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Nutraceutical Properties Of Phycocyanin

- Dec 28, 2017 -

AUTHOR: Berenice Fernández-Rojas a, Jesús Hernández-Juárez b, José Pedraza-Chaverri a,*


a: Departmento de Biología, Facultad de Química, UNAM, Ciudad Universitaria, México, D.F., 04510, México

b: Unidad de Investigación Médica en Trombosis, Hemostasia y Aterogénesis del Hospital General Regional, 

     No. 1 Dr. Dr. Carlos Mac Gregor Sánchez Navarro, IMSS, México, D.F., México



Phycocyanin (PC) is one of the main pigments of the algae Spirulina, which is used as a dietary supplement due to its high protein content. PC is a protein from the phycobiliprotein family characterized by its intense blue color and its structure consists of a protein and non-protein components known as phycocyanobilin. PC scavenges reactive oxygen and nitrogen species (ROS and RNS, respectively) and prevents oxidative damage that may explain, at least in part, its beneficial effects. This review focuses on the beneficial characteristics and properties of PC emphasizing the antioxidant activity on in vitro and in vivo models. The use of PC in clinical trials is warranted.


Keywords: Phycocyanin, Functional food, Scavenging activity, Phycobyliprotein, Nutraceutical



Recently, many scientific papers have focused their attention on health promoting properties of several foods (Avila-Nava et al., 2014; Braithwaite et al., 2014; Tahergorabi, Matak, & Jaczynski, 2014). The role of functional foods and their com-ponents in disease risk reduction, treatment and promotion of human health has been demonstrated (Griffiths, Abernethy, Schuber, & Williams, 2009; Lobo, Patil, Phatak, & Chandra, 2010; Tenore, Novellino, & Basile, 2012; Vulíc et al., 2014; Zhuang, Tang, & Yuan, 2013). Functional foods contain one or more bioactive(s) and as such nutraceutical are defined as compounds or prod-ucts that have been isolated or purified from food sources that possess demonstrated health-promoting properties (Custódio et al., 2009; Lattanzio, Kroon, Linsalata, & Cardinali, 2009; Roberfroid, 2000). In this regard, phycocyanin (PC) is a nutraceutical compound with biological activity isolated and/or purified from seaweeds (De Jesus Raposo, de Morais, & de Morais, 2013; Pangestuti & Kim, 2011). PC has shown anti-inflammatory, antiplatelet, anti-cancer, nephroprotective and hepatoprotective properties that may be explained, at least in part, by its antioxidant activity. The aim of this review is to criti-cally analyze the properties of PC and the results obtained from studies that show the involvement of the antioxidant activ-ity of this protein in its nutraceutical properties.



PC is a protein from the phycobiliprotein (PBP) family (Patel, Mishra, Pawar, & Ghosh, 2005) characterized by its intense blue color. It is a peripheral accessory light-harvesting complex called phycobilisome (PBS), which is assembled on the surface of the thylakoid membrane. Its main function is to transfer the ex-citation energy to the center reaction where the maximum wavelength of absorption is near to 620 nm (Benedetti et al., 2006; De Marsac & Cohen-Bazire, 1977).

PC is one of the main pigments of Mexican algae Spirulina, which is used as a dietary supplement due to its high content of protein, vitamins, minerals and essential fatty acids (Ahsan, Mashuda, Tim, & Mohammad, 2008; Cherng, Cheng, Tarn, & Chou, 2007; Manconia et al., 2009; Thanh-Sang, BoMi,&Se-Kwon, 2013). This pigment is found in cyanobacteria and eukaryotic algae such as Rhodophyta and Cryptomonads (Glazer & Stryer, 1983). PC is classified into three types, C-PC (ob-tained from cyanobacteria), R-PC (obtained from red algae) and R-PCII (obtained from Synechococcus species) (Kuddus, Singh, Thomas, & Al-Hazimi, 2013; Wang et al., 2014). In this review, the general properties of the three types of PC are described. PC has been extracted from different algae sources (Table 1). In the Spirulina algae, PC provides its characteristic green–blue color (Eriksen, 2008; Gantar, Simovic´, Djilas, Gonzalez, & Miksovska, 2012; Silveira, Quines, Burkert, & Kalil, 2008).

