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ORIGINAL ARTICLES
Functional characterization and expression of folate receptor-
α
in T47D human breast cancer cells
J Renukuntla, S Shah, S.H.S. Boddu, AD Vadlapudi, RK Vadlapatla, D Pal, Ashim K Mitra
July-December 2015, 6(2):52-61
DOI
:10.4103/2394-6555.162441
Purpose:
The objective of this study was to investigate the functional and molecular expression of a carrier mediated system responsible for folate uptake in breast cancer (BC) (T47D) cells and to delineate the mechanism of intracellular regulation of this transport system.
Materials and Methods:
[
3
H]-folic acid uptake was studied in T47D cells with respect to time, pH, temperature, sodium and chloride ion dependency. Inhibition studies were conducted in the presence structural analogs, vitamins, metabolic and membrane transport inhibitors. [
3
H]-folic acid uptake was also determined with varying concentrations of cold folic acid. Uptake kinetics was studied in the presence of various modulators of intracellular regulatory pathways; calcium-calmodulin, protein kinases A and C (PKA and PKC) and protein tyrosine kinase (PTK). Molecular evidence was studied by qualitative and quantitative polymerase chain reaction (PCR) and Western blot analysis.
Results:
Linear increase in [
3
H]-folic acid uptake was observed over 30 min. The process followed saturation kinetics with an apparent K
m
of 11.05 nM, V
max
of 1.54 * 10
−8
μmoles/min/mg proteins and K
d
of 9.71 * 10
−6
/min for folic acid. Uptake process was found to be dependent on pH, sodium ions, chloride ions, temperature and energy. Uptake was inhibited in the presence of structural analogs (cold folic acid, methyltetrahydro folate and methotrexate), but structurally unrelated vitamins did not show any effect. Membrane transport inhibitors such as SITC, DIDS, probenecid and endocytic inhibitor colchicine significantly inhibited the [
3
H]-folic acid uptake process. PKA, PTK and Ca
2+
/calmodulin pathways positively regulate the uptake process. Reverse transcriptase polymerase chain reaction (RT PCR) analysis had shown mRNA expression of folate receptor (FR)-α at 407 bp. Quantitative polymerase chain reaction analysis showed significantly higher FR-α mRNA levels in T47D cells compared to MCF-7 cells and Western blot analysis confirmed the FR-α protein expression at 37 kDa.
Conclusions:
This work demonstrated the functional characterization and molecular presence of FR-α in the T47D cell line. The high expression of FRs in T47D human breast carcinoma cells supports their validity as molecular therapeutic targets in BC.
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7
2,496
983
REVIEW ARTICLE
Cichorium intybus
: A concise report on its ethnomedicinal, botanical, and phytopharmacological aspects
Sneha Das, Neeru Vasudeva, Sunil Sharma
January-June 2016, 7(1):1-12
DOI
:10.4103/2394-6555.180157
Cichorium intybus
L. (chicory) is a Mediterranean plant species belonging to the
Asteraceae
family. Chicory is gaining interests because of its culinary features, nutritional values and medicinal characteristics.
C. intybus
has been implemented in folk medicine from North Africa to South Asia for several 100 years. In Indian medicine, it has been used to treat fever, diarrhea, spleen enlargement, jaundice, liver enlargement, gout, and rheumatism. In China, it is valued for its tonic effects upon the liver and digestive tract. In Germany, chicory has been used as a folk medicine for everyday ailments. Thus,
C. intybus
is a plant of great economic potential due to high concentrations of fructooligosacharide, known as inulin, in its roots, used as a replacement ingredient for sugar and fat. The other various phytoconstituents reported in chicory are sucrose, cellulose, proteins, caffeic acid derivatives, flavonoids, polyphenols, carotenoids, anthocyanins, tannins, coumarins, sesquiterpene lactones, fatty acids, pectin, cholins, benzo-isochromenes, alkaloids, vitamins, amino acids, and minerals. The therapeutic investigations reveal that
C. intybus
is useful for maintaining normal health and has nematicidal, antihepatotoxic, antidiabetic, cardioprotective, antiallergic, antihyperlipidemic, anti-inflammatory, antineoplastic, calcium homeostater, bulking agent, immunostimulatory, prebiotic, protective against pancreatitis, antimicrobial, and antioxidant effects. This review encompasses botany, ethnomedicinal uses, phytoconstituents, pharmacological uses, and toxicity studies of
C. intybus
L.
