Spirulina (Arthrospira): An Important Source of Nutritional and Medicinal Compounds

Abstract

Cyanobacteria are aquatic and photosynthetic organisms known for their rich pigments. They are extensively employed as food supplements due to their rich contents of proteins. While many species, such as Anabaena sp., produce hepatotoxins (e.g., microcystins and nodularins) and neurotoxins (such as anatoxin a), Spirulina (Arthrospira) displays anticancer and antimicrobial (antibacterial, antifungal, and antiviral) activities via the production of phycocyanin, phycocyanobilin, allophycocyanin, and other valuable products. This paper is an effort to collect these nutritional and medicinal applications of Arthrospira in an easily accessible essay from the vast literature on cyanobacteria.

Recommended by Horst Felbeck

Department of Chemistry, Ahmadu Bello University, P.M.B. 1069, Zaria, Kaduna, Nigeria

Received 24 January 2013; Revised 13 April 2013; Accepted 16 April 2013

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Cyanobacteria are ancient photosynthetic organisms that are found in various aquatic environments [1-3]. Their photosynthetic pigments confer different colors on them, but they are generally regarded as blue-green. Calling them algae is, however, a misnomer since they are truly prokaryotes that share most of the characteristics of eubacteria. Some of these organisms have nitrogen-fixing potential which makes them important in rice paddy waters [4].

Cyanobacteria form colonies [5] or live as individual cells [6]. They also form coccoid [7] or filamentous structures [8]. The filamentous colonies show the ability to differentiate into three different cell types [9]. Vegetative cells, the normal photosynthetic cells formed under favorable growth conditions; climate-resistant spores in harsh environmental conditions and a thick-walled heterocyst containing the enzyme nitrogenase for nitrogen fixation.

In the last 3.5 billion years, cyanobacterial morphology has been largely maintained as they are very resistant to contamination. Sigler et al. [10] have shown that cyanobacteria form monophyletic taxon. Culture-based morphological characteristics of endolithic cyanobacteria have been extensively described by Al-Thukair and Golubic [11]. Since characterization of microorganisms based on morphology is highly subjective and sometimes very speculative, the shift by genome-based characterization is now gaining momentum. Koksharova and Wolk [12] have presented a good review on the available genetic tools for cyanobacteria studies.

Cyanobacteria are very resistant as they produce protective compounds which shield them against harsh environmental conditions [13]. Some of these compounds also have strong insecticidal activities [14]. Toxic species, including Anabaena species, produce toxins such as microcystins and nodularins which are hepatotoxic, and neurotoxins such as anatoxin a [15, 16].

The Darling River cyanobacterial bloom of 1991 is a clear representation of the environmental hazard that such species pose [17]. However, some species of cyanobacteria possess the ability to produce substances with therapeutic activities such as anticancer and antimicrobial applications [18-22].

Among the myriads of cyanobacteria, Arthrospira platensis is a blue-green cyanobacterium that thrives in elevated alkaline pH [23]. A. platensis is recognized by its peculiar shape of cylindrical trichomes that are arranged in a left-handed helix throughout the filament [24]. The correct taxonomic definitions of Arthrospira have been revealed through the study of the ultrastructural details of its trichomes and 16S rRNA gene sequences [25]. An important ligation detection reaction, in combination with universal array, capable of identifying various cyanobacteria, including Arthrospira , in environmental samples, has been developed [26]. Good understanding of the ecology of this alkaliphilic organism is a catalyst to its mass production and commercial viability as food supplement. By the end of year 2009, its total annual production in Ordos Plateau of Mongolia was in excess of 700 t [27]. With retrospect, the Mexicans [28] and Kanenbu tribe of Chad [29] have been exploiting the protein potentials of S. platensis in their diets for long time now, and about 3000 metric tons of S. platensis is currently produced for commercial purposes [30]. A fed-batch process has been employed in the cultivation of Arthrospira [31], and different solid-liquid separation techniques give various degrees of recovery. Which technique is ultimately selected will depend on the cyanobacterial species, intended concentration of the finished product, and product quality [32]. Cultivation of A. platensis under different trophic modes was shown to affect the product yield [33]. High-value compounds from this organism have been put to assorted uses as cosmaceuticals, nutraceuticals, and as functional foods [34]. Phycocyanin and allophycocyanin, two of such important compounds, have been determined in Spirulina supplements and raw materials by a 2-wavelength spectrophotometric method [35]. Bioactivity and health functions of Arthrospira food supplements have been reviewed [36-38]. Specific functions that have been tested for compounds extracted from this organism are grouped under the following subheadings.

2. Nutritional Functions

Arthrospira (Spirulina ) is among the richest sources of proteins. Its protein content is about 60-70% [39]. In a study that attempted using Spirulina as a protein supplement, it was observed that it can replace up to 40% of protein content in tilapia diets [40]. Rabelo et al. [41] have explained the development of cassava doughnuts enriched with S. platensis biomass.

Unlike many other cyanobacteria that have proven toxicity, no such property has been attributed to Spirulina . While testing for mutagenicity, acute, subchronic, and chronic toxicities and teratogenicity in animal experimentations, Chamorro et al. [42] have shown that Spirulina did not exhibit any potential for organ or system toxicity even though the doses given were elevated above those for expected human consumption. Rather, Spirulina was shown to protect fish from sublethal levels of some chemicals [43]. Likewise, dietary supplementation of Spirulina has helped in alleviating the incidence of anemia experienced during pregnancy and lactation. In the study conducted by Kapoor and Mehta [44], dietary supplementation of S. platensis was found to increase the iron storage of rats, better than achieved from the combination of casein and wheat gluten diets, during the first half of pregnancy and lactation. A review that treats the influence of different compounds from Spirulina on the immune system has been written [45].

