Biotechnology

Year: 2003 | Volume: 2 | Issue: 3 | Page No.: 222-240
DOI: 10.3923/biotech.2003.222.240

Spirulina Species as a Source of Carotenoids and a-Tocopherol and its Anticarcinoma Factors

Hanaa H. Abd El-Baky, Farouk K. El Baz and Gamal S. El-Baroty

Abstract: In the present study blue-green alga Spirulina platensis and Spirulina maxima were grown in batch culture at different nitrogen and NaCl concentrations, respectively. Both species were found to respond to nitrogen deficiency and high NaCl level by accumulation of large amounts of commercially important chemicals such as carotenoids and tocopherols. The higher carotenoids and α-tocopherol contents was obtained when Spirulina sp grown at low nitrogen level (51 ppm N), with values ranged from 19.82 to 24.1 mg g^-1 and from 533.2 to 978.5 μg Kg^-1 (dry weight, d.w.), respectively. Whilst, these values in cells grown in free nitrogen medium were ranged from 30.15 to 31.13 mg g^-1 and from 960.4 to 1325.7 μg Kg^-1 (d. w), respectively. The high performance liquid chromatography (HPLC) elution patter of total caroteniods extracted from S. platensis and S maxima culture contained β-carotene (49.6 to 319.5 μg g-1), lutein (0.06 to17.21 μg g-1), astaxanthin (6.61 to 160.27 μg g-1), zeaxanthin (1.25 to 18.55 μg g-1) and cryptoxanthin (1.41 to 20.13 μg g-1). S. maxima accumulated a large amount of cryptoxanthin and zeaxanthin than that found in S. platensis. The effect of Spirulina species extracts containing various carotenoid compounds and tocopherols on the viability of Ehrlich ascites carcinoma cells (EACC) were evaluated. All algae extracts at different concentration of 200 and 400 ppm significant reduced the cell viability ranged from 89.11 to 5.25%. These extracts did not induce any significant changes in DNA fragmentation of treated EACC compared with untreated cells. But lactate dehydrogenase (LDH) and glutathione-S- transferase (GST) enzyme activities and glutathione level in treated EACC were significantly higher than that in untreated cells. These finding suggest that algae extracts may be reduce cell viability by other mechanism such as membrane lyases instead of apoptosis. Thus, Spirulina extract rich in carotenoids and tocopherols could be used as chemopreventive agents since they are relative non-toxic

Keywords: nitrogen and salt stress, tocopherols, carotenoids, antitumor viability and blue green algae, Spirulina and carotenoids

Introduction

In last decade algae biotechnology has made major advance and many algae metabolites are being production commercially (Borowitzka, 1992 and Morimoto et al., 1995). A number of microalgae such as Duneliella, Chlorella and Spirulina are grown for food, feed or for the extraction of valuable chemical such as carotenoids, antibiotics, fatty acids and antioxidant vitamins (Borowitzka, 1986; El Baz et al., 2002a and b; Abouel Enein et al., 2003; Abd El-Baky et al., 2002).

Carotenoids are naturally occurring pigment in algae that are involved in light-harvesting reaction and protection of algal organelle cells against singlet oxygen- induced damage. The alga species Duneliella and Chlorella show considerable differences in metabolisms of carotenoids. The differences are attributed to unfavorable conditions of growth, especially deficiency of nitrogen and or iron and high light intensity and high salinity (Abd El-Baky et al., 2002; El-Baz et al., 2002a and Borowitzka, 1986) leads to accumulation of secondary carotenoids, mainly ketocarotenoids. However, the increase of ketocarotenoids level in cells of these algae accompanied by the disappearaence of chlorophyll and a decrease in primary carotenoids such as, a and β carotene and lutein (Burczyk, 1987). Spirulina has a higher content of β-carotene, cryptoxanthin and zeaxanthin compared to most other natural source (Mathew et al., 1995). Also, Spirulina offer a wide scope for production of large quantities of natural carotenoids directly in an edible state. It has also been demonstrated to be safe food item (Seshadri et al., 1992 and Mathew et al., 1995).

