) and Glycine-Proline (GP) isolated from the total protein and purified phycoerythrin of P. purpureum respectively, were evaluated for atherogenic foam cell prevention capacity in RAW 264.7 cell line. The IC5O values of peptides were found to be 91.2 ± 1.81 µg/ml (GP), 103.3 ± 4.8 µg/ml (HE) in MTT assay. The two peptides reduce the foam cell formation, intracellular lipid accumulation (cholesterol and triglycerides) and the secretion of TNF-α and IL-6 which are inflammatory cytokines in RAW 264.7 cell line at non-cytotoxic concentrations. A molecular interaction study proposed the binding pose for GP and HE peptides targeting the scavenging receptors CD36, SRA1, and Map Kinase p38 (a protein mediator). CONCLUSIONS: The cell line and molecular docking study indicated that among the two dipeptides, peptide GP has the highest atherogenic foam cell prevention efficiency.

Culture media optimization of Porphyridium purpureum: production potential of biomass, total lipids, arachidonic and eicosapentaenoic acid.

Porphyridium purpureum a red marine microalga is known for phycobiliproteins (PB), polyunsaturated fatty acids and sulphated exopolysaccharides. In the present study, effects of media constituents for the production of different polyunsaturated fatty acids from P. purpureum were considered using a response surface methodology (RSM). A second order polynomial was used to predict the response functions in terms of the independent variables such as the concentrations of sodium chloride, magnesium sulphate, sodium nitrate and potassium dihydrogen phosphate. The response functions were production of biomass yield, total lipid and polyunsaturated fatty acids like arachidonic acid (AA 20:4) and eicosapentaenoic acid (EPA 20:5). Results corroborated that maximum Biomass (0.95 gL(-1)) yield was at the concentrations of sodium chloride (14.89 gL(-1)), magnesium sulfate (3.93 gL(-1)) and sodium nitrate (0.96 gL(-1)) and potassium dihydrogen phosphate (0.09 gL(-1)). Optimum total lipid (17.9 % w/w) and EPA (34.6 % w/w) content was at the concentrations of sodium chloride (29.98 gL(-1)), magnesium sulfate (9.34 gL(-1)) and sodium nitrate (1.86 gL(-1)). Variation in concentration of potassium dihydrogen phosphate for both lipid (0.01gL(-1)) and EPA content (0.20 gL(-1)) was observed. The optimum conditions for biomass, total lipid, AA and EPA varied indicating their batch mode of growth and interaction effect of the salt.

Characterization of vitamin B12 in Dunaliella salina.

Vitamin B12 is one of nature's complex metabolite which is industrially produced using certain bacteria. Algae could be an alternative source of vitamin B12 and in this study, vitamin B12 from a halotolerant green alga, Dunaliella salina V-101 was purified and characterized. The extract of Dunaliella was purified by passing through Amberlite XAD-2 and EASI-extract vitamin B12 immunoaffinity column. The total vitamin B12 content in purified sample fractions was 42 ± 2 µg/100 g dry weight as determined by the chemiluminescence method which was almost close to 49 ± 2 µg/100 g dry weight as estimated by microbiological method. Further quantification of total vitamin B12 using gold nanoparticle (AUNPs) based aptamer showed 40 ± 0.8/100 g dry weight. There was a good correlation among all the methods of quantification. Adenosylcobalamin, a form of vitamin B12 which is a cofactor for methylmalonyl CoA mutase was identified by HPLC. Upon quantification, Dunaliella was found to contain 34 ± 4 µg of adenosylcobalamin for 100 g dry biomass. Authenticity of adenosylcobalmin was confirmed by tandem mass spectrometry (MS/MS), selected ion recording (SIR) and multiple reaction monitoring (MRM) studies.

Methylcobalamin--a form of vitamin B12 identified and characterised in Chlorella vulgaris.

Vitamin B12 is among the most essential biomolecules required for crucial metabolic processes in humans. Vitamin B12 was extracted from Chlorella vulgaris biomass under aqueous conditions, partially purified by passing the extract through amberlite XAD-2, Sep-Pak columns, and further purified by HPLC. The target peak eluent was subjected to characterisation by tandem mass spectrometry (MS/MS), selected ion recording (SIR) and multiple reaction monitoring (MRM) and identified as methylcobalamin (Me-Cbl). Quantification of Me-Cbl was carried out by microbiological and chemiluminescence methods, and found to be 29.87±2 µg/100 g and 26.84±2 µg/100 g dry weight, respectively. The presence of Me-Cbl was further substantiated using gold nanoparticle (AuNPs) based aptamer analysis, and found to be 28.02±2 µg/100 g dry weight. Good similarity was observed among all the methods. Methylcobalamin, a form of vitamin B12 was identified in C. vulgaris and this finding enhances its use as a nutritional supplement.

