Biomedical subjects

G A Ravishankar

Publications and source records attributed to G A Ravishankar.

At least 19 recordsLinked to original sources

PubMed2007

Culture media optimization for growth and phycoerythrin production from Porphyridium purpureum.

Porphyridium spp. is a red micro alga and is gaining importance as a source of valuable products viz., phycobiliproteins (PB), sulfated exopolysaccharides, and polyunsaturated fatty acids with potential applications in the food and pharmaceutical industries. In the present study, the effects of the major media constituents of Porphyridium species were studied using response surface methodology (RSM) on biomass yield, total PB and the production of phycoerythrin (PE). A second order polynomial can be used to predict the PB and PE production in terms of the independent variables. The independent variables such as the concentrations of sodium chloride, magnesium sulfate, sodium nitrate, and dipotassium hydrogen phosphate influenced the total PB and PE production. The optimum conditions showed that total PB was 4.8% at the concentration of sodium chloride 26.1 g/L, magnesium sulfate 5.23 g/L, sodium nitrate 1.56 g/L, and dipotassium hydrogen phosphate 0.034 g/L. In case of optimum PE production (3.3%), the corresponding values are 29.62, 6.11, 1.59, and 0.076 g/L, respectively. PE production depends greatly on the concentrations of chloride, nitrate, and sulfate as well as phosphate of which the former possess the maximum effect.

Biomass↗

PubMed2006

An efficient method for extraction of astaxanthin from green alga Haematococcus pluvialis.

Haematococcus pluvialis is one of the potent organisms for production of astaxanthin, a high value ketocarotenoid. Astaxanthin is accumulated in thick-walled cyst cells of Haematococcus. The thick cell wall is made up of sporopollenin-like material, algaenan, which hinders solvent extraction of astaxanthin. In the present study, an improved method for extraction of astaxanthin without homogenization of cells is reported. Extractability of astaxanthin from cyst cells was evaluated by treating cells with various solvents and pretreating the cells with organic and mineral acids at 70 degrees C followed by acetone extraction. Hydrochloric acid treatment facilitated 86-94% extractability of astaxanthin. Treatment time, temperature, and concentration of the acid were found to be critical factors for maximum extractability. The treatment did not affect the astaxanthin ester profile and the treated cells can be preserved until further use.

Acetone↗

PubMed2006

Characterization of capsaicin synthase and identification of its gene (csy1) for pungency factor capsaicin in pepper (Capsicum sp.).

Capsaicin is a unique alkaloid of the plant kingdom restricted to the genus Capsicum. Capsaicin is the pungency factor, a bioactive molecule of food and of medicinal importance. Capsaicin is useful as a counterirritant, antiarthritic, analgesic, antioxidant, and anticancer agent. Capsaicin biosynthesis involves condensation of vanillylamine and 8-methyl nonenoic acid, brought about by capsaicin synthase (CS). We found that CS activity correlated with genotype-specific capsaicin levels. We purified and characterized CS ( approximately 35 kDa). Immunolocalization studies confirmed that CS is specifically localized to the placental tissues of Capsicum fruits. Western blot analysis revealed concomitant enhancement of CS levels and capsaicin accumulation during fruit development. We determined the N-terminal amino acid sequence of purified CS, cloned the CS gene (csy1) and sequenced full-length cDNA (981 bp). The deduced amino acid sequence of CS from full-length cDNA was 38 kDa. Functionality of csy1 through heterologous expression in recombinant Escherichia coli was also demonstrated. Here we report the gene responsible for capsaicin biosynthesis, which is unique to Capsicum spp. With this information on the CS gene, speculation on the gene for pungency is unequivocally resolved. Our findings have implications in the regulation of capsaicin levels in Capsicum genotypes.

Amino Acid Sequence↗

PubMed2006

Effect of salinity on growth of green alga Botryococcus braunii and its constituents.