3. Antioxidant Functions

Apart from its importance as a food additive for supplementary dietary proteins, there are also a lot of potentials for medical and therapeutic applications [46]. For example, A. platensis plays a hepatoprotective role [47]. This role, which has to do with the antioxidant activity of Spirulina , has been previously asserted by various researchers. The antioxidant activity of Spirulina is ascribed to the presence of two phycobiliproteins: phycocyanin and allophycocyanin, as determined by its action against OH radical generated from ascorbate/iron/H₂ O₂ system. The activity was found to be proportional to the concentration of the phycobiliproteins and was mainly due to the phycocyanin content [48]. As an antioxidant effect, oxygen stress was inhibited by phycocyanin and phycocyanobilin from Spirulina leading to protection against diabetic nephropathy [49]. In an earlier experiment to determine the radical scavenging activity of C-phycocyanin isolate of S. platensis , an intraperitoneally administered C-phycocyanin was found to reduce the peroxide values of CCl₄ -induced lipid peroxidation in rat liver microsomes [50]. Following a study conducted on 60 patients presenting with chronic diffuse disorders in the liver and on 70 experimental animals, Gorban' et al. [51] have found that Spirulina administration prevented the transformation of chronic hepatitis into hepatic cirrhosis. Recently, Paniagua-Castro et al. [52] have demonstrated the protective efficacy of Arthrospira against cadmium-induced teratogenicity in mice.

There are indications that these therapeutic potentials are not the exclusive rights of S. platensis . Spirulina fusiformis also has shown some free radical scavenging activities. In rats, Kuhad et al. [53] have found that radical scavenging activity of S. fusiformis did protect against nephrotoxicity resulting from oxidative and nitrosative stress of the aminoglycoside, gentamicin, an antibiotic commonly used for the treatment of Gram-negative bacterial infections. Pretreatment of mice with Arthrospira maxima effectively led to the reduction in liver total lipids, liver triacylglycerols, and serum triacylglycerols, thus protecting against Simvastatin-induced hyperlipidemia [54]. The hexane extract of Spirulina achieved an impressive 89.7% removal of arsenic from rat liver tissue, which is a better result than obtained with either alcohol or dichloromethane extract [55]. In a more recent finding, aqueous extract of S. platensis showed suppressive potency, through free radical scavenging activity, against cyclophosphamide-induced lipid peroxidation in goat liver homogenates [56].

As a nephroprotective activity, S. platensis extract counteracted the hyperoxaluria experimentally induced by the administration of sodium-oxalate to rats, through stabilization of antioxidant enzymes and glutathione metabolizing enzymes [57]. Protections against mercuric chloride-(HgCl₂ -) induced renal damage and oxidative stress were attributed to the administration of A. maxima to experimental mice [58]. Administration of A. platensis to rats also rendered protection against HgCl₂ -induced testis injury and sperm quality deteriorations [59].

S. platensis biomass preparations have shown some corrective influences on atherosclerotic processes in 68 patients with ischemic heart disease (IHD) and atherogenic dyslipidemia. The patients' immunological states were altered, in addition to changes in lipid spectra [60]. Pretreatment of experimental animals with Spirulina has proved its cardioprotective function, this time against doxorubicin-induced toxicity, as evident from lower mortality, lower degree of lipid peroxidation, decreased ascites, and normalization of antioxidant enzymes, without compromising the antitumor activity of the drug, doxorubicin [61]. The contribution of reactive oxygen species (ROS) to brain injury in neurodegenerative conditions, such as Parkinson's disease, is hampered with proper administration of A. maxima supplement. Following a 40-day pretreatment with 700 mg/kg/day of this supplement, various indicators of toxicity in rat injected with a single dose of 6-hydroxydopamine, 6-OHDA (16 μ g/2 μ L), were decreased [62]. This is an indication of the neuroprotective effect of this supplement against the harmful effect of free radicals. Arthrospira supplement has also a radioprotective effect. This is demonstrated by its free radical scavenging function against gamma-irradiation-induced oxidative stress and tissue damage in rats [63]. Cell death through apoptosis is prevented or delayed by using a cold water extract of S. platensis [64]. Hence, it is suggested that the inclusion of cyanobacterial supplement in beverages and food products should be strongly considered.

4. Antitumor Functions

Strong evidences have shown that S. platensis is also imbued with antitumor and anticancer functions. In this regard, it was discovered that significant to full tumor regression was obtained with intravenous injection of Radachlorin, a new chlorine photosensitizer that was derived from S. platensis [65]. It was shown that hot-water extract of S. platensis facilitated enhanced antitumor activity of natural killer (NK) cells in rats [66]. Recently, complex polysaccharides from Spirulina have brought about suppression of glioma cell growth by downregulating angiogenesis via partial regulation of interleukin-17 production [67]. High production of tumor necrosis factor-α (TNF-α ), in macrophages, was recorded in the presence of acidic polysaccharides from A. platensis [68]. Li et al. [69] have shown that with increased phycocyanin concentration, expression of CD59 proteins in HeLa cells was promoted while Fas protein that induces apoptosis was increased with an attendant decline in the multiplication of HeLa cells. These findings are an evidence for the multidimensional applications of phycocyanin content of S. platensis.

5. Antiviral Functions

Many compounds with antimicrobial activities have been isolated from different marine organisms, and a number of evidences are put forward for the antiviral activity of Spirulina [70, 71]. This antiviral activity, in a large part, is attributable to the richness of S. platensis in vital proteins, fatty acids, minerals, and other important constituents [72].

