Abstract
The cyanobacterium Arthrospira platensis (AP) is a natural source of considerable amounts of ingredients that are relevant for nutra- and pharmaceutical uses. Beyond its nutritionally valuable components, such as carbohydrates, minerals, and proteins, bioactive ingredients extracted from AP have been studied for their therapeutical values.
Background
The cyanobacterium Arthrospira platensis (AP) is a natural source of considerable amounts of ingredients that are relevant for nutra- and pharmaceutical uses. Beyond its nutritionally valuable components, such as carbohydrates, minerals, and proteins, bioactive ingredients extracted from AP have been studied for their therapeutical values [1–4]. The antioxidant potential of AP has been attributed mainly to phycocyanin (PC) [5–7]. Moreover, it has been shown in vitro that the oxygen-free radical scavenging properties of phycocyanin may contribute, at least in part, to its anti-inflammatory activity [8, 9].
While there is a big body of literature about the influence of AP extracts or PC on cancer cells [10], studies about the influence of PC on blood cells are scarce. In this short review, we summarize results from experimental studies about the influence of PC on circulating or resident blood cells.
Erythrocytes
Erythrocytes or red blood cells are anucleate, biconcave cells that transport oxygen and carbon dioxide between the lungs and tissues. As long as sufficient erythrocytes are available for transport, the oxygen supply of the tissue as well as the disposal of metabolic intermediates and end products is guaranteed [11]. During normal metabolic processes, but also via the action of carcinogenic and other harmful substances in the environment, free radicals are continuously produced.
Such free radicals induce chain oxidation of lipids and proteins in erythrocyte membranes [12] and can cause hemolysis proportional to the total flux of peroxyl radicals [13] because they are directly exposed to molecular oxygen and hemoglobin. Oxidative damage is prevented by water-soluble radical scavengers such as uric acid, ascorbic acid, and Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-soluble analog of vitamin E) [14].
It was shown that PC can inhibit erythrocyte hemolysis similar to Trolox and ascorbic acid [15]. Based on IC50 values (concentration of the additive that gave 50% inhibition of peroxidative damage), PC proved to be almost sixteen times more efficient as an antioxidant than Trolox and about twenty times more efficient than ascorbic acid. In addition, PC reduced dose-dependently induced hemolysis and H2O2-induced oxidative DNA damage [16].
These rudimentary studies give first hints at the protective effect of PC against hemolysis induced by peroxyl radicals in human erythrocytes, which seems to be due to the scavenging action of the radicals in the aqueous phase.
Leukocytes
Recently, Romay et al. found that PC (27–81 μM) inhibited luminol-dependent chemiluminescence of opsonized zymosan-stimulated neutrophils. This effect was explained through the capacity of PC to scavenge free radicals and peroxides arising during the respiratory burst of phagocytic cells [17].
These results are in line with a study by Dartsch et al. [18]. Using a cell-based test assay - inducing the formation of intracellular superoxide radicals of functional neutrophils upon stimulation by phorbol-12-myristate-13-acetate as a model – this group investigated the potential of AP-extract to inactivate superoxide radicals. Test concentrations ranging from 50 to 1,000 μg/mL were chosen. The results showed a dose-dependent inactivation of free superoxide radicals characterized by a dose-dependent reduction of the metabolic activity of functional neutrophils and a dose-dependent inactivation of superoxide radicals generated during an oxidative burst.
Cherng et al. reported that PC significantly inhibited the LPS-induced nitrite production and inducible nitric oxide synthase (iNOS) expression accompanied by an attenuation of tumor necrosis factor-α (TNF-α) formation in macrophages [19]. In lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages, PC suppressed the activation of nuclear factor-κB (NF-κB) by preventing the degradation of cytosolic NF-κB inhibitor alpha (IκB-α) [19]. They concluded that the inhibitory activity of PC on LPS-induced NO release might be associated with the suppression of TNF-α formation and NF-κB activation. This may provide an additional explanation for its anti-inflammatory activity and therapeutic effect.
Platelets
Platelets are key actors of primary hemostasis. They are the earliest players in primary hemostasis and are critical in the development of thrombosis [20]. Their main function is stopping hemorrhage following vascular injury by rapidly adhering to the damaged blood vessel wall and then forming a plug by platelet-to-platelet aggregation - sometimes in an overshooting reaction occluding the damaged vessel. To avoid such adverse events, patients at risk for thromboses are treated with platelet inhibitors. There is now preliminary evidence that PC may attenuate or even normalize impaired platelet function.
Koukouraki et al. reported that PC derived from Arthrospira sp. cultures showed a strong inhibitory effect against PAF (platelet activating factor) or thrombin induced aggregation of washed canine platelets [21]. Chiu et al. reported that preincubation (3 minutes) of washed rabbit platelets with PC (1–50 μg/ml) inhibited platelet aggregation induced by collagen (10 μg/ml) or arachidonic acid (100 μM) in a dose-dependent manner [22]. Furthermore, PC suppressed the thromboxane B2 formation significantly due to the suppression of cyclooxygenase and thromboxane B2 synthase activity. At the same time, PC reduced the increase of platelet intracellular calcium level and platelet membrane surface glycoprotein IIb/IIIa expression. In addition, PC significantly increased the cyclic AMP level by inhibiting cyclic AMP phosphodiesterase activity.