Effective inhibition of skin cancer, tyrosinase, and antioxidative properties by astaxanthin and astaxanthin esters from the green alga Haematococcus pluvialis.

Astaxanthin mono- (AXME) and diesters (AXDE) were characterized and examined for anticancer potency with total carotenoids (TC) and astaxanthin (AX) against UV-7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer model in rat. At 200 µg/kg bw, AXDE and AXME reduced UV-DMBA-induced tumor incidences up to 96 and 88%, respectively, when compared to AX (66%) and TC (85%). UV-DMBA has been known to generate high levels of free radicals and tyrosinase enzyme, leading to characteristic symptoms of skin pigmentation and tumor initiation. Intriguingly, ~7-fold increase in tyrosinase and 10-fold decrease in antioxidant levels were normalized by AXDE and AXME as opposed to only ~1.4-2.2-fold by AX and TC, respectively. This result together with the appearance of 72 and 58 ng/mL of retinol in the serum of respective AXE-treated (AXDE + AXME) and AX-treated animals suggested that better anticancer potency of AXEs could be due to increased bioavailability.

Purification, identification, and characterization of methylcobalamin from Spirulina platensis.

The present study reports methylcobalamin in Spirulina platensis using high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), microbiological assay, chemiluminescence assay, liquid chromatography-mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS). Extraction of vitamin B12 from S. platensis was carried out without using cyanide. Partial purification was achieved using Amberlite XAD-2 followed by elution with 80% (v/v) methanol. Activated charcoal facilitated removal of impurities in S. platensis extract and in further purification of vitamin B12. The purified fraction was identified to contain methylcobalamin as analyzed by HPLC and TLC. Authenticity of methylcobalamin was further confirmed by LC-MS and MS/MS. Quantitation of methylcobalamin in a test sample of S. platensis biomass was performed using microbiological assay and chemiluminescence assay and was found to be 38.5±2 and 35.7±2 µg/100 g of dry biomass, respectively.

Ulcer preventive and antioxidative properties of astaxanthin from Haematococcus pluvialis.

The anti-ulcer properties of astaxanthin fractions such as total carotenoid and astaxanthin esters from Haematococcus pluvialis were evaluated in ethanol-induced gastric ulcers in rats. Since oxygen radical release is a pathogenic factor of ethanol-induced gastric damage, astaxanthin - a free radical scavenger, was investigated as a potential ulcer preventive agent. Astaxanthin fractions - total carotenoid and astaxanthin esters were orally administered to experimental rats at 100, 250 and 500 microg/kg b.w. prior to ulcer induction. Alcian blue binding assay indicates that, total carotenoid and astaxanthin esters at 500 microg/kg b.w could protect gastric mucin approximately 40% and 67% respectively. Pre-treatment with astaxanthin esters, also resulted in significant increase in antioxidant enzyme levels - catalase, superoxide dismutase, and glutathione peroxidase in stomach homogenate. Histopathological examination substantiated the protective effect of astaxanthin in pre-treated rats. The increased antioxidant potencies such as free radical scavenging activity with an IC(50) of approximately 8 microg/ml and reducing power abilities (59 x 10(3) U/g) in vitro, reveal that H. pluvialis astaxanthin may protect gastric mucosal injury by antioxidative mechanism. In addition, approximately 23 fold increased lipoxygenase-inhibitory property, in comparison with standard astaxanthin and significant H(+), K(+)-ATPase-inhibitory activity of astaxanthin esters, in comparison with known proton pump blocking anti-ulcer drug - omeprazole, may envisage the potential gastroprotective effect by regulating the gastric mucosal injury and gastric acid secretion by the gastric cell during ulcer disease.

Regulation of carotenoid biosynthetic genes expression and carotenoid accumulation in the green alga Haematococcus pluvialis under nutrient stress conditions.

Haematococcus pluvialis, a green alga, accumulates carotenoids, predominantly astaxanthin, when exposed to stress conditions. In the present work, changes in the pigment profile and expression of carotenogenic genes under various nutrient stress conditions and their regulation were studied. Nutrient stress and higher light intensity in combination with NaCl/sodium acetate (SA) enhanced total carotenoid and total astaxanthin content to 32.0 and 24.5 mg g(-1) of dry biomass, respectively. Expression of carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), lycopene cyclase (LCY), beta-carotene ketolase (BKT), and beta-carotene hydroxylase (CHY) were up-regulated under all the stress conditions studied. However, the extent of expression of carotenogenic genes varied with stress conditions. Nutrient stress and high light intensity induced expression of astaxanthin biosynthetic genes, BKT and CHY, transiently. Enhanced expression of these genes was observed with SA and NaCl/SA, while expression was delayed with NaCl. The maximum content of astaxanthin recorded in cells grown in medium with SA and NaCl/SA correlated with the expression profile of the astaxanthin biosynthetic genes. Studies using various inhibitors indicated that general carotenogenesis and secondary carotenoid induction were regulated at both the transcriptional and the cytoplasmic translational levels. The induction of general carotenoid synthesis genes was independent of cytoplasmic protein synthesis while BKT gene expression was dependent on de novo protein synthesis.