Previously, calcium spirulan (Ca-SP), a novel sulfated polysaccharide that was isolated from hot water extract of S. platensis , has shown antiviral activities against different enveloped viruses such as Herpes simplex virus type-I, measles virus, HIV-I and influenza virus. This high sought for antiviral activity has been suggested to be due to the effect that chelation of calcium ions to sulfate groups has on molecular conformation [73]. Both extracellular and intracellular spirulan-like molecules from the polysaccharide fractions of A. platensis displayed significant antiviral activities against wide range of viruses, including human cytomegalovirus and HIV-I [74]. About 50% and 23% reductions in viral load were recorded for methanolic and aqueous extracts of S. platensis , respectively [75]. Reduction in viral load was attributed to inhibition of HIV-I replication in human T cells, langerhans cells, and peripheral blood mononuclear cells (PBMCs), with up to 50% reduction accorded to PBMCs [76]. Antiviral and immunostimulatory properties of S. platensis preparations were elicited through increased mobilization of macrophages, cytokine production, antibodies generation, accumulation of NK cells, and mobilization of B and T cells [77]. A recent study on the antiviral activity of Spirulina has resulted in the isolation of Cyanovirin-N (CV-N), a novel cyanobacterial carbohydrate-binding protein that inhibits HIV-I and other enveloped viral particles [78]. The Kanenbu tribe of Chad and most people in Korea and Japan, who consume Spirulina diet daily, have been shown to display lower cases of HIV/AIDS than their surrounding neighbors who do not take such diet. Therefore, it is expected that consistent intake of diets containing Spirulina can help in reducing the prevalence of HIV/AIDS [79]. Antiherpetic activities were noted for the crude extracts of S. fusiformis [80]. While Hernández-Corona et al. [81] have reported antiviral activity of S. maxima against HSV-2, Shalaby et al. reported similar activity for S. platensis against HSV-I [82].

6. Antibacterial Functions

Spirulina is not without antibacterial activity. In 3-week-old chicks injected with either Escherichia coli or Staphylococcus aureus suspensions, 0.1% Spirulina was found to enhance their bacterial clearance abilities, as shown by the improvement in the activities of different phagocytotic cells, including heterophils, thrombocytes, macrophages, and monocytes in the chickens [83]. Microalgal cultures of A. platensis have displayed significant antibacterial activity against six Vibrio strains: Vibrio parahaemolyticus, Vibrio anguillarum, Vibrio splendidus, Vibrio scophthalmi, Vibrio alginolyticus , and Vibrio lentus [84]. Antibacterial activity against Streptococcus pyogenes and/or S. aureus was proven for the phycobiliproteins isolated from A. fusiformis [85]. Purified C-phycocyanin from S. platensis markedly inhibited the growth of some drug resistant bacteria: E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and S. aureus [86]. This shows the potentials of compounds isolated from these cyanobacterial species in the fight against drug resistance.

7. Antifungal Functions

Recently, Spirulina has also exhibited antifungal activities [87]. Activity of 13 mm was recorded against Candida glabrata in the butanol extract of Spirulina sp. [88]. The immunostimulatory effect of S. platensis extract was tested in Balb/C mice infected with candidiasis [89]. In this experiment, pretreatment of the mice with 800 mg/kg of the extract for 4 days before intravenous inoculation with C. albicans resulted in increased production of cytokines TNF-α and interferon-gamma (IFN-γ ), leading to increased survival time and better fungal clearance than in control groups. Glucosamine production was reduced by about 56% when the antifungal activity of the methanolic extract of S. platensis was tested against Aspergillus flavus [90]. Contrary to these findings, S. platensis grown in Zarrouk media, DB₁ media and papaya skin extract media did not show any antifungal activity [91]. In some instances, extracts from S. platensis may display a stimulatory effect toward cultured microorganisms. It was found by Gorobets et al. [92] that different doses of S. platensis, when added to culture fluid, displayed important stimulatory and inhibitory effects on the cultured microorganisms due to the presence of complex metabolites that were active in the prepared nutrient agar. Similarly, S. platensis biomass was used to maintain the counts of starter organisms in acidophilus-bifidus-thermophilus (ABT) milks at satisfactory levels during whole duration of storage. This is a novel opportunity for the production and maintenance of functional dairy foods [93].

8. Miscellaneous Functions

Many compounds produced from marine organisms, including cyanobacteria, have important protective functions against various allergic responses such as asthma, atopic dermatitis, and allergic rhinitis [94].

Powders of S. platensis have inhibited anaphylactic reaction resulting from antidinitrophenol IgE-induced histamine release or from TNF-α of rats [95]. Spirulina was also found effective against allergic rhinitis [96]. In an earlier human feeding study conducted in this regard, Spirulina -based dietary supplement was found effective in suppressing the level of interleukin- (IL-) 4 [23]. Zymosan-induced upsurge in the level of beta-glucuronidase of experimental mice was significantly reduced following the administration of phycocyanin [97]. This antiarthritic action may be due to the combination of various mechanisms such as free radical scavenging, inhibition of arachidonic acid metabolism as well as inhibition of TNF-α within the mice.

S. platensis has also neuroprotective ability. Its neuroprotective effect was demonstrated in adult Sprague-Dawley rats through a significant reduction in the volume of cerebral cortex infarction and increased poststroke locomotor activity. Hence, it is suggested that chronic treatment with Spirulina can reduce ischemic brain damage [98]. A report has shown that lead-induced increase in mast cells in rat ovary, during estrous cycle, is curtailed by using Spirulina at 300 mg/kg [99].

Another important compound synthesized by Spirulina , which equally has a lot of vital applications, is polyhydroxyalkanoates. These are polyesters produced by bacterial fermentation of sugars or lipids. According to Campbell et al. [100], S. platensis stores about 6% of its total dry weight and this value decreases during stationary phase of its growth profile. However, Jau et al. [101] have posited that this value can be increased to 10% when the organisms are grown under nitrogen-deficient, mixotrophic culture medium. The use of recombinant E. coli , due to its fast rate of growth and minimal nutrient requirements, to overproduce polyhydroxyalkanoates, has the potential of increasing the number of polyhydroxyalkanoates inclusions per cell [102]. Polyhydroxyalkanoates hold an assuring promise in therapeutic applications as drug carriers that display a release pattern similar to those of monolithic devices; an early rapid release followed by a prolonged, but slower release pattern. This type of drug-release behavior is normally required for depositing adequate concentration of drug of interest at the site of infection [103]. Among the polyhydroxyalkanoates, 4-hydroxybutyrate has long been advocated for the treatment of alcohol withdrawal syndrome in alcohol-dependent subjects [104].