Furthermore, PBPs are the most abundant proteins in many cyanobacteria and algae. However, it has been postulated that PBPs are not essential for the cell function because they are degraded when nitrogen is deprived. Therefore, PBPs are con-sidered as a source of nitrogen storage (Sloth, Wiebe, & Eriksen,2006).

PBPs are water soluble; they have bright color and are highly fluorescent. Also, they exhibit different and unique qualita-tive and quantitative features including a broad spectrum of visible light absorption and high absorption coefficient (Chattopadhyay et al., 2012; Glazer & Stryer, 1983).

PC purity is evaluated based on the absorbance ratio A620/A280. The absorbance at 620 and 280 nm correspond to PC and total protein, respectively (Patil, Chethana, Sridevi, & Raghavarao, 2006). PC is considered food grade when A620/A280 is ≤0.7, reagent grade when A620/A280 is between 0.7 and 3.9 and analytical grade when A620/A280 is ≥4.0 (Antelo, Anschau, Costa, & Kalil, 2010; Kuddus et al., 2013; Patil et al.,2006).

Equation (1) is used to determine the PC concentration (mg/mL) in crude extracts (Antelo et al., 2010; Bennett & Bogorad, 1973; Patel et al., 2005; Silveira et al., 2008).

Equation is used to determine the PC concentration .jpg 

The optical density at 652 nm corresponds to allophycocyanin;another PBP. The lethal dose 50 (LD50) of PC analytic grade has not been established yet.The higher dose used with

no observed adverse effect level (NOAEL) was studied by Romay, Ledón, and González (1998b); they found a NOAEL of 3 g/kg. Currently it has been shown that NOAEL of PC given orally is 5 g/kg

 (Ou, Lin, Pan, Yang, & Cheng, 2012). The NOAEL of 70 mg/kg by intraperitoneal (i.p.) route was used in rats, showing no side effects (Gupta, Dwivedi, & Khandelwal, 2011). However, the higher dose of 200 mg/kg i.p. has been used in experi-ments with Wistar rats (González et al., 2003; Kumari & Anbarasu, 2014; Vadiraja, Gaikwad, & Madyastha, 1998). PC is classified in category 5 according to the International Labor Or-ganization and based on the oral dose of 5 g/kg. Additional research with higher doses, given orally or i.p., of analytical grade PC is needed to definitively establish this compound as non-toxic. Further information about the metabolites produced and/or generated by different routes of administra-tion is required because it is an issue that has not yet been explored.

PC food grade is generally recognized as safe (GRAS) food used as dye in the formulation of food products such as des-serts, sweets, cake decoration, milkshakes, gum, jellies and ice cream as well in cosmetics, alcoholic beverages, biotechnol-ogy and in medicine (drugs) (Antelo et al., 2010; FDA, 2012; Kim, Ravichandran, Khan, & Kim, 2008; Yoshida, Takagaki, & Nishimune, 1996).

The isoelectric point of PC varies between 4.1 and 6.4 de-pending on the algae source and methods employed for extraction and purification (Chen & Wong, 2008; Silveira et al., 2008; Wang et al., 2014); its extinction coefficient (E1%1cm)at 620 nm is of 770,000 M−1 cm−1 (Benedetti et al., 2004). Formerly, it was believed that PC was not light sensitive (Abeliovich & Shilo,1972) but recent evidences indicate that it is light sensitive and must be kept in darkness (Benedetti et al., 2006; Wang et al., 2014). In fact, PC produces reactive oxygen species (ROS) under light conditions as it is described later. Moreover, this protein must be purified between 4 and 5 °C (Benedetti et al., 2006; Niu, Wang, Lin, & Zhou, 2007; Patil, Chethana, Madhusudhan, & Raghavarao, 2008) because is heat sensitive (Benedetti et al., 2006; Chaiklahan, Chirasuwan, Loha, Tia, & Bunnag, 2011; Oliveira, Rosa, Moraes, & Pinto, 2009; Silveira et al., 2008), besides its purity decreases through the time (Gantar et al., 2012).



