Over Production of Phycocyanin Pigment in Blue Green Alga Spirulina
sp. and It`s Inhibitory Effect on Growth of Ehrlich Ascites Carcinoma Cells
Hanaa H. Abd El-Baky
Two species of blue green algae Spirulina platensis and Spirulina maxima were grown in nutrient medium containing different nitrogen and salt levels. In both species increasing nitrogen levels led to increase in phycocyanin pigments from 12.08 to 22.3% and soluble protein content from 29.7 to 86.1 mg g -1. Also, Spirulina has great variety in composition of phycocyanin pigments ranging from C-phycocyanin (C-PC) from 1.65 to 4.02%, allophycocyanin (A-PC) from 2.53 to 6.11% and R-phycocyanin (R-PC) from 5.75 to 12.35% as a results, of changing nitrogen contents and salt stress. Spirulina platensis at high nitrogen level gave highest percentage of total phycocyanin 9.94% and R-CP 5.75% was the predominate among phycocyanin pigments. The increasing in NaCl levels in nutrient medium led to production significant in phycocyanin contents and soluble protein in Spirulina platensis and Spirulina maxima cells. The composition of phycocyanin pigment was changed markedly as results of increasing in NaCl level. Both algal species grown under combined stress (nitrogen deficient and high NaCl level) produced higher amount of phycocyanin than control. The anti-carcinoma activity of Spirulina towered Ehrlich Ascites Carcinoma Cells (EACC) was evaluated by cell viability, DNA fragmentation and enzymes assay. Phycocyanin significantly inhibited the growth of EACC in a dose-dependent manner. Phycocyanin did not induce DNA fragmentation in EACC, (no ladder of DNA fragments). However, glutathione (GST), the activity of glutathione S- transferase (GST) and lactate dehydrogenase (LDH) were significantly increased over the control level. These findings indicate that phycocyanin may be able to inhibit the growth of EACC by membrane destructor, which led to increase the leakage of cell constituent and increase LDH and GST enzyme activities. Therefore, algal phycocyanin may have antitumor activity and could be used as a chemoprventive agent.
Spirulina is one of the most promising microalgae, which be utilized for the production of cyanocobalamine (B12), antioxidant pigment like β-carotene, tocopherols and γ-linolenic acid, and can be used as raw material for single cell protein (SCP) (Belay et al., 1996 and Ortega-Calvo et al., 1993). Several fine compounds such as essential fatty acids like γ-linolenic acid (GLA), essential amino acids, antioxidant vitamins like tocopherols , minerals and proteins (Richmond, 1980 and Roughan, 1989), are found in Spirulina species at relatively high concentration and command a high market value (Santillan, 1982 and Cohen, 1995). The deep blue color of phycocyanin and other extractable pigment including myxoxanthophyl and zeaxanthin extracted from microalga Spirulina has been widely used as a naturally occurring colorant for food additive purposes (Hirata et al., 2000 and Kato, 1994). Phycocyanin had anticancer, antioxidant, antiviral and anti-inflammatory activities (Romay et al., 1998; Gonzalez et al., 1999; Hirata et al., 2000 and Mathew et al.,1995). Also, phycocyanin is a powerful tonic agent for the immune system in animals and human, which providing protection from variety of diseases (Liu et al., 2000).
Great variability in the chemical gross composition of Spirulina species have been shown as a result of several factors such as genotype, the stage in growth cycle, the source and concentration of nitrogen be used in the culture medium ( Ruengjitchatchawalya et al., 2002). These environmental variability can used for producing cells with biochemical contents that can be previously determined as a function of nitrogen source and concentration.
The phycocyanin content in Spirulina can be also effected by the source and concentration of nitrogen in the culture medium. This pigment may serve as a nitrogen storage material since the phycocyanin content is highest when Spirulina is cultivated under favorable nitrogen level (Richmond, 1980). In the present work the effect of nitrogen and NaCl on accumulation of Phycocyanin in Spirulina maxima and Spirulina plantensis were studied. Also, the antitumor activity of these compounds was evaluated.