9. Conclusions

In the foregoing essay, various nutritional and medicinal potencies have been attributed to metabolites from the cyanobacteria, Spirulina (Arthrospira) sp. In the present clamor for alternative medicine, these organisms serve as very viable potential sources of bioactive products with commercial imports. Therefore, more should be done in the study, culture, isolation, and purification of these organisms to enable beneficial harvest of their important inclusions.

References

[1] J. A. Downing, S. B. Watson, E. McCauley, "Predicting cyanobacteria dominance in lakes," Canadian Journal of Fisheries and Aquatic Sciences , vol. 58, no. 10, pp. 1905-1908, 2001.

[2] T. Miyatake, B. J. MacGregor, H. T. S. Boschker, "Depth-related differences in organic substrate utilization by major microbial groups in intertidal marine sediment," Applied and Environmental Microbiology , vol. 79, no. 1, pp. 389-392, 2013.

[3] L. . Charpy, B. E. Casareto, M. J. Langlade, Y. Suzuki, "Cyanobacteria in coral reef ecosystems: a review," Journal of Marine Biology , vol. 2012, 2012.

[4] M. Singh, N. K. Sharma, S. B. Prasad, S. S. Yadav, G. Narayan, A. K. Rai, "Freshwater cyanobacterium Anabaena doliolum transformed with ApGSMT-DMT exhibited enhanced salt tolerance and protection to nitrogenase activity, but changed its behavior to halophily," Microbiology , vol. 159, pp. 641-648, 2013.

[5] M. Zhang, X. Shi, Y. Yu, F. Kong, "The acclimative changes in photochemistry after colony formation of the cyanobacteria Microcystis aeruginosa ," Journal of Phycology , vol. 47, no. 3, pp. 524-532, 2011.

[6] M. Sabart, D. Pobel, E. Briand, B. Combourieu, M. J. Salençon, J. F. Humbert, D. Latour, "Spatiotemporal variations in microcystin concentrations and in the proportions of microcystin-producing cells in several Microcystis aeruginosa populations," Applied and Environmental Microbiology , vol. 76, no. 14, pp. 4750-4759, 2010.

[7] J. Kazmierczak, W. Altermann, B. Kremer, S. Kempe, P. G. Eriksson, "Mass occurrence of benthic coccoid cyanobacteria and their role in the production of Neoarchean carbonates of South Africa," Precambrian Research , vol. 173, no. 1-4, pp. 79-92, 2009.

[8] N. Morin, T. Vallaeys, L. Hendrickx, L. Natalie, A. Wilmotte, "An efficient DNA isolation protocol for filamentous cyanobacteria of the genus Arthrospira," Journal of Microbiological Methods , vol. 80, no. 2, pp. 148-154, 2010.

[9] I. V. Kozhevnikov, N. A. Kozhevnikova, "Taxonomic studies of some cultured strains of Cyanobacteria (Nostocales) isolated from the Yenisei River basin," Inland Water Biology , vol. 4, no. 2, pp. 143-152, 2011.

[10] W. V. Sigler, R. Bachofen, J. Zeyer, "Molecular characterization of endolithic cyanobacteria inhabiting exposed dolomite in central Switzerland," Environmental Microbiology , vol. 5, no. 7, pp. 618-627, 2003.

[11] A. A. Al-Thukair, S. Golubic, "Five new Hyella species from Arabian Gulf," Algological Studies , vol. 64, pp. 167-197, 1991.

[12] O. Koksharova, C. Wolk, "Genetic tools for cyanobacteria," Applied Microbiology and Biotechnology , vol. 58, no. 2, pp. 123-137, 2002.

[13] D. D. Wynn-Williams, H. G. M. Edwards, "Proximal analysis of regolith habitats and protective molecules in situ by Laser Raman Spectroscopy. Overview of terrestrial Antarctic habitats and Mars analogs," Icarus , vol. 144, no. 2, pp. 486-503, 2000.

[14] P. G. Becher, F. Jüttner, "Insecticidal compounds of the biofilm-forming cyanobacterium Fischerella sp. (ATCC 43239)," Environmental Toxicology , vol. 20, no. 3, pp. 363-372, 2005.

[15] M. . Pírez, G. Gonzalez-Sapienza, D. Sienra, G. Ferrari, M. Last, J. A. Last, B. M. Brena, "Limited analytical capacity for cyanotoxins in developing countries may hide serious environmental health problems: simple and affordable methods may be the answer," Journal of Environmental Management , vol. 114, pp. 63-71, 2013.

[16] S. Klitzke, C. Beusch, J. Fastner, "Sorption of the cyanobacterial toxins cylindrospermopsin and anatoxin-a to sediments," Water Research , vol. 45, no. 3, pp. 1338-1346, 2011.

[17] L. C. Bowling, P. D. Baker, "Major cyanobacterial bloom in the Barwon-Darling River, Australia, in 1991, and underlying limnological conditions," Marine and Freshwater Research , vol. 47, no. 4, pp. 643-657, 1996.

[18] L. T. Tan, "Filamentous tropical marine cyanobacteria: a rich source of natural products for anticancer drug discovery," Journal of Applied Phycology , vol. 22, no. 5, pp. 659-676, 2010.

[19] F. Heidari, H. Riahi, M. Yousefzadi, M. Asadi, "Antimicrobial activity of cyanobacteria isolated from hot spring of geno," Middle-East Journal of Scientific Research , vol. 12, no. 3, pp. 336-339, 2012.

[20] R. Prasanna, A. Sood, P. Jaiswal, S. Nayak, V. Gupta, V. Chaudhary, M. Joshi, C. Natarajan, "Rediscovering cyanobacteria as valuable sources of bioactive compounds (Review)," Applied Biochemistry and Microbiology , vol. 46, no. 2, pp. 119-134, 2010.

[21] R. B. Volk, F. H. Furkert, "Antialgal, antibacterial and antifungal activity of two metabolites produced and excreted by cyanobacteria during growth," Microbiological Research , vol. 161, no. 2, pp. 180-186, 2006.