Differential expression of carotenogenic genes and associated changes in pigment profile during regeneration of Haematococcus pluvialis cysts.

Haematococcus pluvialis is a green alga known to accumulate astaxanthin in extra-plastidic lipid vesicles under stress conditions. The present study revealed the influence of few cultural parameters and temperature treatments on regeneration efficiency of red cysts along with changes in pigment profile and expression of carotenogenic genes during regeneration. Regeneration efficiency has been improved by incubating less aged cyst cells in a medium containing ammonium carbonate, 16:8 light-dark cycle with a light intensity of 30 mumol m(-2) s(-1). During regeneration, there was a decrease in total astaxanthin, total carotenoids, and carotenoid to chlorophyll ratio, and increase in beta-carotene, lutein, total chlorophyll, and chlorophyll a to b ratio. Expression analysis revealed the presence of transcripts of carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), lycopene cyclase (LCY), beta-carotene ketolase (BKT), and beta-carotene hydroxylase (CHY) in cyst cells, and these transcripts were up regulated transiently upon transfer to favorable conditions. As the culture growth progressed, carotenogenic gene expressions were decreased and reached basal expression levels of green motile vegetative cells. In addition, this is the first report of detection of carotenogenic gene transcripts in red cysts, and their differential expression during regeneration. The present study suggests the use of red cysts as alternate inoculum for mass cultivation to combat protozoan predation.

Isolation and characterization of hydrocarbon producing green alga Botryococcus braunii from Indian freshwater bodies

Samples of green colonial unicellular microalga Botryococcus braunii were collected from Bear Shola Falls at Kodaikanal (latitude 10.31 N and longitude 77.32 E), Tamil Nadu, India. Specimens were isolated, cultured and examined for its hydrocarbon content, morphological features and DNA structural resemblance with the known strain to confirm its identity. Inter simple sequence repeats (ISSR) finger printing revealed strong genetic similarity among the authentic strain (B. braunii N-836) and the Indian isolated strain (B. braunii CFTRI- Bb1) from Kodaikanal. The type of hydrocarbons produced by the Kodaikanal isolates were analyzed and identified as saturated hydrocarbons in the range of C21 to C33 by GCMS. Tetracosane and octacosane were found as the major components among the saturated hydrocarbons produced by this alga, constituting 17.6 percent and 14.8 percent respectively. Hydrocarbon content of the organism was in the range of 13-18 percent of its dry biomass. The fat content of the organism was found to be 22 percent (w/w). Palmitic and oleic acids were found to be major fatty acids produced by the alga. Lutein and beta-carotene were found to be the major carotenoids and constituted about 64.1 percent and 25.1 percent respectively of the total carotenoids. Based on ISSR finger printing and hydrocarbon analyses the new algal strain from Kodaikanal was identified as Botryococcus braunii.

Antioxidant activity of Botryococcus braunii extract elucidated in vitro models.

Botryococcus braunii is a green colonial microalga that is used mainly for the production of hydrocarbons, exopolysaccharides, and carotenoids. In the present study, the antioxidant properties of acetone extracts of B. braunii were evaluated using in vitro model systems such as 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxy radical scavenging, and lipid peroxidation in human low-density lipoprotein and rat tissues. Acetone extracts of B. braunii (equivalent to 10 ppm total carotenoid) exhibited 71 and 67% antioxidant activity in DPPH and hydroxyl radical scavenging model systems, respectively. Similarly, the extract also showed 72, 71, and 70% antioxidant activity in the liver, brain, and kidney of rats. Low-density lipoprotein oxidation induced by Cu2+ ions was also protected (22, 38, and 51%) by the algal extract in a dose-dependent manner (4, 6, and 8 ppm levels of total carotenoid). Thiobarbituric acid reactive substances concentration in the blood, liver, and kidney of rats was also significantly decreased in B. braunii treated samples compared with those of control. Carotenoids (violaxanthin, astaxanthin, lutein, zeaxanthin, chlorophylls a and b, and alpha, beta-carotene) identified in the B. braunii acetone extract may be exhibiting antioxidant activity. Among the carotenoids, lutein represents more than 75% of the total carotenoids. B. braunii extract was shown to be effective for protecting biological systems against various oxidative stresses in vitro. This is the first report on the antioxidant properties of B. braunii.