Materials and Methods
In the present work the effect of nitrogen and NaCl on accumulation of Phycocyanin
in Spirulina maxima and Spirulina plantensis were obtained from
the culture collection of Texas University, Austin, USA.
Algae was cultivated (in National Research Center during 2003) in Zarrouks
medium (Zarrouk, 1966). NaNO3 was used as a nitrogen source with
four different concentrations 410 ppm N (control), 205 ppm N, 102.5 ppm N and
51 ppm N and zero nitrogen. Also NaCl was used at different concentrations 0.02
M (control), 0.1 M and 0.2 M and medium contain 102.5 ppm N and 0.1 M NaCl.
Algae was cultivated in 2L flasks. The cultures were gassed with 0.03% CO2
in air and algae were cultivated at 25°C±3, pH 10.5. The cultivated
flasks were illuminated by continuous cool white fluorescent lamps at 400 W.
The growth of Spirulina maxima and Spirulina plantensis was
measured by dry weight methods and optical density at 450 nm (Vonshak, 1997).
Stationary-phase cells were harvested at 4°C by centrifugation at 6000
rpm for 10 min.
Extraction and determination of phycocyanin
The concentration of blue phycocyanin pigment including: allophycocyanin
(APC), phycocyanin (PC) and R-phycocyanin (R-PC) were determined spectrophotometrically
at 650 and 618 nm; 618 and 650 nm and 498, 615 and 650 nm respectively as reported
by Kursar and Alberte (1983).
Viability of Ehrlich Ascites Carcinoma Cells (EACC)
The tumor cell line
The original tumor cells was obtained from Cell Biology Department, National
Cancer Institute, Cairo University, Egypt. The tumor cells were maintained
in female mice as cell line in Biochemistry Department, Faculty of Agriculture,
Cairo University. The mice were injected with aliquot 0.2 ml (for each mice)
of a 10% suspension of minced tumor cell line saline.
Viability of tumor cells
The viability percentages of tumor cells were measured by the modified cytotoxic
trypan blue exclusion technique of Bennett et al. (1976).
Determination of glutathione (GSH)
The GSH content was determined in tumor cells solution (2 ml containing
4x106 cells) incubated with and without the test extracts as well
as control. The reaction is based on the reaction with 5, 5` dithiobis
2- nitrobenzoic (DTNB) reagent to give a compound that absorbed a light
at 412 nm (Silber et al., 1992). GSH was expressed as μg 10-6
Determination of glutathione-S- transferase activity (GST)
The activity of GST in tumor cells were determined according to method of
Habig et al. (1974).
Determination of lactate dehydrogenase (LDH)
LDH activity was determined in tumor cells after incubation with algal extract
as described by Bergmeyer (1974) using biosystems kit.
DNA fragmentation assay
After EACC treatment with algal extracts for 2 h, a portion of treated cells
were washed three times with cold phosphate buffersaline (pH 7.8) and
then, they were lysed with a lysis buffer (50 mM Tris-HCl, (pH 8.0), 0.2 mM
EDTA, 10 mM NaCl , 2% SDS, 50 mg L -1 proteinase) at 50°C for
more than 4 h and then chilled in ice. Proteins were precipitated by saturated
NaCl and removed by centrifugation at 1500 g, for 10 min, the supernatant contained
DNA fragments (Liu et al., 2000). Then the DNA fragment was evaluated
spectrophotometrically 200 μl of supernatant were transferred to test tube
containing a 200 μl diphenyl amine (0.088 M DPA, 98 v/v glacial acetic
acid, 1.5% v/v sulfuric acid and 0.5% acetyladehyde) then kept at 4°C for
48 h. The developed bluish color was recorded at 600 nm (Perandones et al.,
Data represent the means±SD. Results were analyzed by one-way ANOVA and Scheffe F-test to identify significant differences between groups. P-values < 0.01 were considered significant. All analyses performed using Co Stat software version 4 (Abacus Concepts, Berkeley, CA).