Growth of Botryococcus braunii (race 'A') and production of its constituents viz, hydrocarbon, carbohydrate, fatty acid, and carotenoids were influenced by different levels of salinity. Under salinity at 34 mM and 85 mM, 1.7-2.25-fold increase in the relative proportion of palmitic acid and two fold increase in oleic acid were observed. A twofold increase in carotenoid content was noticed at 85 mM salinity with lutein (75% of total carotenoid) as the major carotenoid followed by beta-carotene. The increase in biomass yields and changes in other constituents indicated the influence of salinity and the organism's adaptability to the tested levels of salinity (17 mM to 85 mM).

Biomass↗

PubMed2006

Evaluation of PCR-based methods for isolating flanking regions of genes.

Several polymerase chain reaction (PCR)-based methods are available for isolation of unknown genomic fragments. In the present study, a comparative evaluation of a few methods of ligation-mediated PCR methods and a ligation-independent one were made by isolating promoter fragment for N-methyltransferase gene involved in the caffeine biosynthetic pathway of Coffea canephora. The benefits of tertiary PCR and the effects of a 4-base cutting restriction endonuclease on the size of the PCR products obtained were demonstrated in one of the ligation-mediated PCR methods. The methods adopted in this study differed in the sizes of the 5'-flanking regions obtained. The efficiencies of various methods used reflect the inherent limitations of the PCR-based methods for isolation of unknown flanking regions.

5' Flanking Region↗

PubMed2006

Antioxidant property of Decalepis hamiltonii Wight & Arn.

Aromatic edible root of D. hamiltonii was subjected to the extraction of the antioxidant rich fraction. Different parts of root namely whole tuber, peel, tuber without peel and medullary portion were extracted with dichloromethane (European Patent No. W02005063272). The extract was found to contain flavor compound 2-hydroxy-4-methoxybenzaldehyde (2H4MB), which was identified by TLC and GC. Medullary portion was found to be rich in 2H4MB, (73.73 mg g(-1) dry tissue) followed by peel, containing 68.34 mg g(-1) 2H4MB. Different concentration of dichloromethane extracts were subjected for antioxidant assay by DPPH (1,1 dihydroxy 2-picryl hydrazyl) method, this has shown 44, 46.7% radical scavenging activity in case of medullary, peel extracts and 67.3% in case of pure 2-hydroxy-4-methoxybenzaldehyde at 100 ppm concentration, whereas ascorbic acid used as standard showed 94.3% activity. In beta-carotene linoleate model system (b-CLAMS) 43.46 and 45.7% antioxidant activity was observed in medullary and peel extracts at 100 ppm concentrations respectively, whereas standard 2-hydroxy-4-methoxybenzaldehyde exhibited 69.64% at 100 ppm and BHA (butylated hydroxyl anisole) 90.1% activity also at 100-ppm level. Similarly hydroxyl radical scavenging activity was found to be 48.36, 46.86, 48.26 and 73.60% in whole tuber, medullary, peel and standard 2-hydroxy-4-methoxy benzaldehyde respectively at 100 ppm levels. This is the first report on the antioxidant activity of D. hamiltonii. Results have shown that 2H4MB is one of the major constituents responsible for antioxidant activity. Hence the extract of D. hamiltonii can be utilized for the production of antioxidant rich fractions required for various health benefits.

Antioxidants↗

PubMed2005

Stable transformation and direct regeneration in Coffea canephora P ex. Fr. by Agrobacterium rhizogenes mediated transformation without hairy-root phenotype.

A system for genetic transformation of Coffea canephora by co-cultivation with Agrobacterium rhizogenes harbouring a binary vector has been developed. The objective of the present study was the genetic transformation and direct regeneration of transformants through secondary embryos bypassing an intervening hairy root stage. Transformants were obtained with a transformation efficiency up to 3% depending on the medium adjuvant used. A. rhizogenes strain A4 harbouring plasmid pCAMBIA 1301 with an intron uidA reporter and hygromycin phosphotransferase (hptII) marker gene was used for sonication-assisted transformation of Coffea canephora. The use of hygromycin in the secondary embryo induction medium allowed the selection of transgenic secondary embryos having Ri T-DNA along with the T-DNA from the pCAMBIA 1301 binary vector. In addition transgenic secondary embryos devoid of Ri-T-DNA but with stable integration of the T-DNA from the binary vector were obtained. The putative transformants were positive for the expression of the uidA gene. PCR and Southern blot analysis confirmed the independent, transgenic nature of the analysed plants and indicated single and multiple locus integrations. The study clearly demonstrates that A. rhizogenes can be used for delivering transgenes into tree species like Coffea using binary vectors with Agrobacterium tumefaciens T-DNA borders.