Epidemiological and animal studies indicate the inhibitory effect of micronutrients, such as vitamin E, C and carotenoids on carcinogensis. Studies have also shown that Spirulina extracts exert chemoprephylactic actions on certain cancers (Schwartz and Shklar, 1987; Schwartz et al., 1988; Govannucci, 1999; Abd El-Baky et al., 2002). For example, the extracts of Spirulina and Dunaliella algae have been demonstrated to be capable of regressing carcinomas of the hamster buccal pouch. The preventive potential of micronutrients should preferably be realized by modulation of diet rather than in the form of pharmacological supplements (Mathew et al., 1995).

In the present work the effect of nitrogen and NaCl on accumulation of caroteniods and tocopherols in Spirulina maxima and Spirulina plantensis were studied. Also, the antitumor activity of these compounds was evaluated.

Materials and Method

Algae source: Spirulina maxima and Spirulina plantensis were obtained from the Culture Collection of Texas University, Austin, USA.

Growth conditions
Algae was cultured in 2 L Erlenmeyer flasks containing Zarrouk’s medium as described by Zarrouk, (1966). NaNO3 was used as a nitrogen (N) source at 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. The cultures were gassed with 0.03% CO2 in air and the algae were cultivated at 25ºC±3. The pH of the medium was adjusted to 10.5 with 1 M NaOH prior to autoclaving. The cultivated flasks were illuminated by continuous cool white fluorescent lamps at 400 W.

Growth measurements
The growth of Spirulina maxima and Spirulina plantensis was measured by dry weight methods and optical density at 450 nm (Vonshak, 1997).

Harvesting
After 2 weeks (end of logarithmic-phase), the algae cells were harvested at 4°C by centrifugation at 10000 x g for 10 min.

Extraction of carotenoids and tocopherols
Carotenoids and tocopherols were extracted from algal cells with tetrahydrofuran (THF) 1: 10 (w/v) in present 30 mg L^-1 of BHT (2,6 ditertra-butyl-p-cresol) and magnesium carbonate (0.1 g-1 sample). After 24 h, the aliquot of the clear extracted pigments was filtered and evaporated to 5 ml under a stream of nitrogen. The extracted pigments were saponified with 25 ml of 10% (v/w) methanolic potassium hydroxide for 2 h at room temperature, then the carotenoids and tocopherols were extracted with dichloromethane. The solvent layer was then separated by separatory funnel, washed several times with distilled water and dried on NaSO4. The organic solvent was evaporated to dryness under nitrogen (Farag et al., 1998).

Determination of algal carotenoids
The total carotenoids were determined by spectrophotometric method at 450 nm, β-carotene served as a standard compound and was used for preparing the calibration curve (Semenko and Abdullaev, 1980).

Identification of carotenoids
The thermoseparation Liquid Chromatography system consisted of a spectra system UV 2000 detector and Spectra System P2000 pump was used. The carotenoids pigments were separated on a 250x4.6 mm (i.d) column packed with Chromosil C₁₈ material, 5 µm particle size and eluted with 80:10 (v/v) acetonitrile : methanol. Flow rate was 1 ml min^-1 and detection was carried out at 438 nm. Some available standard carotenoids: β-carotene, zeaxanthin, lutein, astazenthin and cryptoxanthin (Sigma), were also run by the same HPLC method (Honya et al., 1994).

Determination of algal tocopherol
Tocopherols were determined by high performance liquid chromatography (HPLC) equipped with Spectra System UV2000 detector at 290 nm. Tocopherol were separated on a 250x4.6 mm (i.d) column packed with Vydac and eluted with 90:10 acetonitrile: methanol (v/v) at flow rate was 1 ml min^-1 and α- tocopherol standard (Sigma) was ran under the same conditions.