Determination of protein
Protein content of treated tumor cells was extracted by phosphate buffer
and determined spectrophotometrically at 595 nm, using comassein blue G 250
as a protein binding dye (Bradford, 1976). Bovine serum albumin (BSA) was used
as a protein standard. Data represent the means±SD. Results were analyzed
by one-way ANOVA and Scheffe F-test to identify significant differences
between groups. P-values <0.01 were considered significant. All analyses
performed using Co Stat software version 4 (Abacus Concepts, Berkeley, CA).
Results and Discussion
In Table 1 and 2 the two of blue green alga Spirulina species are compared in phycocyanin production at different growth conditions. Decreasing the nitrogen concentration in the nutrient medium led to a decrease in the phycocyanin content (total phycocyanin). The most significant decrease was observed when Spirulina algae was grown in free nitrogen medium (0.0%N). Under this conditions total phycocyanin content in S. plantensis and S. maxima was 3.31 and 1.7% (D.W), respectively and with increasing nitrogen concentration these quantities were increased slowly and reached to high values 12.08 and 9.94%, respectively and with increasing nitrogen concentration 410 ppm N. as NaNO3, However, at comparable nitrogen levels, the S. plantensis algae generally produced higher amount of phycocyanin than in the S. maxima (12.89-9.94%). Phycocyanin: composed of C-PC, APC and RPC were determined by spectrophotometric method. Both Spirulina species have a great variety of phycocyanin pigments ranging from C-PC, A-PC and R-PC. The percentages of these pigments changed markedly by nitrogen concentrations variation. By decreasing of nitrogen concentration, Spirulina species mainly produced R-PC and lower amount of C-PC. At nitrogen levels were (51 and 410 ppm), the % of C-PC, A-PC and R-PC in S. plantensis were 0.71 (2.7), 1.78 (3.57) and 2.22% (6.80%), respectively. While, in S. maxima were 0.53(1.65), 0.83 (2.53) and 1.53% (5.75), respectively at the same nitrogen level.
Effect of NaCl stress
In Table 1 and 2 the S. plantensis
and S. maxima are compared in production of phycocyanin became pigments
when grown in medium containing 5 at (0.1 M NaCl ) and 10 at 0.2 M NaCl fold
level became of NaCl over than the optimum NaCl level (0.02 M NaCl). Increasing
NaCl in nutrient medium led to produced significant amount of phycocyanin content
when compared to the control. The amount of total phycocyanin in S. plantensis
grown under NaCl stress (0.1 and 0.2 ppm) were 1.38(22.3) and 1.85(16.63%),
respectively times over the control (12.08%). Whereas, these levels were 1.46
(14.47) and 1.89(18.87), respectively of the control (9.94%), in S. maxima
algae. However, composed phycocyanin pigment have great variety in both
species and the amount of each pigment was changed markedly as a results of
increased NaCl concentration. S. plantensis and S. maxima grown
under combined stress of nitrogen deficient (102 ppm N) and high NaCl concentration
(0.1 MnaCl), produced lower amount of phycocyanin content than that grown in
medium containing enough nitrogen and high NaCl level concentration.