[22] R.-B. Volk, "Antialgal actitiy of several cyanobacterial exometabolites," Journal of Applied Phycology , vol. 18, no. 2, pp. 145-151, 2006.

[23] T. K. Mao, J. Van De Water, M. E. Gershwin, "Effects of a Spirulina-based dietary supplement on cytokine production from allergic rhinitis patients," Journal of Medicinal Food , vol. 8, no. 1, pp. 27-30, 2005.

[24] L. M. Colla, C. Oliveira Reinehr, C. Reichert, J. A. V. Costa, "Production of biomass and nutraceutical compounds by Spirulina platensis under different temperature and nitrogen regimes," Bioresource Technology , vol. 98, no. 7, pp. 1489-1493, 2007.

[25] C. Sili, G. Torzillo, A. Vonshak, B. A. Whitton, "Arthrospira (Spirulina)," Ecology of Cyanobacteria II , pp. 677-705, Springer, Dordrecht, The Netherlands, 2012.

[26] B. Castiglioni, E. Rizzi, A. Frosini, K. Sivonen, P. Rajaniemi, A. Rantala, M. A. Mugnai, S. Ventura, A. Wilmotte, C. Boutte, S. Grubisic, P. Balthasart, C. Consolandi, R. Bordoni, A. Mezzelani, C. Battaglia, G. De Bellis, "Development of a universal microarray based on the ligation detection reaction and 16S rRNA gene polymorphism to target diversity of cyanobacteria," Applied and Environmental Microbiology , vol. 70, no. 12, pp. 7161-7172, 2004.

[27] Y. M. Lu, W. Z. Xiang, Y. H. Wen, "Spirulina (Arthrospira) industry in Inner Mongolia of China: current status and prospects," Journal of Applied Phycology , vol. 23, no. 2, pp. 265-269, 2011.

[28] N. Kumar, S. Singh, N. Patro, I. Patro, "Evaluation of protective efficacy of Spirulina platensis against collagen-induced arthritis in rats," Inflammopharmacology , vol. 17, no. 3, pp. 181-190, 2009.

[29] O. Ciferri, "Spirulina, the edible microorganism," Microbiological Reviews , vol. 47, no. 4, pp. 551-578, 1983.

[30] A. Belay, "The potential of Spirulina (Arthrospira) as a nutritional and therapeutic supplement in health management," Journal of the American Nutraceutical Association , vol. 5, no. 2, pp. 27-49, 2002.

[31] J. C. M. Carvalho, R. P. Bezerra, M. C. Matsudo, S. Sato, J. W. Lee, "Cultivation of Arthrospira (Spirulina) platensis by Fed-Batch Process," Advanced Biofuels and Bioproducts , pp. 781-805, Springer, New York, NY, USA, 2013.

[32] S. L. Pahl, A. K. Lee, T. Kalaitzidis, P. J. Ashman, S. Sathe, D. M. Lewis, M. A. Borowitzka, N. R. Moheimani, "Harvesting, thickening and dewatering microalgae biomass," Algae for Biofuels and Energy , vol. 5, pp. 165-185, Springer, Dordrecht, The Netherlands, 2013.

[33] L. Trabelsi, H. Ben Ouda, F. Zili, N. Mazhoud, J. Ammar, "Evaluation of Arthrospira platensis extracellular polymeric substances production in photoautotrophic, heterotrophic and mixotrophic conditions," Folia Microbiologica , vol. 58, pp. 39-45, 2013.

[34] M. A. Borowitzka, "High-value products from microalgae-their development and commercialization," Journal of Applied Phycology , vol. 1, pp. 1-14, 2013.

[35] N. Yoshikawa, A. Belay, "Single-laboratory validation of a method for the determination of c-phycocyanin and allophycocyanin in spirulina (Arthrospira) supplements and raw materials by spectrophotometry," Journal of AOAC International , vol. 91, no. 3, pp. 524-529, 2008.

[36] M. Filomena de Jesus Raposo, R. M. Santos Costa de Morais, A. M. Miranda Bernado de Morais, "Bioactivity and applications of sulphated polysaccharides from marine microalgae," Marine Drugs , vol. 11, no. 1, pp. 233-252, 2013.

[37] T. G. Sotiroudis, G. T. Sotiroudis, "Health aspects of Spirulina (Arthrospira) microalga food supplement," Journal of the Serbian Chemical Society , vol. 78, no. 3, pp. 395-405, 2013.

[38] J. Villa, C. Gemma, A. Bachstetter, Y. Wang, I. Stromberg, P. C. Bickford, M. E. Gershwin, A. Belay, "Spirulina, aging, and neurobiology," Spirulina in Human Nutrition and Health , pp. 271-291, CRC Press, Taylor & Francis Group, Boca Raton, Fla, USA, 2007.

[39] Y. Ishimi, F. Sugiyama, J. Ezaki, M. Fujioka, J. Wu, "Effects of spirulina, a blue-green alga, on bone metabolism in ovariectomized rats and hindlimb-unloaded mice," Bioscience, Biotechnology and Biochemistry , vol. 70, no. 2, pp. 363-368, 2006.

[40] M. A. Olvera-Novoa, L. J. Domínguez-Cen, L. Olivera-Castillo, C. A. Martínez-Palacios, "Effect of the use of the microalga Spirulina maxima as fish meal replacement in diets for tilapia," Aquaculture Research , vol. 29, no. 10, pp. 709-715, 1998.

[41] S. F. Rabelo, A. C. Lemes, K. P. Takeuchi, M. T. Frata, J. C. Monteiro de Carvalho, E. D. G. Danesi, "Development of cassava doughnuts enriched with Spirulina platensis biomass," Brazilian Journal of Food Technology , vol. 16, no. 1, pp. 42-51, 2013.

[42] G. Chamorro, M. Salazar, L. Favila, H. Bourges, "Pharmacology and toxicology of the alga Spirulina," Revista de Investigacion Clinica , vol. 48, no. 5, pp. 389-399, 1996.