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6
4,555
694
SYMPOSIUM
In vitro
α-glucosidase and α-amylase inhibition by aqueous, hydroalcoholic, and alcoholic extract of
Euphorbia hirta
L.
Manjur Ali Sheliya, Rayhana Begum, Kolappa Krishna Pillai, Vidhu Aeri, Showkat Rasool Mir, Abuzer Ali, Manju Sharma
January-June 2016, 7(1):26-30
DOI
:10.4103/2394-6555.180156
Background:
Euphorbia hirta
L. (Euphorbiaceae), commonly known as Dudhani, is distributed in the warm region of India and China. Traditionally, it is used in respiratory and gastrointestinal disorders. In addition, the antidiabetic property of the plant was also reported in the literature. This study was designed to evaluate the effect of aqueous, hydroalcoholic, and methanolic extracts (MEHs) of
E. hirta
on α-glucosidase and α-amylase
in vitro
.
Materials and Methods:
Aqueous, hydroalcoholic, and MEHs of
E. hirta
were prepared as per application program interface. In α-glucosidase activity, α-glucosidase (0.1 μ/mL) and substrate, 2.5 mM p-nitrophenyl-α-D-glucopyranoside was used; absorbance was recorded at 405 nm. In α-amylase activity, α-amylase solution (1.0 μ/mL) and substrate, 0.25% starch were used, and absorbance was measured at 540 nm. The IC
50
values were calculated by linear regression.
Results:
All the extracts showed α-glucosidase inhibitory activity comparable to acarbose with MEH having highest inhibitory activity among tested extracts. The observed IC
50
values were 213.63, 146.9, 78.88, and 8.07 μg/mL for aqueous, hydroalcoholic, MEH, and acarbose, respectively. All the extracts have shown mild α-amylase inhibitory activity compared to acarbose. Lineweaver–Burk plot has shown that the MEH is a mixed noncompetitive inhibitor for α-glucosidase enzyme.
Conclusion:
The results from this
in vitro
study clearly indicated that MEH of
E
.
hirta
had strong inhibitory activity against α-glucosidase and mild inhibitory activity against α-amylase. It can be used for management of postprandial hyperglycemia with lesser side effects, and provide a strong rationale for further animal and clinical studies.
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2,782
388
ORIGINAL ARTICLES
Antibiotic susceptibility pattern of
Salmonella enterica
serovar typhi and
Salmonella enterica
serovar paratyphi A with special reference to quinolone resistance
Shoorashetty Manohar Rudresh, T Nagarathnamma
July-December 2015, 6(2):70-73
DOI
:10.4103/2394-6555.162448
Background
and
Objectives:
Typhoid fever is endemic in India. Extensive use of first-line antibiotics has led to the emergence of multi-drug resistant (MDR) Salmonella typhi. Ciprofloxacin has become empirical therapy of choice against MDR salmonellae. Recent year's emergence of low-level ciprofloxacin resistance in salmonellae resulted in delayed response and serious complications. Nalidixic acid (NA) screen test is used as surrogate marker for detection low-level ciprofloxacin resistance. In this study, we evaluated prevalence of MDR and low-level ciprofloxacin resistant S. typhi and Salmonella paratyphi A.
Materials
and
Methods:
A total of 50 blood culture isolates of S. typhi and S. paratyphi A were tested for antibiotic susceptibility according to Clinical Laboratory Standards Institute (CLSI) method. Minimal inhibitory concentration (MIC) to ciprofloxacin was carried out by E-test and agar dilution method.
Results:
Among the 50 salmonella isolates, 80% were S. typhi and 20% were S. paratyphi A. MDR was found in 2% S. typhi. NA resistant salmonellae showed ciprofloxacin MIC ranging from 0.25 to 0.75 μg/ml. One isolate of S. typhi showed ciprofloxacin MIC of 32 μg/ml and was also resistant to ceftriaxone. NA screen test for low-level ciprofloxacin resistance was 100% sensitive and 97.9% specific.
Interpretation
and
Conclusion:
NA resistant isolates should be tested for ciprofloxacin MIC to decide therapeutic options. The current CLSI breakpoints may have to be re-evaluated for salmonellae.