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 a 0.2 ml aliquot (for each mice) of a 10% suspension of minced tumor cell line in 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). The cells incubated with and without the test extracts as well as control based on the reaction with 5,5 ` dithiobis –2- nitrobenzoic (DTNB) reagent to give a compound that absorbed at 412 nm (Silber et al., 1992). GSH was expressed as µg10^-6 cells.

Determination of glutathione-S- transferase activity (GST)
The activity of GST in treated and untreated 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).

DNA fragmentation assay
After EACC treated with algal extracts for 2 h a portion of treated cells were washed three times with cold phosphate buffer (pH 8.0). Then the cells 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 K) at 50 °C for more than 4 h and chilled in ice. Proteins were precipitated with saturated NaCl and removed by centrifugation at 15000 x g for 10 min, leaving the supernatant containing DNA fragments (Liu et al., 2000). The DNA fragment was evaluated by spectrophotometric method at 600 nm. As described by Perandones et al. (1993).

Determination of protein
Protein content of treated tumor cells was extracted by phosphate buffer (pH 8.0) 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.

Statistical analysis
The data are presented as mean with standard deviation (SD). All results were analyzed by one- way ANOVA and Scheffe’ F-test to identify significant differences between groups. P < 0.01 were considered significant. All analyses were performed using Co Stat software version 4 (Abacus Concepts, Inc., Berkeley, CA, USA).

Results and Discussion

The concentration of nitrogen used in the culture medium affected in the carotenoids content of blue green alga Spirulina plantensis and Spirulina maxima at the end of logarithmic phase is shown in Table 1, 2 and Fig. 1. The higher levels of carotenoids produced by both species were obtained when grown at low nitrogen level (51 ppm N), with values of 19.82 and 24.21 mg g^-1 (d w), respectively. Whereas, the highest levels of carotenoids were occurred in cells grown in free nitrogen medium, with value of 31.13 and 30.15 mg g^-1 (d.w), respectively. In contrary, S. plantensis and S. maxima grown in medium contained high nitrogen level (410 ppm N) produced lower amount of carotenoids, with value 4.75 and 5.31 mg g^-1 (d.w), respectively.

Therefore, the variation in carotenoids content in blue green algae occurred as a function of nitrogen levels. However, at all nitrogen levels the S. maxima contained relatively large amount of total caroteniods than in S. plantensis.

Identification of carotenoids pigment
Figure 1 shows the HPLC elution pattern of carotenoids pigment extracted from the S. plantensis and S. maxima that had been grown in culture medium containing different concentration of nitrogen. The results revealed that at all nitrogen levels, both of algae species grown contained β-carotene (49.6 to 319.5μg g-1), lutein (0.06-17.21μg g-1), astaxanthin (6.6 to 160.3μg g-1), caryptoxanthin (1.4 to 20.1μg g-1) and zeaxanthin (1.2 to 18.5μg g-1) (Table 1 and 2).

The amount of these carotenoids varies with nitrogen concentration in medium. However, both S. plantensis and S. maxima had the capacity to accumulate large amount of β-carotene by decrease nitrogen levels. Among all carotenoids, lutein was accumulated most in both species in free N medium, followed by astaxanthin.. However, the S. maxima was accumulated large amount of caryptoxanthin and zeaxanthin than in S. plantensis.

Influence of salinity stress on carotenoids
The effect of NaCl stress on quantity and quality of S. plantensis and S. maxima carotenoids are shows in Table 1 and 2. The values vary with different NaCl and nitrogen levels. At higher NaCl, the grown cells contained higher significant amount of carotenoids reaching to 3% of the cells d.w. For instance, the carotenoid content of S. plantensis and S. maxima grown at 0.1 and 0.2 (in parentheses) M NaCl were 7.82 (13.2) and 9.72 mg g^-1 (19.52 mg g^-1) of the cells d.w, respectively. Whereas, these values were 34.84 and 38.98 mg g^-1 (d.w) in cells grown under combination condition of low N level (102.5 ppm nitrogen) and 0.1M NaCl.