Blotting, Southern↗

PubMed2005

Digital image processing-an alternate tool for monitoring of pigment levels in cultured cells with special reference to green alga Haematococcus pluvialis.

A method for analyzing carotenoid content in Haematococcus pluvialis, a green alga was developed using digital image processing (DIP) and an artificial neural network (ANN) model. About 90 images of algal cells in various phases of growth were processed with the tools of DIP. A good correlation of R(2)=0.967 was observed between carotenoid content as estimated by analytical method and DIP. Similar correlation was also observed in case of chlorophyll. Since the conventional methods of carotenoid estimation are time consuming and result in loss of pigments during analysis, DIP method was found to be an effective online monitoring method. This method will be useful in measurement of pigments in cultured cells.

Algorithms↗

PubMed2005

Isolation of promoter for N-methyltransferase gene associated with caffeine biosynthesis in Coffea canephora.

N-Methyltransferases (NMTs) catalyze the three SAM dependent sequential methylation of xanthosine, producing caffeine in Coffea species. In the present work, a PCR based genome walking method was adopted to isolate and clone the promoter for the NMT gene. Inspection of the promoter sequence revealed the presence of several motifs important for the regulation of the gene expression. The whole fragment was fused to the beta-glucuronidase (gus) reporter gene and used in Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. GUS assays proved that the isolated promoter was able to direct the expression of the reporter gene in transgenic tobacco. Based on the promoter sequence, primer was designed and the genomic fragment comprising the promoter and its corresponding gene was amplified and cloned. Sequencing of one of the genomic clones revealed the presence of four exons and three introns in NMT gene. The differences in the restriction pattern among the genomic clones were studied using PCR-RFLP. This is the first report of cloning of the promoter for a gene involved in caffeine biosynthetic pathway and it opens up the possibility of studying the molecular mechanisms that regulate the production of caffeine.

Agrobacterium tumefaciens↗

PubMed2005

Chemical composition, iron bioavailability, and antioxidant activity of Kappaphycus alvarezzi (Doty).

Kappaphycus alvarezzi, an edible seaweed from the west coast of India, was analyzed for its chemical composition. It was found that K. alvarezzi is rich in protein (16.24% w/w) and contains a high amount of fiber (29.40% w/w) and carbohydrates (27.4% w/w). K. alvarezzi showed vitamin A activity of 865 mug retinal equivalents/100 g of sample. It contained a higher quantity of unsaturated fatty acids (44.50% of the total), in which relative percentage of oleic acid was 11%, cis-heptadecanoic acid 13.50%, and linoleic acid 2.3% and 37.0% of saturated fatty acids (mainly heptadecanoic acid). K. alvarezziwas also found to be good source of minerals, viz 0.16% of calcium, 0.033% of iron, and 0.016% of zinc, which are essential for various vital biological activities. Bioavailability of iron by in vitro methods showed a higher efficiency in intestinal conditions than in stomach conditions. Ascorbic acid influenced higher bioavailability of iron. Successive extracts of n-hexane, acetone, ethyl acetate, ethanol, and direct extractables of chloroform/methanol (1:1 and 2:1) were screened for antioxidant activity using a beta-carotene linoleic acid model system (B-CLAMS), DPPH (alpha,alpha-diphenyl-beta-picrylhydrazyl) model system and hydroxyl radical scavenging activity. The chloroform/methanol (2:1) extract has shown 82.5% scavenging activity at 1000 ppm. Acetone fraction extracts at the 1000 ppm level showed 63.31% antioxidant activity in beta-carotene linoleic acid system. The acetone extract showed 46.04% scavenging activity at 1000 ppm concentration. In the case of hydroxyl radical scavenging activity, all the extracts showed better activity at the concentrations of 25 and 50 ppm, where at the 50 ppm level ethyl acetate extract showed 76.0%, acetone 75.12%, and hexane 71.15% activity, respectively. Results of this study suggest the utility of K. alvarezzi (Eucheuma) for various nutritional products, including antioxidant for use as health food or nutraceutical supplement.