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2
2,627
314
In-vitro
antimicrobial screening and molecular docking studies of synthesized 2-chloro-N-(4-phenylthiazol-2-yl)acetamide derivatives
Rahmat Ali, Suresh Kumar, Obaid Afzal, Sandhya Bawa
July-December 2015, 6(2):79-87
DOI
:10.4103/2394-6555.162452
Introduction:
Glucosamine-6-phosphate (GlcN6P) synthase biosynthetic pathway has been identified as potential targets for the development of new antimicrobial agents. Aim: A series of 2-chloro-N-(42-phenylthiazol-25-yl)acetamide derivatives (3a-r) was synthesized and evaluated their antimicrobial activity.
Materials and Methods:
The 2-chloro-N-(Para substituted phenylthiazol-25-yl) acetamide (2a-c) were synthesized by stirring intermediates (1a-c) with 2-chloroacetylchloride in dichloromethane in the presence of K2CO3. The intermediate (2a-c) were further reacted with different secondary amine such as pyrrolidine, N-methyl piperazine, N-ethyl piperazine, thiomorpholine, morpholine, piperidine etc in ethanol in presence of TEA Triethylamine (TEA) to get desired compounds (3a-r). Compounds were characterized by a spectroscopic technique such as Fourier transform infraredFTIR,
1
H-NMR,
13
C-NMR, and mass spectrometry. The synthesized thiazole derivatives (3a-r) were screened for anti-bacterial and anti-fungal activity against Escherichia coli, Staphylococcus aureus NCTC 6571, Pseudomonas aeruginosa NCTC 10662, CandidaC. albicans (MTCC-183), AspergillusA. niger (MTCC 281) NCTC 10418 and AspergillusA. flavus (MTCC 277).
Result and Conclusion:
The results of anti-bacterial screening revealed that among all the screened compounds, eight compounds viz. 3b, 3c, 3d, 3e, 3i, 3j, 3k, and 3p showed moderate to good anti-bacterial and antifungal activity having minimum inhibitory concentration (MIC) between 6.25- and 25 µg/ml. While compound 3d showed the most promising antibacterial activity against E. coli and S. aureus, while the compound 3j showed promising antifungal activity with MIC value 6.25 µg/ml against C. albicans, A. niger and A. flavus. In addition, all these eight potential molecules were also examined for possible binding on enzyme GlcN6Pglucosamine-6-phosphate synthase by molecular docking studies on (PDB ID 1JXA).
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2,544
452
Amlodipine potentiates the protective effect of zonisamide on pentylenetetrazol-induced kindling in mice
Mahfooz Alam, Bhulan Kumar Singh, Vinay Kumar
July-December 2015, 6(2):88-92
DOI
:10.4103/2394-6555.162453
Background:
Zonisamide (ZON) and amlodipine (AML) were studied in different experimental models of epilepsy individually. However, combination of ZON and AML has not been explored yet. Hence, this study was aimed to evaluate the combination therapy of ZON and AML on pentylenetetrazol (PTZ)-induced kindling in mice.
Materials and Methods:
Swiss albino mice of either sex were randomly divided into five groups and each group containing 8 mice. Kindling was induced by PTZ (45 mg/kg/day on 8
th
, 10
th
and 12
th
and 75 mg/kg/day on 14
th
day). After treatment animals were observed for 30 min for behavioral analysis then animals were sacrificed and brain was taken out for biochemical analysis.
Results:
PTZ-significantly induced seizure was characterized by alteration in seizure score and latency as well as significantly increased in brain thiobarbituric acid reactive substance (TBARS) levels and significantly decreased in reduced glutathione, catalase (CAT) and superoxide dismutase (SOD). Treatment with ZON and AML significantly (P < 0.05, P < 0.01 and P < 0.001) resorted seizure score, latency, TBARS, reduced glutathione, CAT and SOD near to normal compared with pentylentetrazol treated rats.
Conclusion:
This study provides experimental evidence that treatment with both ZON and AML attenuated seizure and oxidative stress in PTZ-induced kindling mice.