Table 1, 2 and Fig. 1 shown the carotenoids profile of S. maxima and S. plantensis grown under NaCl stress. The carotenoids profile was varied in both species as results to increase NaCl level in nutrient medium. At higher NaCl level, the grown cells contained higher significant amount of β-carotene than other carotenoids. The accumulated values of β-carotene in S. plantensis and S. maxima (in parentheses) grown at 0.1 and 0.2 M NaCl concentration being 6.1 (4.57) and 5.26, (5.84), respectively times as high as that at 0.02 M NaCl. In addition of β-carotene, these algae cells accumulated significant amount of astaxanthin 5.03 (3.1) and 11.53 (5.73), lutein 22.2 (2.4) and 23.7 (4.0), zeaxanthin 2.17 (1.9) and 2.38 (2.16) and carpytoxanthin 1.41 (1.19) and 1.79 (1.35), respectively times as great as that found in cells grown at 0.02 M NaCl.

Influence of nitrogen on the production of α- tocopherol
Influence of nitrogen on the production of α-tocopherol from S. plantensis and S. maxima is shown in Table 3 and Fig. 1 (HPLC profile). The proportion of α-tocopherol in S. plantensis and S. maxima increased with decreasing nitrogen level in nutrient medium. The maximum values of a-tecopherol in S. plantensis and S. maxima were obtained when grown in free N medium, with values 960 and 1325μg Kg^-1 (d.w), respectively. Whereas, minimum values were found in cells grown in adequate nitrogen medium (410 ppm N), with values 144.8 and 266.5μg Kg^-1 (d.w).

Influence of salt stress on the production of α- tocopherol
Table 3 shown the influence of NaCl stress on tocopherol content. In both algae S. plantensis and S. maxima, the a-tecopherol content increased with increasing NaCl concentrations. The a-tecopherol values were 641.5 and 878.9μg Kg^-1 and 427.3 and 532.5μg Kg^-1 in S. plantensis and S. maxima grown in 0.2 M and 0.1 M NaCl culture, respectively.

However, these amounts decreased significantly with decreasing nitrogen concentration to 102.5 ppm N in 0.1 M NaCl medium (Table 3 and Fig. 1).

It seems that the mechanism suggested by Arad et al. (1993); Rice et al. (1994) and El-Baz et al. (2002) for the acclimation of carotenoids in Chlorella and Dunaliella to growth under stress condition also applies to Sprinulina. It seems that the division of algae cells grown under N starvation are blocked, while photosynthesis continues, leading to storage specific compounds such as carotenoids, carbohydrates and triglycerides. The accumulation of these compounds was attributed to the fact that carbohydrates and carotenoids do not require nitrogen for their synthesis. Also, it has previously been suggested that the carbohydrates and carotenoids synthesizing enzymes may be less susceptible to disorganization than in the system responsible for other compounds synthesis (Fogg, 1975).

Early studies on vitamin E production by microorganism showed that most of the prokaryotes and yeast showed little or no tocopherol (Hughest and Tove, 1982 ). But, some microalgae such as Euglena gracilis and Dunaliella are considered a potential source for tocopherols (with vitamin E, values between 1.12 – 1.35 mg g^-1 d.w) under condition of oxygen or nitrogen starvation and light intensity (El-Baz et al., 2002 a; Venkataraman et al., 1994 and Ogbonna et al., 1998). In the present study, α- tocopherol (vitamin E) content of Spirulina grown under condition of nitrogen stress, varied between 1.72- 2.0% of the dry weight and this content probably can be increased under other conditions. However, this content is higher than in conventional food traditionally considered rich in vitamin E (Fabregas and Herrero, 1990 ; El-Baz et al., 2002a). Finally, the metabolic pathway for accumulation of fatty material leading to catabolism by β-oxidation to produce the excess of acetyl CoA. Thus, the biosynthesis of carotenoids and tocopherol in Spirulina cells cultivated under nitrogen starvation increase, which acetyl CoA serves as a precursor for synthesis of tocopherol and carotenoids. In other words, the accumulation of β-carotenoids and α-tocopherol in algae cells grown under nitrogen starvation may be due to stimulation of lipolysis process which produced a large amount of acetyl CoA (Abd El-Baky, 1999).