Antioxidants↗

PubMed2005

Uptake and degradation of DDT by hairy root cultures of Cichorium intybus and Brassica juncea.

Hairy root cultures of Cichorium intybus and Brassica juncea were used for their ability to uptake and degrade DDT (1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane). After 24 h of 14C DDT treatment, only 12-13% of the total applied radioactivity was detected in the culture media, indicating the efficient uptake of DDT by the hairy roots. The majority of the applied radioactivity was associated with the roots. DDT was degraded to various other products such as DDD, DDE and DDMU, along with some unknown compounds by hairy root cultures, which were detected by thin layer chromatography (TLC) and autoradiography. The rate of in situ degradation was found to be higher during the initial stages of culture and the residual 14C DDT in the roots was found to decrease from 77% to 61% over a period of 10-days. There was no spontaneous degradation of 14C DDT in media lacking hairy root cultures or in media with autoclaved hairy roots. This suggests that endogenous root enzymes play a role in the breakdown of 14C DDT. These results suggest the potential applicability and advantage of using these plant species for phytoremediation of persistent xenobiotics such as DDT in an eco-friendly and efficient manner for environmental clean up.

Biodegradation, Environmental↗

PubMed2005

Protective effect of Dunaliella salina-A marine micro alga, against carbon tetrachloride-induced hepatotoxicity in rats.

This is the first report on the hepatoprotective potentials of marine micro algae Dunaliella species. Dunaliella salina, halotolarent green alga was cultivated in modified autotrophic medium. The alga was subjected to light and nutrient stress in order to accumulate (beta-carotene along with other carotenoids. Such beta-carotene enriched yellow cells were fed to rats by mixing with regular feed at the dose of 2.5 and of 5.0gkg(-1) b.w. for 2 weeks. The degree of hepatoprotection was measured up on challenging animals with toxin (2.0gkg(-1) of carbon tetrachloride) by estimation of biochemical parameters like, serum transaminases (serum aspartate transaminase (S)AST and serum alanine transaminase (S)ALT), serum alkaline phosphatase and total protein. The results were compared to animals on normal diet and with group fed with 100mugkg(-1) b.w. of standard all trans beta-carotene. Among the three test groups the group fed with algae of 5.0gkg(-1) body weight, showed maximum protection. The levels of (S)AST and (S)ALT was found to be 61.3+/-6.4 and 80.7+/-5.6%, against 90.8+/-10.5 and 144.7+/-13.9% in case of standard beta-carotene. The protein contents were increased in case of control to 6.1+/-0.7 and the same was found to be significantly less in case of 5.0gkg(-1)Dunaliella fed group, which shown 5.6+/-0.8% total protein. However, the activity of 2.5gkg(-1) was also significant comparatively (P<0.05). The results indicate that Dunaliella, which contains isomeric forms of beta-carotene can act as good antihepatotoxic when compared to synthetic all trans beta-carotene. Dunaliella has shown the presence of both cis and trans isomeric forms of beta-carotene, where as synthetic compounds contain only trans isomer. Hepatoprotectivity may be due to presence of various isomeric forms of carotene and other oxygenated carotenoids (xanthophylls) in algae.

Journal Article↗

PubMed2005

Methyl jasmonate modulated biotransformation of phenylpropanoids to vanillin related metabolites using Capsicum frutescens root cultures.