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2
1,816
196
Hepatoprotective effects of aqueous root bark extracts of
Anogeissus leiocarpus
,
Terminalia avicennioides
, and their mixture in diethylnitrosamine-treated rats
Amadu Kayode Salau, Musa Toyin Yakubu, Adenike Temidayo Oladiji
July-December 2015, 6(2):93-100
DOI
:10.4103/2394-6555.162454
Introduction:
This study investigated the hepatoprotective activity of aqueous root bark extracts of Anogeissus leiocarpus, Terminalia avicennioides, and their 1:1 mixture in diethylnitrosamine (DEN)-treated rats.
Materials and Methods:
Rats were orally pretreated with extracts and 1:1 mixture (200, 400, and 800 mg/kg body weight) for 2 weeks before being challenged with DEN (30 mg/kg body weight) once a week on weeks 3 and 4. Curcumin (200 mg/kg body weight) was used as reference antioxidant agent, and distilled water was used as a control. The whole experiment lasted for 4 weeks. The activities of liver and serum alkaline phosphatase (ALP), aspartate and alanine aminotransferases (AST and ALT), and gamma-glutamyltransferase (GGT), as well as the levels of serum total protein, albumin, total, and conjugated bilirubin were evaluated.
Results:
The extracts and their mixture significantly (P < 0.05) prevented the DEN-induced alterations in the activities of ALP, AST, ALT, and GGT in the liver and serum of the animals; as well as the altered levels of serum total and conjugated bilirubin.
Conclusion:
Overall, the results of this study revealed that the extracts possess hepatoprotective activities against DEN-induced liver damage in rats.
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2
1,605
242
SYMPOSIUM
Quantification of vasaka alkaloids in
in vitro
cultures and in natural leaves from Indian subcontinents by reversed phase- high performance liquid chromatography
Madhukar Garg, Ennus Tajuddin Tamboli, YT Kamal, SH Ansari, Sayeed Ahmad
January-June 2016, 7(1):51-54
DOI
:10.4103/2394-6555.180165
Background:
The present study was designed to develop an efficient protocol for studying the enhancement of
in vitro
production of secondary metabolites in
Adhatoda vasica
leaves collected from the varied geographical locations of Indian sub-continents by a validated simultaneous high-performance liquid chromatography gradient method for the quantification of vasicine and vasicinone in the developed calli and crude samples.
Materials and Methods:
The analysis was carried out on a reverse phase C
18
column using 0.1% of orthophosphoric acid: Acetonitrile in gradient manner and carried out the detection at 280 nm wavelength keeping the flow rate of 1.0 mL/min.
Results:
The simultaneous method was found linear with regression coefficient
r
2
= 0.991 in a wide range (100–500 μg/mL) precise, accurate, and robust for quinazoline alkaloids in samples. Results clearly showed a significant increase in the concentration of alkaloids in
in vitro
cultures as compared to natural ones. The proposed method was validated as per the International Conference on Harmonization guidelines for accuracy, precision, robustness, limit of detection, and limit of quantitation.
Conclusion:
The developed method was found suitable for quality control of
A. vasica
and for the analysis of vasicine and vasicinone in any type of sample.
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2
1,749
179
ORIGINAL ARTICLE
Doxorubicin prodrug for cytoplasmic and nuclear delivery in breast cancer cells
Animikh Ray, Kishore Cholkar, Ravinder Earla, Ashim K Mitra
January-June 2016, 7(1):13-19
DOI
:10.4103/2394-6555.180158
Purpose:
The objective of this study was to develop a novel peptide prodrug of doxorubicin which can evade over-expressed efflux pumps in breast cancer cells. This approach may lead to increased uptake and higher drug accumulation in nuclei of cancer cells.
Materials and Methods:
L-val-L-val doxorubicin prodrug was synthesized following standard f-moc chemistry. The prodrug was analyzed for stability, cellular and nuclear uptake and interaction with efflux and peptide transporters. Breast cancer cells (T47D) were grown on polystyrene 12-well plates.
Result:
The prodrug Val-Val-doxorubicin was found to be very stable in breast cancer cell homogenate. It was able to evade efflux pumps. The prodrug penetrated cytoplasm and nucleus of cancer cells by interacting with peptide transporters.These transporters (pepT1 and pepT2) are expressed both on plasma and nuclear membrane of breast cancer cells. Uptake of prodrug was found to be 10 times more than parent drug.
Conclusion:
Peptide prodrug derivatization of doxorubicin has potential to evade efflux pumps and increase availability and nuclear accumulation of doxorubcin in breast cancer cells.