However, acetyl CoA could be incorporated to phytyl pyrophosphate. It is known that tocopherol is synthesized by condensing the phytyl pyrophosphate with homogentistic acid to methyphyty-l quinones and subsequently methylation (Jsniszowska, 1987; Ogbonna et al., 1998).

The effect of algal extracts on viability of Ehrlich ascites carcinoma cells
The effect of algal extracts, β-carotene, astaxanthin, α-tocopherol and their mixture on viability of Ehrlich ascites carcinoma cells (EAAC) were examined by the trypan blue exclusion method. As shown in Table 4 and 5, treatment of cells with 200 and 400 ppm of β-carotene and astaxanthin (in parentheses) significant reduced cell viability to 34.9 (39.2) and 10.4% (24.7%), respectively. While, their mixture of β-carotene and astaxanthin (1: 1, w/w) reduced cell viability greater than those did by each one alone.

The α-tocopherol at 200 and 400 ppm significantly reduced the cell viability, with 37.2 and 15.4 %, respectively. The mixture of β-carotene, astaxanthin and a tocopherol (1:1:1, w/w/w) at 200 and 400 ppm was significant reduced the cell viability over than those obtained by each one alone, with 10.3 and 0.0%. Thus, cell viability was dependent of carotenoids and α-tocopherol concentration as well as the carotenoid structure.

Algal extracts of S. plantensis and S. maxima significantly reduced the viability of EACC is compared with untreated cells. The algae extracts of S. plantensis and S. maxima grown in free nitrogen medium which contained relatively large amounts of carotenoids and α-tocopherol were significantly reduced the cell viability greater than those did by other algae extracts. At a concentration of 200 and 400 ppm (in parentheses), these extracts significantly reduced cell viability to 22.20 % (10.70%) and 18.33 (5.25%), respectively. The extracts obtained from Spirullina sp. grown under NaCl stress condition that contained lower amount of carotenoids and α-tocopherol reduced the cell viability, but less than those alga extracts contained large amount carotenoids and α-tocopherol contents.

Thus, cell viability was depended on the of carotenoids and tocopherol content in these alga extracts.

DNA fragment assay
Exposure of Spirullina extracts, β-carotene, astaxanthin and a tocopherol and those mixtures at 200 and 400 ppm to the EACC clearly did not induce any significant change in DNA fragmentation compared with untreated cell (Table 6). This indicates that carotenoids (β-carotene and astaxanthin), α-tocopherol and algae extracts may not be able to induce apoptosis in the EACC. However, treated of EACC with cis-platinum (apotosis agent) at 50 mM clearly induced the morphological changes characterized by reduction of cell volume (data not shown) and increased the DNA fragmentation to 7.25% of the control (Duthie et al.,1997).

Enzymes induction
To further examine the role of carotenoids, α-tocopherol and algae extracts on the reduction of EACC viability, the intercellular enzyme activity of lactate dehydrogenase (LDH) and glutathine S-transferase (GST) was assayed. Cells treated with algae extracts significantly enhanced enzyme activities when compared with untreated cells. In general, EAC treated cells with S. plantensis and S. maxima extracts at 200 and 400 ppm had increased LDH, GST activities (Table 7, 8 and Fig. 2).

The LDH and GST activities in cells incubated with algal extracts obtained from S. plantensis and S. maxima (in parentheses) grown in free N medium (the most algae extract rich in carotenoids) were markedly increased and dose dependently, in rang 200 and 400 ppm, with values 535.8 (578.9) and 918.4 U L-1 (982.5 U L-1), respectively. Compared with that of β-carotene, α-tocopherol, astaxanthin and their mixtures, all algae extracts increased the level of LDH and GST of treated EACC. Treated of EAC cells with β-carotene, astaxanthin, α-tocopherol and their mixture increased at 200 and 400 ppm (in parentheses) significantly (P<0.05) increased LDH level, with 3.0 (5.6), 2.3 (3.7), 2.5(6.2) and 6.2 (11.6), respectively time over than that in untreated cells. Also, the GST level was increased, with 5.8 (10.0), 4.0 (7.16), 7.68 (12.4) and 8.5 (15.1) time as high as that in increased.