[43] K. P. Sharma, N. Upreti, S. Sharma, S. Sharma, "Protective effect of Spirulina and tamarind fruit pulp diet supplement in fish (Gambusia affinia Baird & Girard) exposed to sublethal concentration of fluoride, aluminum and aluminum fluoride," Indian Journal of Experimental Biology , vol. 50, pp. 897-903, 2012.

[44] R. Kapoor, U. Mehta, "Supplementary effect of spirulina on hematological status of rats during pregnancy and lactation," Plant Foods for Human Nutrition , vol. 52, no. 4, pp. 315-324, 1998.

[45] S. A. Kedik, E. I. Yartsev, I. V. Sakaeva, E. S. Zhavoronok, A. V. Panov, "Influence of spirulina and its components on the immune system (review)," Russian Journal of Biopharmaceuticals , vol. 3, no. 3, pp. 3-10, 2011.

[46] T. Hirahashi, M. Matsumoto, K. Hazeki, Y. Saeki, M. Ui, T. Seya, "Activation of the human innate immune system by Spirulina: augmentation of interferon production and NK cytotoxicity by oral administration of hot water extract of Spirulina platensis ," International Immunopharmacology , vol. 2, no. 4, pp. 423-434, 2002.

[47] M. Samir, P. S. Amrit, "A review of pharmacology and phytochemicals from Indian medicinal plants," The Internet Journal of Alternative Medicine , vol. 5, no. 1, pp. 1-6, 2007.

[48] J. E. Piero Estrada, P. Bermejo Bescós, A. M. Villar del Fresno, "Antioxidant activity of different fractions of Spirulina platensis protean extract," IL Farmaco , vol. 56, no. 5-7, pp. 497-500, 2001.

[49] J. Zheng, T. Inoguchi, S. Sasaki, Y. Maeda, M. McCarty, M. Fujii, N. Ikeda, K. Kobayashi, N. Sonada, R. Takayanagi, "Phycocyanin and phycocyanobilin from Spirulina platensis protect against diabetic nephropathy by inhibiting oxidative stress," American Journal of Physiology , vol. 304, no. 2, pp. R110-R120, 2013.

[50] V. B. Bhat, K. M. Madyastha, "C-Phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro," Biochemical and Biophysical Research Communications , vol. 275, no. 1, pp. 20-25, 2000.

[51] E. M. Gorban', M. A. Orynchak, N. G. Virstiuk, L. P. Kuprash, T. M. Panteleimonova, L. B. Sharabura, "Clinical and experimental study of spirulina efficacy in chronic diffuse liver diseases," Likars'ka Sprava , no. 6, pp. 89-93, 2000.

[52] N. Paniagua-Castro, G. Escalona-Cardoso, D. Hernández-Navarro, R. Pérez-Pastén, G. Chamorro-Cevallos, " Spirulina (Arthrospira) protects against cadmium-induced teratogenic damage in mice," Journal of Medicinal Food , vol. 14, no. 4, pp. 398-404, 2011.

[53] A. Kuhad, N. Tirkey, S. Pilkhwal, K. Chopra, "Effect of Spirulina, a blue green algae, on gentamicin-induced oxidative stress and renal dysfunction in rats," Fundamental and Clinical Pharmacology , vol. 20, no. 2, pp. 121-128, 2006.

[54] J. L. Blé-Castillo, A. Rodríguez-Hernández, R. Miranda-Zamora, M. A. Juárez-Oropeza, J. C. Díaz-Zagoya, " Arthrospira maxima prevents the acute fatty liver induced by the administration of simvastatin, ethanol and a hypercholesterolemic diet to mice," Life Sciences , vol. 70, no. 22, pp. 2665-2673, 2002.

[55] S. K. Saha, M. Misbahuddin, R. Khatun, I. R. Mamun, "Effect of hexane extract of spirulina in the removal of arsenic from isolated liver tissues of rat," Mymensingh Medical Journal , vol. 14, no. 2, pp. 191-195, 2005.

[56] S. Ray, K. Roy, C. Sengupta, "In vitro evaluation of antiperoxidative potential of water extract of Spirulina platensis (blue green algae) on cyclophosphamide-induced lipid peroxidation," Indian Journal of Pharmaceutical Sciences , vol. 69, no. 2, pp. 190-196, 2007.

[57] S. M. Farooq, D. Asokan, R. Sakthivel, P. Kalaiselvi, P. Varalakshmi, "Salubrious effect of C-phycocyanin against oxalate-mediated renal cell injury," Clinica Chimica Acta , vol. 348, no. 1-2, pp. 199-205, 2004.

[58] R. Rodríguez-Sánchez, R. Ortiz-Butrón, V. Blas-Valdivia, A. Hernández-García, E. Cano-Europa, "Phycobiliproteins or C-phycocyanin of Arthrospira (Spirulina) maxima protect against HgCl₂ -caused oxidative stress and renal damage," Food Chemistry , vol. 135, no. 4, pp. 2359-2365, 2012.

[59] G. E. El-Desoky, S. A. Bashandy, I. M. Alhazza, Z. A. Al-Othman, M. A. M. Aboul-Soud, K. Yusuf, "Improvement of mercuric chloride-induced testis injuries and sperm quality deteriorations by Spirulina platensis in rats," PLoS ONE , vol. 8, no. 3, 2013.

[60] V. A. Ionov, M. M. Basova, "Use of blue-green micro-seaweed Spirulina platensis for the correction of lipid and hemostatic disturbances in patients with ischemic heart disease," Voprosy Pitaniia , vol. 72, no. 6, pp. 28-31, 2003.

[61] M. Khan, J. C. Shobha, I. K. Mohan, M. U. R. Naidu, C. Sundaram, S. Singh, P. Kuppusamy, V. K. Kutala, "Protective effect of Spirulina against doxorubicin-induced cardiotoxicity," Phytotherapy Research , vol. 19, no. 12, pp. 1030-1037, 2005.