PBSs are disintegrated when they are extracted in buffer; as a result, the PBPs lose their capacity to transfer electrons in the photosynthetic process conferring to PBPs a fluorescent property (Eriksen, 2008). Compared to other fluorophores, PBPs have a high molar extinction coefficient and high fluores-cence quantum field. The PC maximum excitation wavelength is close to 620 nm with an emission peak at 640 nm.

PBPs are conjugated to immunoglobulins, protein A and avidin. This property has been used for histological applica-tions, fluorescence microscopy, immunoassays and fluorescence-activated cell sorting. However, PC loses their fluo-rescence and absorbance when is denatured.



PC is composed of a protein and a non-protein component known as phycocyanobilin (PCB). Figure 1 shows how PBPs and their complex form the PBS located at the thylakoid outer mem-brane; they are adjacent to the reaction center of photosystem II light harvesting apparatus (Patel et al., 2005). Its main func-tion is to collect light efficiently when chlorophyll absorbs and transfer poorly the light energy to chlorophyll in the thyla-koid membrane (Liron et al., 2014; MacColl, 2004; Zilinskas & Greenwald, 1986). The structure of PCB is similar to that of bilirubin and biliverdin (Fig. 2)(Lakshmi, Maheswari, & Annamalai, 2008). PCB is an open-chain tetrapyrrole respon-sible for the intense blue color of PC.

The protein component of PC consists of two homologous subunits, α and β chain types globin covalently linked to type PCB by thioether linkage (Fig. 3).

According to Scheer and Zhao (2008), α chain is attached to one PCB via cysteine 84 and β chain is joined to two PCBs via cysteines 84 and 155 by thioether linkages. In contrast, Tang et al. (2012) described that it is bound to the cysteine 195 and Shen et al. (2006) suggested that PCBs are bound to the cys-teines 82 and 153 (Shen et al., 2006; Shen, Schluchter, & Bryant, 2008). Both chains have α-helix structure and the three-dimensional structures are similar among all organisms (Scheer & Zhao, 2008). According to its primary structure, α subunit has two cysteines and two methionine residues. The β subunit con-tains three cysteines and five methionine residues (Huang, Guo, Wong, & Jiang, 2007).

X-ray crystallographic analyses of PC have shown that the chiral C3 carbons of the PCB chromophores attached to cys-teines α 84 and β 82 are in the R configuration, whereas the C3 carbon of the PCB at cysteine β 153 is in the S configuration (Schirmer, Bode, Huber, Sidler, & Zuber, 1985; Shen et al., 2006).

The monomer of PC is formed by the α and β chains, these form aggregates naturally as trimer (α3β3) or hexamer (α6β6), the latter is the functional form of PC and usually forms blocks into the antenna complex (Padyana, Bhat, Madyastha, Rajashankar, & Ramakumar, 2001; Scheer & Zhao, 2008). The α3β3 is composed of nine PCB units assembled by three equal parts (Womick & Moran, 2009). The molecular weight of sub-units varies depending on the algae source and methods for extraction and purification. The α subunit is between 13 and 20.5 kDa and β subunit is between 11 and 24.4 kDa. The protein Data Bank describes that the molecular weight of PC ob-tained from Spirulina maxima is 121 kDa (α3β3); the molecular weight for the α-chain is 17,362.6 Da and for the β-chain is 17,827.2 Da (Satyanarayana, Patel, Mishra, Ghosh, & Suresh, 2007). According to the database, the apoprotein contains 20 amino acids and provides the fluorescent activity of PC. Besides, it is able to chelate Fe+2 and Hg+2 (Liron et al., 2014; Suresh, Mishra, Mishra, & Das, 2009). PC trimers have a disk shape of a diameter of 11 nm, a thickness of 3 nm and a cavity of 3.5 nm in diameter at the center (Sun, Wang, & Qiao, 2006).