|| Influence of nitrogen and salt stress on phycocyanin pigment
in Spirulina plantensis
|| Influence of nitrogen and salt stress on phycocyanin pigment
in Spirulina maxima
|Values represents are mean of three replicates and based on
dry weight, All values are significant at (P<0.5)
||Inhibitory effect of phycocyanin extract from Spirulina
plantensis on the viability of Ehrlich Ascites Carcinoma Cells (EACC)
||Inhibitory effect of phycocyanin extract from Spirulina
maxima on the viability of Ehrlich Ascites Carcinoma Cells (EACC)
|2ml of cell solution containing 4x106 cells
In both algae Spirulina species, the total soluble protein was increased
with the increased of nitrogen and high NaCl level (Table 1
and 2). At comparable nitrogen levels 0, 51, 102.5, 205 and
410 ppm N in the medium, the soluble protein content in S. plantensis and
S. maxima (in parenthesis) were 10.1 (4.51), 15.3 (9.21), 20.6 (14.3),
27.7 (18.5) and 29.7 (22.1 mg g-1), respectively. Also, the soluble
protein content of Spirulina species were increased as results of NaCl
increase in present of sufficient nitrogen levels in nutrient medium (Table
1 and 2).
By varying the concentration of the nitrogen in nutrient medium, S. plantensis and S. maxima can be manipulated with respect to their total phycocyanin and soluble protein content. The Spirulina grown in medium with high nitrogen levels yielded a maximum phycocyanin pigment (up to 12.2%), whereas with decreasing nitrogen level the phycocyanin content of Spirulina cells was dropped (Becker, 1994). Piorreck et al. (1984) grew Spirulina platensis and other three unicellular algae at different nitrogen levels and they observed significant changes in pigment and total protein content. Which decreasing nitrogen concentration led to decrease in chlorophyll and protein content due to breakdown of the whole chloroplast apparatus. However, Spirulina protein can accumulate in considerable amount (up to 70%) in stationary-phase cells when grown in nutrient medium rich in nitrogen. However, the protein fraction of Spirulina species was containing up to 20% of cyanophycin granules, a water-soluble blue pigment (Becker, 1994 and Ciferri, 1983).
Effect of phycocyanin extract of Spirulina species on viability of
Phycocyanin extracts of two species of blue green microalga Spirulina
maxima and Spirulina plantensis on the viability of EACC were examined
by means of the trypan blue exclusion method. After 2 h incubation of tumor
cells in fresh medium with or without algal phycocyanin, the cell viability
was measured. As shown in Table 3 and 4,
treatment of cells with Spirulina -phycocyanin caused significant reduction
in cell viability. Generally as the concentration of phycocyanin algal extract
increased, the viability of EACC were reduced, which suggested that the effect
of PC-S. maxima and S. plantensis on the growth of EACC was dose
dependent. Further, the increase of phycocyanin content (% of dry weight) in
the phycocyanin algal extracts led to a great decrease in cell viability. The
most significant decreases in cell viability were observed in phycocyanin algal
extract of S. plantensis and S. maxima containing total phycocyanin
22.3 and 18.88%, respectively, which reduced the cell viability to 23.6 and
26.2%, respectively. In contrast, the extract of S. plantensis and S.
maxima contain less level of phycocyanin, 3.3 and 2.89% did not, produce
any significant change in cell viability at concentration level of 200 ppm,
whereas at 400 ppm these extracts gave significant effect on reduction of cell
viability. Thus, the cell viability was depended on phycocyanin content and
Cells constituents and enzyme levels
The levels of glutathione (GSH) and activities of glutathione S-transferase
(GST) and lactic dehydrogenase (LDH) were determined in treated EACC, in relation
to reduction of tumor cells viability with algal phycocyanin. As shown in Table
(5 and 6) all algal extracts were markedly increased the
level of cellular GSH and GST and LDH activities in the tumor cells when compared
with the control, especially with S. Plantensis extracts rich in phycocyanin
(22% of DW). Thus, as the concentration of algal extracts increased, the level
of GSH and enzyme levels were increased, which suggest that the effect of algal
extracts on the cellular constituents of EACC were dose dependent.