[62] J. C. Tobon-Velasco, V. Palafox-Sanchez, L. Mendieta, E. Garcia, A. Santamaria, G. Chamorro-Cevallos, I. Daniel Limon, "Antioxidant effect of Spirulina (Arthrospira) maxima in a neurotoxic model caused by 6-OHDA in the rat striatum," Journal of Neural Transmission , 2013.

[63] R. Makhlouf, I. Makhlouf, "Evaluation of the effect of Spirulina against Gamma irradiation-induced oxidative stress and tissue injury in rats," International Journal of Applied Sciences and Engineering Research , vol. 1, no. 2, pp. 152-164, 2012.

[64] W. L. Chu, Y. W. Lim, A. K. Radhakrishnan, P. E. Lim, "Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals," BMC Complementary and Alternative Medicine , vol. 10, article 53, 2010.

[65] V. A. Privalov, A. V. Lappa, O. V. Seliverstov, A. B. Faizrakhmanov, N. N. Yarovoy, E. V. Kochneva, M. V. Evnevich, A. S. Anikina, A. V. Reshetnicov, I. D. Zalevsky, Y. V. Kemov, T. J. Dougherty, "Clinical trials of a new chlorin photosensitizer for photodynamic therapy of malignant tumors," in Optical Methods for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy XI, vol. 4612, of Proceedings of SPIE, pp. 178-189, January 2002.

[66] Y. Akao, T. Ebihara, H. Masuda, Y. Saeki, T. Akazawa, K. Hazeki, O. Hazeki, M. Matsumoto, T. Seya, "Enhancement of antitumor natural killer cell activation by orally administered Spirulina extract in mice," Cancer Science , vol. 100, no. 8, pp. 1494-1501, 2009.

[67] Y. Kawanishi, A. Tominaga, H. Okuyama, S. Fukuoka, T. Taguchi, Y. Kusumoto, T. Yawata, Y. Fujimoto, S. Ono, K. Shimizu, "Regulatory effects of Spirulina complex polysaccharides on growth of murine RSV-M glioma cells through Toll-like receptor-4," Microbiology and Immunology , vol. 57, no. 1, pp. 63-73, 2013.

[68] M. L. Parages, R. M. Rico, R. T. Abdala-Díaz, M. Chabrillón, T. G. Sotiroudis, C. Jiménez, "Acidic polysaccharides of Arthrospira (Spirulina) platensis induce the synthesis of TNF-α in RAW macrophages," Journal of Applied Phycology , vol. 24, pp. 1537-1546, 2012.

[69] B. Li, X. Zhang, M. Gao, X. Chu, "Effects of CD59 on antitumoral activities of phycocyanin from Spirulina platensis ," Biomedicine and Pharmacotherapy , vol. 59, no. 10, pp. 551-560, 2005.

[70] J. Yasuhara-Bell, Y. Lu, "Marine compounds and their antiviral activities," Antiviral Research , vol. 86, no. 3, pp. 231-240, 2010.

[71] A. M. S. Mayer, A. D. Rodríguez, R. G. S. Berlinck, M. T. Hamann, "Marine pharmacology in 2005-6: marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action," Biochimica et Biophysica Acta , vol. 1790, no. 5, pp. 283-308, 2009.

[72] L. P. Blinkova, O. B. Gorobets, A. P. Baturo, "Biological activity of Spirulina," Zhurnal Mikrobiologii Epidemiologii i Immunobiologii , no. 2, pp. 114-118, 2001.

[73] T. Hayashi, K. Hayashi, M. Maeda, I. Kojima, "Calcium spirulan, an inhibitor of enveloped virus replication, from a blue-green alga Spirulina platensis ," Journal of Natural Products , vol. 59, no. 1, pp. 83-87, 1996.

[74] S. Rechter, T. König, S. Auerochs, S. Thulke, H. Walter, H. Dörnenburg, C. Walter, M. Marschall, "Antiviral activity of Arthrospira-derived spirulan-like substances," Antiviral Research , vol. 72, no. 3, pp. 197-206, 2006.

[75] S. M. Abdo, M. H. Hetta, W. M. El-Senousy, R. A. Salah El Din, G. H. Ali, "Antiviral activity of freshwater algae," Journal of Applied Pharmaceutical Science , vol. 2, no. 2, pp. 21-25, 2012.

[76] S. Ayehunie, A. Belay, T. W. Baba, R. M. Ruprecht, "Inhibition of HIV-1 replication by an aqueous extract of Spirulina platensis (Arthrospira platensis) ," Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology , vol. 18, no. 1, pp. 7-12, 1998.

[77] Z. Khan, P. Bhadouria, P. S. Bisen, "Nutritional and therapeutic potential of Spirulina," Current Pharmaceutical Biotechnology , vol. 6, no. 5, pp. 373-379, 2005.

[78] J. Balzarini, "Carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses?," Antiviral Chemistry and Chemotherapy , vol. 18, no. 1, pp. 1-11, 2007.

[79] J. Teas, J. R. Hebert, J. H. Fitton, P. V. Zimba, "Algae-a poor man's HAART?," Medical Hypotheses , vol. 62, no. 4, pp. 507-510, 2004.

[80] M. Sharaf, A. Amara, A. Aboul-Enein, S. Helmi, A. Ballot, A. Astani, P. Schnitzler, "Molecular authentication and characterization of the antiherpetic activity of the cyanobacterium Arthrospira fusiformis ," Die Pharmazie , vol. 65, no. 2, pp. 132-136, 2010.

[81] A. Hernández-Corona, I. Nieves, M. Meckes, G. Chamorro, B. L. Barron, "Antiviral activity of Spirulina maxima against herpes simplex virus type 2," Antiviral Research , vol. 56, no. 3, pp. 279-285, 2002.

[82] E. A. Shalaby, S. M. M. Shanab, V. Singh, "Salt stress enhancement of antioxidant and antiviral efficiency of Spirulina platensis ," Journal of Medicinal Plant Research , vol. 4, no. 24, pp. 2622-2632, 2010.