Structure of phycobilisome (PBS) phycocyanin.jpg 

Fig. 1 – Structure of phycobilisome (PBS), the major complex light collection in cyanobacteria and red algae.PBSs are composed of hundreds of seemingly similar chromophores, which are proteins. They are assembled in a fashion that enables highly efficient transfer of energy

unidirectionally to the center reaction. Several phycobiliproteins (PBPs) are involved in a cascade of energy transfer from phycoerythrin (PE, red cylinders) to phycocyanin (PC, blue cylinders) and allophycocyanin (APC, green sphere) and finally to the reaction center in photosystems II (purple ellipse) and I (orange ellipses). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)



It is well established that antioxidants are able to prevent ROS production and/or to scavenge them (Halliwell, 1996; Samaranayaka & Li-Chan, 2011; Šliumpaite˙ , Venskutonis,

Murkovic, & Pukalskas, 2013; Wiriyaphan, Chitsomboon,Roytrakul, & Yongsawadigul, 2013).The imbalance between antioxidant systems and ROS production is called oxidative stress.

This latter is related to various diseases including atherosclerosis,

neurodegenerative diseases, cancer, diabetes mellitus,inflammatory diseases, as well as aging processes (Durackovα,2010).

– Chemical structure of phycocyanobilin (PCB) phycocyanin.jpg 


In 1998, Cuban researchers described for the first time the antioxidant activity of PC (Romay et al., 1998a). These authors determined that PC was able to scavenge hydroxyl radical (OH), alkoxyl radical (RO) and superoxide anion (O2•−). PC was also able to inhibit lipid peroxidation (Romay et al., 1998a). In addition they described for the first time the anti-inflammatory properties of PC (Romay et al., 1998b). It took a little longer for other researchers worldwide to confirm previous results or to discover that PC scavenged singlet oxygen (1O2) (Tapia, Galetovic,Lermp, Pino, & Eduardo, 1999), hypochlorous acid (HOCl) (Bermejo, Pinero, & Villar, 2008; Romay, Gonzalez, Pizarro, &Lissi, 2000), peroxyl radical (ROO) (Benedetti, Benvenuti,Scoglio,&Canestrari, 2010; Romay et al., 2000),OH (Bermejo et al., 2008),peroxynitrite (ONOO) (Bhat & Madyastha, 2001), nitric oxide(NO) (Thangam et al., 2013) and hydrogen peroxide (H2O2) (Fernαndez-Rojas et al., 2014) (Fig. 4). PC is also able to scavenge the non-natural radicals 2,2-azinobis(3-ehtylbenzothiazolin-6-sulphonic acid) diammonium salt (ABTS•+) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Chen &Wong, 2008; Huang et al., 2007).The O2•−, OH, DPPH and ABTS•+  scavenging activity of the purified PC from selenium-enriched Spirulina platensis algae (PC-Se) was slightly increased (Chen &Wong, 2008; Huang et al., 2007).

Currently, some mechanisms have been proposed to explain how PC stabilizes ROS. Both components are involved, the apoprotein (α and β subunits) and PCB (Pleonsil, Soogarun, &

Suwanwong, 2013).

Apoprotein has been separated using methanol and denaturing agents such as sodium dodecyl sulphate (SDS), urea and trypsin. These agents have been used to demonstrate that

apoprotein contributes to the OH scavenging activity. In fact, a change in pH from 7 to 11 increased the •OH scavenging activity because the changes in protein charge modified its

configuration (Huang et al., 2007). Romay et al. (2000) reported that apoprotein scavenges HOCl by reacting with cysteine and methionine residues. Other amino acids such as tryptophan,

tyrosine and histidine were able to scavenge ROO(Patelet al., 2005).