Normal root cultures of Capsicum frutescens biotransform externally fed precursors, like caffeic acid and veratraldehyde, to vanillin and other related metabolites. The bioconversion of caffeic acid to further metabolites--viz. vanillin, vanillylamine, vanillic acid--was shown to be elicited by treating the cultures with 10 microM methyl jasmonate (MJ). Root cultures treated with MJ accumulated (1.93 times) more of vanillin (20.2 microM on day-3) than untreated ones. A concomitant increase in enzymatic activity of caffeic acid O-methyl transferase (CAOMT, EC 2.1.1.68) was obtained in MJ treated cultures, compared to untreated cultures. After 24 h of MJ treatment, a 13.7-fold increase in CAOMT activity was recorded in root cultures of C. frutescens. Cultures treated with veratraldehyde accumulated more vanillin (78 microM) than caffeic acid fed cultures, 6 days after precursor addition. Capsaicin did not accumulate even after addition of precursors. The efficiencies of biotransformation with caffeic acid and veratraldehyde were 2.2% and 9% with respect to vanillin formation, indicating a possible diversion of the phenylpropanoid pathway towards other secondary metabolites.

Acetates↗

PubMed2005

Comparative evaluation of hepatoprotective activity of carotenoids of microalgae.

The present study deals with evaluation of the hepatotoprotective activity of carotenoids from two well-known microalgae, Spirulina platensis and Dunaliella salina. Carotenoids were extracted in hexane:isopropyl alcohol (1:1 vol/vol) and fed orally in olive oil to Wistar albino rats at a dose of 100 microg/kg of body weight/day (in terms of carotenoids). The degree of hepatoprotection was measured by estimation of biochemical parameters like serum transaminases [serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT)], serum alkaline phosphatase, total albumin, and total protein. The results were compared with those for a control group, a CCl4-induced hepatic damage group, and a group treated with synthetic beta-carotene (all-trans) at the same dose. The protein content of the CCl4-treated group, which received normal diet and a dose of toxin, showed a significant decrease, i.e., 3.92 mg/mL, whereas the protein levels were higher, i.e., 6.96 and 6.32 mg/mL, in the case of the Dunaliella and Spirulina, respectively, carotenoid-treated groups. The CCl4-treated group shown higher activity of transaminases (128.68 units/mL SGPT and 171.52 units/mL SGOT). However, the activity of SGPT was 62.83 units/mL for Dunaliella and 76.83 units/mL for Spirulina, i.e., carotenoids of Dunaliella showed a higher degree of protection. For serum alkaline phosphatase, the standard beta-carotene value was 81.52 units/mL, compared with 84.46 units/mL for the CCl4-treated group; however, natural algal carotenoids yielded 38.45 units/mL (D. salina) and 44.73 units/mL (Spirulina). The total albumin value diminished with CCl4 treatment (2.46 mg/mL); the effect was highest for Dunaliella, followed by the Spirulina carotenoid-treated group. The results clearly indicate that carotenoids from Dunaliella possess better hepatoprotection compared with those from Spirulina. High-performance liquid chromatography of the carotenoids indicated that Spirulina contains only beta-carotene and Dunaliella contains other carotenoids and xanthophyll. The increase in protection with Dunaliella indicates that mixed carotenoids exhibit better biological activity than beta-carotene alone. The results of this study indicate that carotenoids obtained from an algal source have a higher antihepatotoxic effect, compared with synthetic beta-carotene and with beta-carotene alone from a natural source.

Alanine Transaminase↗

PubMed2004

Phytoremediation--a novel and promising approach for environmental clean-up.

Phytoremediation is an eco friendly approach for remediation of contaminated soil and water using plants. Phytoremediation is comprised of two components, one by the root colonizing microbes and the other by plants themselves, which degrade the toxic compounds to further non-toxic metabolites. Various compounds, viz. organic compounds, xenobiotics, pesticides and heavy metals, are among the contaminants that can be effectively remediated by plants. Plant cell cultures, hairy roots and algae have been studied for their ability to degrade a number of contaminants. They exhibit various enzymatic activities for degradation of xenobiotics, viz. dehalogenation, denitrification leading to breakdown of complex compounds to simple and non-toxic products. Plants and algae also have the ability to hyper accumulate various heavy metals by the action of phytochelatins and metallothioneins forming complexes with heavy metals and translocate them into vacuoles. Molecular cloning and expression of heavy metal accumulator genes and xenobiotic degrading enzyme coding genes resulted in enhanced remediation rates, which will be helpful in making the process for large-scale application to remediate vast areas of contaminated soils. A few companies worldwide are also working on this aspect of bioremediation, mainly by transgenic plants to replace expensive physical or chemical remediation techniques. Selection and testing multiple hyperaccumulator plants, protein engineering ofphytochelatin and membrane transporter genes and their expression would enhance the rate of phytoremediation, making this process a successful one for bioremediation of environmental contamination. Recent years have seen major investments in the R&D, which have also resulted in competition of filing patents by several companies for economic gains. The details of science & technology related to phytoremediation have been discussed with a focus on future trends and prospects of global relevance.