In general, EAC treated cells with S. plantensis and S. maxima extracts at 200 and 400 ppm had increased LDH and GST activities. Earlier studies have demonstrated that carotenoid treatment of human oral carcinoma cells increased some antioxidant enzyme activity (Palozza et al., 2000). Also, Abd El-Baky, (2003) reported that algal extracts rich in phycocayanin, β-carotene and α-tocopherol may modulates the activities of GST and GSH in EACC line. In animal studies that examined the chemopreventive effect of algae Dunalliell and Chlorella on chemically induced carcinogensis such as Benzo-a-pyrene (B-a-P) and Dimethyl antherathine (DAMA), the antioxidant enzyme levels correlated with reduced incidences of tumor formation (Shklar and Schwartz, 1988). Furthermore, we have previously suggested that the increased in GST, SOD, LDH and GSH levels contribute to the anticarcinogesis effect of algal extracts in Benzo-a-pyrene-induced carcinogesis in rats (Abd El-Baky et al., 2002).

GSH level
The influence of algal extracts and β-carotene, α-tocopherol, astazanthin at 200 and 400 ppm on GSH level in EACC as shown in Table 7, 8 and Fig. 2. Algal extracts significant increased GSH level compared with untreated cells. The GSH level of untreated cells was 6.1µg10^-6 cells. Whilst, treated of EACC cells with S. plantensis and S. maxima (in parentheses) extracts (obtained from free N culture) at 200 and 400 ppm increased the GSH level to 70.32 (92.33) and 100.73 (171.34) µg 10^-6 and β-caratene and α-tocopherol, respectively. However, all algae extracts, β-carotene and α-tocopherol increased GSH concentration in treated cells compared with untreated cells.

Recently, Liu et al., (2000) and Abd El-Baky, (2003) reported that algae extract of S. plantensis and S. maxima may be able to inhibit the growth of Leukemia-blast crisk 562 and EACC by pathway other than apoptosis. Moreover, Gabai, et al., (1995), observed that EACC has low susceptibility to apoptosis due to the propensity of these cells to accumulate those heat shock proteins on exposure to apoptosis inducing agents. It is also possible that, the majority of apoplotic cell are not phagocytzed but enter secondary necrosis (Vahrmeijer et al., 1999). In the present study, carotenoids and α- tocopherol act only as antioxidant but not as prooxidant. The prooxidant action of carotenoids has been suggested to induce apoptosis in tumor cells (Palozza et al., 2001).

People in East Asian countries, in which the mortality rate from cancer is low (Hsing et al., 2000) ingest astazanthin, fucoxanthin and nexzanthin through edible algae such as Undaria pinnatilida. Thus, consumption of algae might reduce the risk of cancer (Kotake-Nara et al., 2001). Schwartz and Shklar, (1987) has reported that extracts of Sp. plantensis (rich in β- carotene and other caroteniods) induced tumor regression and stimulated a large number of tumor necrosis factor alpha-positive (TNF-a) macrophages at the tumor site which may be a mechanism for the tumor degeneration and regression.

Finally, this study revealed that the blue-green algae Spirulina sp. offer a wide scope for accumulation of large quantities and qualitative natural carotenoids and tocopherols. Spirulina extracts was found to reduce EAC cell viability through other mechanism rather than apoptosis induction. Thus, these results suggest those ingestion Spirulina rich in β-carotene, astaxanthin and other carotenoids and α-tocopherol might have the chemopreventive potential. Therefore, Spirulina could be easily incorporated into daily dietary practices since they are relatively non- toxic.

REFERENCES