Phycocyanin (PC) structure.jpg 

PCB scavenges most radicals (Pleonsil et al., 2013; Zhan-Ping et al., 2005). 1O2 is stabilized by oxidation of double bonds of the tetrapyrrole (Romay et al., 2000). PCB also scavenges ONOO(Bhat & Madyastha, 2001), HOCl (Lissi, Pizarro, Aspee, & Romay,2000), OH (Huang et al., 2007) and ROO (Patel et al., 2005). Other assays have been used to determine the scavenging activity of PC including oxygen radical absorbance capacity (ORAC), ferrous ion oxidation xylenol orange (FOX), ferric reducing ability of plasma (FRAP) assay, reducing power assay and intracellular reactive oxygen levels using 2′,7′ dichlorofluorescein diacetate (DCFH-DA) (Madhyastha, Sivashankari, & Vatsala, 2009). During the scavenging activity of PC, its color gradually fades and its absorbance at 620 nm is reduced, its fluorometric intensity disappears and two new bands are observed at approximately 640 nm (Benedetti et al., 2004; Lissi et al., 2000; Patel et al., 2005;Tapia et al., 1999).

Under different light conditions PC has a bi-functional activity,it can generate •OH, whereas in the dark it traps them. The ROS production is counteracted when concentration of PC increased (Zhan-Ping et al., 2005). However, Madhyastha et al. (2009) have shown that blue light increases the scavenging activity against DPPH,OH and HOCl as well as in the ORAC and FRAP assays due to the induction of a conformational change in α and β chains altering the presence of cysteines (Madhyastha et al., 2009).

Once the antioxidant properties of PC were demonstrated; this protein was subjected to several oxidizing conditions to evaluate its biological efficacy against some pathological disorder in in vitro and in in vivo models. Currently, it is well established that PC is able to trap several ROS. Besides, PC may prevent the development of some diseases in which oxidative stress is involved. The results obtained from these in vivo and in vitro studies will be discussed later.


Several studies have evaluated the effect of PC in cancer cells and in non-cancer cells from several organs under different protocols and oxidative challenges (Table 2, Table 3 and Fig. 5).


6.1 Cancer cells

PC-Se has shown to prevent the cellular proliferation of human melanoma A375 cells and human breast adenocarcinoma MCF-7 cells by the induction of the intrinsic pathway of apoptosis (Table 2). This latter was characterized by a reduction in the mitochondrial membrane potential in cancer cells but not in normal human fibroblast Hs68 cells (Chen & Wong, 2008, Fig. 5A). Therefore, these data suggest the use of PC as a pro-phylactic agent for cancer diseases.

On the other hand, PC showed beneficial effects in terms of drug resistance. It is known that the expression of multidrug resistance protein (MDR-1), involved in the resistance of dis-tinct anticancer drugs such as doxorubicin (DOX), is regulated by ROS and cyclooxygenase-2 (COX-2) expression in human he-patocellular carcinoma (HepG2) cells. As described earlier, PC may prevent MDR-1 induction because it is able to scavenge ROS and inhibit COX-2 expression. In fact, PC prevents the re-sistance to DOX in HepG2 cells (Nishanth et al., 2010, Fig. 5A). Interestingly, does not interfere with the antineoplastic activ-ity of DOX in human ovarian cancer cells (Khan et al., 2006b).


Phycocyanin scavenges several reactive oxygen species (ROS).jpg 


Fig. 4 – Phycocyanin scavenges several reactive oxygen species (ROS). The sequential univalent reduction of oxygen is shown in the lower panel. PC is able to scavenge all ROS tested in different studies. OH, hydroxyl radical; RO, alkoxyl radical; O2 •, superoxide anion; 1O2, singlet oxygen; HOCl, hypochlorous acid; ROO•, peroxyl radical; ONOO, peroxynitrite; NO, nitric oxide; H2O2, hydrogen peroxide; ABTS•+, 2,2-azinobis(3-ehtylbenzothiazolin-6-sulfonic acid) diammonium salt; DPPH, 1,1-diphenyl-2-picrylhydrazyl.


The use of PC has been proposed as a photosensitizer in HepG2 cells when these are pre-treated with PC and irradi-ated with laser (He–Ne).These cells initiate the apoptotic process through the intrinsic pathway (Wang et al., 2012); PC also induces the loss of mitochondrial membrane potential, the release of cytochrome c by the ROS increase, the activation of caspase-3 and the arrest of the cell cycle in G2/M, but not in the normal human liver cell line HL7702 (Wang et al., 2012, Fig. 5). When confocal microscopy was used, PC was located inside mitochondria (Wang et al., 2012), which may explain its apoptotic effects.