[83] M. A. Quereshi, R. A. Ali, R. Hunter, "Immuno-modulatory effects of Spirulina platensis supplementation in chickens," in Proceedings of the 44th Western Poultry Disease Conference, pp. 117-121, Sacramento, Calif, USA, 1995.

[84] F. Kokou, P. Makridis, M. Kentouri, P. Divanach, "Antibacterial activity in microalgae cultures," Aquaculture Research , vol. 43, no. 10, pp. 1520-1527, 2012.

[85] H. M. Najdenski, L. G. Gigova, I. I. Iliev, P. S. Pilarski, J. Lukavsky, I. V. Tsvetkova, M. S. Ninova, V. K. Kussovski, "Antibacterial and antifungal activities of selected microalgae and cyanobacteria," International Journal of Food Science and Technology , 2013.

[86] D. V. L. Sarada, C. S. Kumar, R. Rengasamy, "Purified C-phycocyanin from Spirulina platensis (Nordstedt) Geitler: a novel and potent agent against drug resistant bacteria," World Journal of Microbiology and Biotechnology , vol. 27, no. 4, pp. 779-783, 2011.

[87] A. Duda-Chodak, "Impact of water extract of Spirulina (WES) on bacteria, yeasts and molds," ACTA Scientiarum Polonorum Technologia Alimentaria , vol. 12, pp. 33-39, 2013.

[88] J. Sivakumar, P. Santhanam, "Antipathogenic activity of Spirulina powder," Recent Research in Science and Technology , vol. 3, no. 4, pp. 158-161, 2011.

[89] M. Soltani, A.-R. Khosravi, F. Asadi, H. Shokri, "Evaluation of protective efficacy of Spirulina platensis in Balb/C mice with candidiasis," Journal of Medical Mycology , vol. 22, no. 4, pp. 329-334, 2012.

[90] M. Moraes de Souza, L. Prietto, A. C. Ribeiro, T. Denardi de Souza, E. Badiale-Furlong, "Assessment of the antifungal activity of Spirulina platensis phenolic extract against Aspergillus flavus ," Ciencia e Agrotecnologia , vol. 35, no. 6, pp. 1050-1058, 2011.

[91] N. Akhtar, M. M. Ahmed, N. Sarker, K. R. Mahbub, M. A. Sarker, "Growth response of Spirulina platensis in papaya skin extract and antimicrobial activities of Spirulina extracts in different culture media," Bangladesh Journal of Scientific and Industrial Research , vol. 47, no. 2, pp. 147-152, 2012.

[92] O. B. Gorobets, L. P. Blinkova, A. P. Baturo, "Stimulating and inhibiting effect of Spirulina platensis on microorganisms," Zhurnal Mikrobiologii Epidemiologii i Immunobiologii , no. 6, pp. 20-24, 2001.

[93] L. Varga, J. Szigeti, R. Kovács, T. Földes, S. Buti, "Influence of a Spirulina platensis biomass on the microflora of fermented ABT milks during storage (R1)," Journal of Dairy Science , vol. 85, no. 5, pp. 1031-1038, 2002.

[94] T.-S. Vo, D.-H. Ngo, S.-K. Kim, "Marine algae as a potential pharmaceutical source for anti-allergic therapeutics," Process Biochemistry , vol. 47, no. 3, pp. 386-394, 2012.

[95] N. H. Yang, E. H. Lee, H. M. Kim, " Spirulina platensis inhibits anaphylactic reaction," Life Sciences , vol. 61, pp. 1237-1244, 1997.

[96] C. Cingi, M. Conk-Dalay, H. Cakli, C. Bal, "The effects of spirulina on allergic rhinitis," European Archives of Oto-Rhino-Laryngology , vol. 265, no. 10, pp. 1219-1223, 2008.

[97] D. Remirez, A. González, N. Merino, R. González, O. Ancheta, C. Romay, S. Rodríguez, "Effect of phycocyanin in zymosan-induced arthritis in mice--phycocyanin as an antiarthritic compound," Drug Development Research , vol. 48, no. 2, pp. 70-75, 1999.

[98] Y. Wang, C. F. Chang, J. Chou, H. L. Chen, X. Deng, B. K. Harvey, J. L. Cadet, P. C. Bickford, "Dietary supplementation with blueberries, spinach, or spirulina reduces ischemic brain damage," Experimental Neurology , vol. 193, no. 1, pp. 75-84, 2005.

[99] T. Karaca, N. Simsek, "Effects of spirulina on the number of ovary mast cells in lead-induced toxicity in rats," Phytotherapy Research , vol. 21, no. 1, pp. 44-46, 2007.

[100] J. Campbell, S. E. Stevens, D. L. Balkwill, "Accumulation of polyhy-B-hydroxybutyrate in Spirulina platensis ," Journal of Bacteriology , vol. 1499, no. 1, pp. 361, 1982.

[101] M. H. Jau, S. P. Yew, P. S. Y. Toh, A. S. C. Chong, W. L. Chu, S. M. Phang, N. Najimudin, K. Sudesh, "Biosynthesis and mobilization of poly(3-hydroxybutyrate) [P(3HB)] by Spirulina platensis ," International Journal of Biological Macromolecules , vol. 36, no. 3, pp. 144-151, 2005.

[102] A. J. Anderson, E. A. Dawes, "Occurrence, metabolic role and industrial uses of bacterial polyhydroxyalkanoates," Microbiological Reviews , vol. 54, no. 4, pp. 450-474, 1990.

[103] G. Q. Chen, Q. Wu, "The application of polyhydroxyalkanoates as tissue engineering materials," Biomaterials , vol. 26, no. 33, pp. 6565-6578, 2005.

[104] L. Gallimberti, M. Ferri, S. D. Ferrara, F. Fadda, G. L. Gessa, "Gamma-hydroxybutyric acid in the treatment of alcohol dependence: a double-blind study," Alcoholism , vol. 16, no. 4, pp. 673-676, 1992.