Biodegradation, Environmental↗

PubMed2004

Efficient clonal propagation method for Decalepis hamiltonii, an endangered shrub, under the influence of phloroglucinol.

A highly efficient two stage protocol was developed for induction of multiple shoots from single node in vitro shoot tip explants of Decalepis hamiltonii. It was found that phloroglucinol (PG) had synergistic effect on shoot multiplication when added with N6-benzyladenine and gibberellic acid. This protocol uses PG for both multiple shoot induction from nodal explants, elongation of primary shoots and initiation of adventitious shoot formation from primary shoots, which was more in presence of triacontanol (TRIA). Maximum number of shoots per culture was observed on the medium containing N6-benzyladenine (1.1 microM; BA), GA3 (5.8 microM) and PG (800 microM). Sub-culturing of the shoots onto MS medium containing optimum concentration of BA (5.6 microM), PG (200 microM) and TRIA (0.011 microM) produced elongated shoots along with secondary shoot formation. The long shoots were rooted on alpha-naphthalene acetic acid (5.38 microM; NAA) and PG (400 microM) containing medium. The rooted plantlets were hardened and their field survival rate was 80-90%.

Culture Media↗

PubMed2004

Production of 2-hydroxy-4-methoxybenzaldehyde in roots of tissue culture raised and acclimatized plants of Decalepis hamiltonii Wight & Arn., an endangered shrub endemic to Southern India and evaluation of its performance vis-a-vis plants from natural habitat.

Axillary buds obtained from field grown plants of D. hamiltonii were used to initiate multiple shoots on Murashige and Skoog's medium (MS) supplemented with 2 mg L(-1) 6-benzyl aminopurine (BA) and 0.5 mg L(-1) indole-3-acetic acid (IAA). Profuse rooting was achieved when the actively growing shoots were cultured on MS medium supplemented with 1.0 mg l(-1) indole-3-butyric acid (IBA). Regenerated plants were grown successfully in the plains, in contrast to wild growth in high altitudes and rocky crevices of hilly regions. Roots of different sizes from one-year-old tissue culture raised field grown plants had the same profile of 2-hydroxy-4-methoxybenzaldehyde as that of wild plants. A maximum of 0.14% and 0.12% 2-hydroxy-4-methoxybenzaldehyde was produced in roots of one year old tissue culture derived plants and greenhouse grown plants respectively.

Benzaldehydes↗

PubMed2004

In vitro clonal propagation of bird eye chilli (Capsicum frutescens Mill.).

An efficient and highly reproducible protocol for micropropagation of bird eye chilli Capsicum frutescens was attempted. Murashige and Skoog (MS) medium containing 0.5-3.0 mgl(-1) of 6-benzyladenine (BA), 2-isopentenyl adenine (2iP), kinetin and 0.5-2.0 mg l(-1) of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) along with 1 gl(-1) activated charcoal (AC) were used for shoot regeneration from both shoot tip and nodal explants. Shoot tip explants (100%) grew well on medium containing 1 mgl(-1) of kinetin and 1 mgl(-1) of IBA. Shoot proliferation (1-3) from nodal explants was effective on this medium. The regenerated shoots with 4-7 nodes had further growth upon sub-culturing onto kinetin (1 mgl(-1)) and IBA (1 mgl(-1)) and rooted simultaneously. The rooted plants were transferred to pots after hardening under controlled conditions. The survival percentage in pots was 80-90%.

Capsicum↗