In colon cancer HT-29 cells and lung adenocarcinoma A549 cells, PC induced, in a dose dependent manner, a reduction in cell viability by inducing apoptosis. In addition, other authors have previously described that PC treatment increases nuclear condensation in cancer cells in a dose-dependent manner. Also, DNA fragmentation increases and cells are arrested at the G0/G1 phase of cell cycle, probably as a checkpoint to prevent cell replication (Thangam et al., 2013).

Finally, the use of PC has been proposed as an agent in the photodynamic therapy that in combination with He–Ne light could provide a possible tumor therapy (Li, Chu, Gao, & Li, 2010; Wang et al., 2012). Indeed, PC has been suggested as a complementary agent to the traditional anticancer drugs, designed to limit the growth of cancer cells, and for re-ducing the toxic side effects of these drugs. The knowledge of PC photodynamic properties, mainly its binding capacity to cancer cells, has suggested the use of this protein in the co-localization of tumors in vivo (Morcos, Berns, & Henry, 1988; Thangam et al., 2013).

 Anticancer properties of phycocyanin (PC) on in vitro studies.jpg 


Antioxidant protective effect of phycocyanin (PC) in several in vitro models.jpg 

6.2  Macrophage cells

In the mouse leukemic monocyte macrophage RAW 264.7 cells, PC inhibited the inflammation induced with lipopolysaccha-ride by the inhibition of •NO production and of the inhibitor of κB(IκB-α). Also it inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and tumor necrosis factor-α (TNF-α) formation (Cherng et al., 2007,

Fig. 5).


6.3  Blood cells

PC extracts attenuated the 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced hemolysis, lipid peroxidation and oxidation of glutathione (GSH) in erythrocytes and plasma,

from humans and rats (Benedetti et al., 2004).

In the field of hemostasis, Hsiao et al. (2005) showed that PC is a potent inhibitor of agonist-induced human platelet aggregation because it prevents the effects of ROS as second

messengers in the initial phase of platelets activation (Fig. 5C).

According to these results, PC may be a useful agent in the antiplatelet therapy in patients diagnosed with arterial thrombosis (Hsiao et al., 2005).

When human lymphocytes are irradiated with 2 Gy (137Cs gamma source, dose rate 1.92 Gy/min) a reduction on antioxidant enzymes was observed. Nevertheless, the pre-treatment with PC for 2 h prevented the reduction in the levels of catalase (CAT), superoxide dismutase (SOD) and glutathione-Stransferase (GST) (Ivanova et al., 2010) (Table 3).


6.4  Neuronal cells

The knowledge of agents that decrease the undesirable effects on the brain represents one of the main research areas worldwide.

PC has shown beneficial effects in neuronal cells. For example, it acts as a neuroprotective agent (prevention for 24 h) against apoptosis induced by potassium and serum (K/S) removal.

These effects were achieved by inhibiting ROS induced by K/S deprivation in cerebellar granule cells (Rimbau et al., 2001).

On the other hand, PC has shown to induce favorable effects in human neuroblastoma SH-SY5Y cells because it attenuates, in a dose-dependent manner, tert-butyl hydroperoxideinduced

cell death. This effect is explained, at least in part, by its antioxidant activity and iron chelating which stop lipid peroxidation and damage (Bermejo et al., 2008; Marín-Prida et al., 2012). Microglial BV-2 cells are another example where PC decreased the lipopolysaccharide-induced mRNA levels for inducible nitric oxidative synthase (iNOS), COX-2, TNF-α and interleukin 6 (IL-6) (Chen et al., 2012, Fig. 5B). These findings suggest the use of PC as a preventive agent in neurodegenerative disorders where oxidative stress is involved. Nevertheless, it is necessary to design clinical studies to know whether PC may or may not represent an alternative for the treatment of neurological diseases.