|| Phycocyanin extract from Spirulina plantensis enhanced
glutathione level , glutathione S- transferase
||Phycocyanin extract from Spirulina maxima enhanced
glutathione level , glutathione S- transferase activity and lactate dehydrogenase
activity of Ehrlich Ascites Carcinoma Cells (EACC)
|± S.D, 2ml of cell solution containing 4x106 cells,
All values are significant at ( P< 0.5), Values represents are mean of
Phycocyanin of S. plantensis and S. maxima grow in medium contain
0.2 M NaCl at 400 ppm increase in GSH, GST and LDH levels about 27.4 (18.13),
12.9 (10.16) and 11.8 (9.5), respectively, times as that found in untreated
cells. Whereas, the algal extracts of S. plantensis and S. maxima
grown in free nitrogen medium at 400 ppm significant increase GSH, GST and
LDH to, 8.02 (6.04), 4.2 (2.52) and 3.0 (2.39) time over the in untreated cells.
|| Effect of phycocyanin extract from Spirulina plantensis
on DNA fragmentation
||Effect of phycocyanin extract from Spirulina maxima on
Alga Spirulina have a higher content of phycocyanin other than plant source (Vadiraja et al., 1998). Phycocyanin has various medical properties, which may inhibit the growth of much type of tumor cells by pathways other than apopotosis (Liu et al., 2000). Because phycocyanin has characteristic stability and solubility in aqueous solution and non-toxicity, it has been used in many research applications. Cyanobacteria-phycocyanin (C-PC) could reduce the viability of mouse myeloma cells, when cultured with 250 mg C-PC for 3 days ( Morcos et al., 1988 ). Also, Liu et al. (2000) reported that phycocyanin of S. plantensis inhibited the growth of human Leukemia K 562 cells in a dose and time dependent manner by a potential pathway other than apoptosis.
In this study the phycocyanin extracts of two algal species inhibited the growth of EACC in a dose-dependent, the algal extracts contain a large amount of phycocyanin had a higher destructive effect on EACC. From this observation, it is clear that the anticarcinoma or antitumor activity of algae extracts were mostly due to phycocyanin compound present in these extracts. The EACC were killed in treated solution, 2 h after incubation, these indicated that phycocyanin extracts did not induce apoptosis. Similar finding were obtained by Liu et al. (2000) who found that phycocyanin of alga S. plantensis and S. maxima kiled the human leukemia K 562 cells by a potential pathway other than apoptosis. This study revealed that the S. plantensis algae extracts may induce cell death of EACC by membrane destruction, which lead to increase the leakage of cell constituent (GSH and LDH and GST enzymes).
The whether of phycocyanin algal extracts could induce apoptosis in EACC was performed using calorimetric method. After DNA was extracted from the treated EACC, the percentage of DNA fragment was calculated as shown in Table 7 and 8. Apoptosis-induce cis- platinum (50 nM) produced 7.25%. DNA fragmentation. Compared with apoptosis induce treat, the phycocyanin algal extract of S. plantensis and S. maxima content high level of phycocyanin 22.3 and 18.88% were most significant decreased the DNA fragment to 0.18 and 0.23%, respectively. The algal extracts S. plantensis and S. maxima contents less phycocyanin% were produced DNA-fragment with 2.99 and 4.13%, respectively. This suggests that phycocyanin algal extracts may not be able to induce the apoptosis in the EACC. Consecontly, the phycocyanin algal extracts apparently reduce cell viability by anther mechanisms such as cell membrane lyases. However, the results revealed that after 2 h of incubation of algae extract with tumor cells clear showed lower DNA fragment, than control. These mean that no intranucleosm degradation of DNA (Ladder DNA) was occurred in treated EACC Reddy et al. (1997) reported that DNA fragmentation (Ladder DNA) was not essentially produced as a results of apoptosis pathways. In addition the algae extracts may induce chromosomal abnormalities in EACC (Duthie et al., 1997). Finally, the phycocyanin had an antitumor activity, and could be used chemo-preventive agent.
In conclusion, S. plantensis and S. maxima can be manipulated a big yield of phycocyanin when grown in medium containing 0.2 M NaCl. These, phycocyanin pigments inhabited the growth of EACC in a dose depended manner by a potential pathway other than apoptosis.
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