Microscopic view on a layer of normal erythrocytes (primary magnification: 1:40). (LEICA DMRX, Camera: Sony3CCD, light source: halogen light; image size 760×570 pixel equivalent to 100×75 μm side length, Wetzlar, Germany).
Glutardialdehyde Induced Fluorescence Staining of activated and fully spread platelets (Micrographs were taken using Stimulated emission depletion (STED) microscopy; 100-fold primary magnification with a ZEISS LSM800 in the high resolution AIRYSCAN-mode).
Triple staining of an endothelial cell monolayer two days after seeding. Endothelial cells were characterized by von Willebrand factor staining (in red), nuclei (in blue), and actin fibers (in green). (LSM800 confocal Laser Scanning Microscope system and ZEN software for image post processing, Carl Zeiss Microscopy GmbH, Jena, Germany).
The first study using human platelets was performed by Hsiao [23]. In this study, PC (0.5–10 nM) was pre-incubated for three minutes with washed platelets and inhibited aggregation following the induction with collagen (1 μg/mL), U46619 (1 μM), thrombin (0.05 U/mL), and arachidonic acid (60 μM) in a concentration-dependent manner. Concentrations of 4 and 8 nM PC inhibited thromboxane A2 formation and intracellular Ca2 + mobilization in the platelets.
A first in vivo study in humans treated with a very low dose of an aqueous AP extract of 2.3 g/d [24] over two weeks revealed no changes in markers for platelet activation (P-selectin expression) or serum P-selectin levels. However, subjective observations by some study participants included one case of increased bruising and three cases of incidents of bleeding gums. This led to the discussion by the authors of the study that daily consumption of high doses of the AP extract may have a mild inhibiting effect on the blood clotting system.
Overall, these findings demonstrate that PC seems to inhibit platelet aggregation, which may be associated with mechanisms including inhibition of thromboxane A2 formation, intracellular calcium mobilization, and platelet surface glycoprotein IIb/IIIa expression accompanied by increasing cyclic AMP formation.
The endothelium is the innermost cellular layer of all blood vessels. It is a passive barrier between the circulation and interstitial tissue and plays a secretory, synthetic, metabolic, and immunologic role in regulating multiple physiological processes, such as membrane permeability, vascular tone, angiogenesis, and haemostasis [25]. Therefore, in addition to platelets, they are also an essential player in thrombotic processes.
As with platelets, AP has been shown to have a beneficial effect on vascular endothelial cells. The formation of a monolayer of human umbilical venous endothelial cells (HUVEC) was significantly accelerated after supplementing the HUVEC culture with AP [26]. This effect might be caused by an AP-induced increase in the expression of endothelial nitric oxide synthase (eNOS) [27, 28]. This is associated with the amelioration of endothelial NO production [29], which would stimulate endothelial cell proliferation [30]. In addition, the anti-inflammatory capacity of AP might contribute to this effect. It could be shown that AP can lower TNF-α [29] and TGF-ß concentration, which can reduce the inhibition of the proliferation, entailing a higher proliferation of endothelial cells [31, 32].
A subsequent study showed that this effect also depended on the production conditions of AP [33]. The biomass composition and especially the influence on HUVEC proliferation differed significantly (up to 109%) between AP powders from five different sources. Furthermore, the detachment of HUVEC after LPS addition was dramatically reduced by AP [33].
Pre-clinical toxicity testing
Up to now, toxicological studies in healthy animals have not revealed any toxic effects of Arthrospira on hematological parameters, kidney, liver, reproductive system, and body physiology during and after the administration of acute or chronic doses [16, 35]. No toxicity, no adverse effects, and no mortality have been reported during sub-chronic (5 mg phycoerythrin per kg b.w./day [34] and acute toxicity tests of PC in healthy rats [16, 37] and healthy mice [16, 37] even at 2,000 [36], 3,000 [17] or 5,000 [37, 38] mg/kg oral administration. In addition, also intraperitoneal administration of 70 mg/kg [39] and even 200 mg/kg PC also showed no adverse effect in rats [40].
Due to its long history as a food source and its favorable safety profile in animal studies, AP is generally recognized as safe (GRAS) for human consumption [41–45]. A safety evaluation by the United States Pharmacopoeia concluded that there is not sufficient evidence to suggest harm associated with AP so that – for A. maxima and A. platensis – a Class A safety assignment was designated [46]. The dosage recommended for adults is in the range of 3–10 g of AP per day. So far, AP can only be commercialized as dietary supplement.
Conclusion
The available body of knowledge reveals promising effects of AP-derived PC on vascular cells. However, many questions are still unanswered. There is an urgent need to elucidate the molecular mechanism(s) behind the observed effects e.g. on platelets as major ‘cellular’ players in thrombotic processes. The influences of PC on cells of the vascular tissue beyond the endothelial origin – such as smooth muscle cells and fibroblasts – remain to be studied as well.
Sophisticated in vitro test systems and especially in vivo research are required to elucidate the complex interplay between circulating blood cells (e.g. platelets and leukocytes), cells of the vascular wall, and, finally, the crosstalk between both.