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2,181
334
ORIGINAL ARTICLES
Evaluation of antifungal activity of aqueous extracts of some medicinal plants against
Aspergillus
flavus,
pistachio aflatoxin producing fungus
in
vitro
Sahar Omidpanah, Hamid Sadeghi, Mehdi Mohamadian Sarcheshmeh, Azadeh Manayi
July-December 2015, 6(2):66-69
DOI
:10.4103/2394-6555.162446
Background:
Contamination with aflatoxin, by Aspergillus flavus, is one the major challenges in agriculture and food industry. Preparation of organic products using natural components is widely considered these days.
Aims:
In this study, effects of aqueous extracts of five medicinal herbs, including thyme, senna, mentha, basil, and safflower on the growth of the A. flavus were investigated.
Mterials and Methods:
The extracts with different concentrations (200-800 µg/mL) and polyethylene glycol with the equal osmotic potential of plant extracts were added to the potato dextrose agar medium to evaluate fungus growth after 7 days using agar dilution method. Benomyl, a fungicide, was used as a positive standard. The tests were performed in triplicate, and the mean diameters of fungus growth were calculated as well.
Results and Conclusion:
All concentrations of the plants extracts significantly inhibited the fungus growth in comparison with each other and control treatments, while the extracts of thyme and safflower manifested the most effective prohibition compared to benomyl with minimum inhibitory concentration of 200 and 400 µg/mL, respectively.
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1
3,040
715
Phytochemical evaluation of the ethanolic extracts of some Nigerian herbal plants
Enegide Chinedu, Arome David, Solomon F Ameh
January-June 2015, 6(1):11-14
DOI
:10.4103/2394-2002.148882
Background:
Herbs basically, are plants or plant parts employed due to their scent, medicinal properties or flavor. Herbal medicines have longed been used in the management and treatment of various ailments even before the arrival of modern medicine. Herbal medicines are still being used today, as it has even gained a new momentum in the field of medicine. Phytochemicals are responsible for eliciting definite physiological effects of various herbs in the body. In Nigeria, various plants are being used traditionally in the treatment of divers ailments. Some of these plants include Tapinanthus bangwensis, Tamarindus indica, Ocimum gratissimum, Allium sativum, Kigelia africana, Azadirachta indica, Solanum virginianum, Myrianthus serratus and Vernonia amygdalina. Though there have been claims of success in their traditional usage, it is however important to carry-out phytochemical assessment on them.
Aim:
The aim was to evaluate the phytochemical constituents of the ethanolic extract of these plants.
Materials and Methods:
The plant materials were extracted, and the plant extracts were screened for the presence of various phytochemical constituents such as alkaloids, steroids, glycosides, cardiac glycosides, flavonoids, carbohydrate, saponins, tannins and anthraquinones using the standard methods.
Result:
The experimental result revealed the presence of the various bioactive phytochemicals in the different plant extracts investigated. These phytochemicals were however present in different proportions in the various plant extracts.
Conclusion:
The experimental result vindicates the usage of these plants traditionally for medicinal purposes.
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1
2,871
412
Validated RP-HPLC-PDA method for simultaneous determination of Zidovudine, Lamivudine, and Nevirapine in pharmaceutical formulation
B Mohammed Ishaq, K Vanitha Prakash, G Krishna Mohan
January-June 2015, 6(1):27-32
DOI
:10.4103/2394-2002.148890
Aim and Objectives:
A simple, rapid, and sensitive high performance liquid chromatographic method with ultraviolet detection has been developed and validated according to the International Conference on Harmonization (ICH) guidelines for the quantitation and qualification of zidovudine (ZID), lamivudine (LAM), and nevirapine (NEV) in pharmaceutical dosage forms.
Materials and Methods:
The proposed method was based on the separation of the drugs in reversed phase mode using Water's C18 250 cm × 4.6 mm, 5 μ particle size column maintained at an ambient temperature. The optimum mobile phase consisted of Water: Methanol (70:30 v/v), pH adjusted to four with orthophosphoric acid (OPA). The flow rate of mobile phase was set 1.0 mL min
-1
and photodiode array detection was performed at 275 nm with a total run time of 8 min which is very short for accurate analysis of simultaneous estimation of three analytes. The method was validated according to ICH guidelines.
Results:
The method was linear over the concentration range of 25-75 μg mL
-1
with limit of quantifications (LOQ) of 13, 0.49, and 0.40 ng mL
-1
for ZID, LAM and NEV respectively and limit of detection (LOD) of 4, 0.14, and 0.12 ng mL
-1
for ZID, LAM and NEV respectively. Accuracy (% recovery studies) and precision values of both inter and intraday obtained from six different replicates for all the analytes ranged from 99.00% to 100.00% and % relative standard deviation of precision (assay) was between 0.64 and 1.28, respectively. All the three analytes and their combination drug product were exposed to thermal, photolytic, hydrolytic, reductive, oxidative and peroxide stress conditions and the stressed samples were analyzed by the proposed method. There were no interfering peaks from excipients, impurities or degradation products due to variable stress conditions and the proposed method is specific for the simultaneous estimation of ZID, LAM and NEV in the presence of their degradation products.
Conclusion:
The proposed method can be successfully applied in the quality control and stability samples of pharmaceutical dosage forms.
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1
2,630
307
SYMPOSIUM
Simultaneous determination of 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid in
Glycyrrhiza glabra
root by reversed phase high-performance liquid chromatography
Ambika Chamoli, Makhmur Ahmad, Mojeer Hasan, Bibhu Prasad Panda
January-June 2016, 7(1):59-62
DOI
:10.4103/2394-6555.180168
Background:
The aim of the present research work is to develop a high-performance liquid chromatography (HPLC) method for simultaneous analysis of 18α-glycyrrhetinic acid (18α-GA) and 18β-GA (18β-GA) of
Glycyrrhiza glabra
.
Materials and Methods:
About 20 μL aliquots of each 18α-GA and 18β-GA were analyzed using reversed-phase C-18 column. The mobile phase was acetonitrile:tetrahydrofuran:water (10:80:10, v/v/v). The run time was 10 min at flow rate of 1 ml/min. Ultraviolet detection was carried out at 254 nm.
Results:
18α-GA and 18β-GA were well resolved in reversed phase C-18 column using mobile phase acetonitrile: tetrahydrofuran: water (10:80:10, v/v/v, pH 7.9). The R
t
of 18α-GA and 18β-GA was detected at 2.091 and 2.377 min, respectively.
Conclusion:
The developed chromatography method could be extended for potential quantification or simultaneous determination of these markers in plant as well as in herbal formulation.
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1
1,810
197
Strain improvement of
Phaffia rhodozyma
for astaxanthin production
Hina Nangia, Mojeer Hasan, Mohd. Azhar , Prakash Chandra Bhatt, Bibhu Prasad Panda
January-June 2016, 7(1):63-68
DOI
:10.4103/2394-6555.180169
Objective:
The aim of the present study is to evaluate and optimize different carbon sources at different temperatures in shake flask for cell growth and astaxanthin accumulation in ultraviolet (UV) mutant
Phaffia rhodozyma
. The focus is to make the process cost-effective and fruitful with higher productivity.
Materials and Methods:
Adaptive strain development was carried out so that the
P. rhodozyma
can give the best astaxanthin at a higher temperature (35°C), increasing the process economy.
P. rhodozyma
was cultivated with three broad spectrum antibacterial drugs - streptomycin, gentamycin, and ampicillin (200 μL) and with antifungal drug - fluconazole (200 μL) to determine the effect on yeast growth and astaxanthin production. Rice, cane juice, and sucrose were selected as carbon source and compared with dextrose at different fermentation temperatures - 22°C, 30°C, 33°C, and 35°C for astaxanthin production.
Results:
P. rhodozyma
was resistant to antibiotic drugs inhibiting bacterial and fungal contaminants. Rice being more economical was selected as carbon source, but the strain was not able to utilize starch-rich substrate leading to its rejection. When
P. rhodozyma
was grown in cane juice, biomass is highest (OD 2.77) at 30°C and 610 nm wavelength, whereas astaxanthin productivity is highest (OD 2.64) in dextrose media at 30°C and 450 nm. Comparative metabolic and growth results for UV mutants showed significantly higher biomass and astaxanthin productivity in comparison to wild strain.
Conclusion:
The most efficient carbon source in terms of penny-pinching and astaxanthin productivity was found to be dextrose containing media at 30°C.
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3,184
433
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Online since 10 April, 2014