6.5  Thymocytes

Tributyltin is a member of the organotin family; it is extensively used as a biocide. However, this agent is associated with thymus damage and oxidative stress. In isolated thymocytes, PC decreased tributyltin induced oxidative damage and apoptosis (Gupta et al., 2011). PC increased GSH levels, and prevented the loss of mitochondrial membrane potential, activation of caspase-3 activity, induction of Bax (an apoptosis promoter) and increased the Bax/Bcl-2 ratio (Gupta et al., 2011).

Therefore,it was postulated that the beneficial effects of PC were related to both its antioxidant activity and its iron chelating activity since iron is the mediator of tributyltin-induced damage (Gupta et al., 2011; Suresh et al., 2009).


6.6  Liver cells

PC has shown beneficial effects in liver, kidney and heart cell lines. PC showed protective effects in immortalized normal hepatocyte cell line LO2 treated with carbon tetrachloride (CCl4)

and PC for 6 h (Table 3). Briefly, PC prevented, in a dosedependent manner, the following alterations induced by CCl4: cell viability lost, disruption of nuclear envelope and cytoplasm,

alanine transaminase (ALT) leakage, ROS production,decrease in the activity of SOD and of GSH levels and in secondary oxidation products represented by malondialdehyde (MDA) levels (Ou, Zheng, Lin, Jiang, & Yang, 2010).


6.7  Renal cells

The pre-treatment of Madin–Darby canine kidney (MDCK) cells with PC for 1 h prevented the reduction of the mitochondrial membrane potential, the increased ROS production, lipid

peroxidation and reduced ATP synthesis induced by oxalate (Farooq et al., 2014) (Table 3).


6.8  Cardiac cells

DOX induces cardiac damage associated to the increased ROS production. Primary cultures of cardiomyocytes were pretreated with PC and subsequently with DOX for 24 h (Table 3).

As a consequence, ROS productionwas attenuated. In addition,PC prevented apoptotic cell death, reduced Bax and increased Bcl-2 levels (an apoptotic inhibitor) and prevented cytochromec release and caspase-3 activation (Khan et al., 2006b).


6.9  PC crosses through the cell membrane?

The location of PC inside cells is controversial. Using confocal laser fluorescence, PC was localized inside HepG2 cells when these latter were exposed 24 h to a PC concentration between

1 and 100 mM (Nishanth et al., 2010). Conversely, when immunofluorescence was used PC was located into cytosol of K562 cells, but it could not be localized inside cell nucleus after 48 h

of treatment with PC. It is important to highlight that K562 cells were treated with saponin to permeabilize the cell membrane allowing the uptake of polyclonal antibodies against PC

(Subhashini et al., 2004). Wang et al. (2012) used a similar method to immunolocalize PC inside HepG2 mitochondria (Wang et al., 2012). Furthermore,Wu, Lin, Yang,Weng, and Tsai

(2011) used polyclonal rabbit antibodies to immunolocalize PC (0.1 mg/mL) insidemurine metastatic melanoma (B16F10) cells.

Ten minutes after the treatment, PC was found inside cells,and after 30 min the protein was localized inside nuclei and some minutes later in the cytoplasm (Wu et al., 2011). Another

study took advantage of the PC fluorescence characteristics (excitation: 584 nm; emission: 640nm) to determine whether this protein entered to thymocytes after 24 and 48 h of incubation

(Gupta et al., 2011). However, results were not clear to establish whether the fluorescence observed was due to the interaction between PC and microplate or between PC and cells.

It is unknown whether PC requires a transport protein carrier to enter cells. Further studies should be performed to explore this area in the future.



Most research related to PC has been conducted on in vivo models, mainly in rats and mice. Since the antioxidant activity of PC was determined by in vitro assays, this protein has been employed in several animal models where the oxidative stress plays an important role (Table 4 and Fig. 6).


7.1  Anti-inflammatory

Inflammation is a physiological complex process that provides to the organism a response to tissue damage caused by mechanical,chemical or microbial action.ROS can initiate and also perpetuate inflammatory cascades and induce subsequent tissue damage (Kaplan et al., 2007). In this way, antioxidants may be useful to attenuate inflammation.PC was first evaluated in a damage model induced by glucose oxidase (GO). The treatment with PC 1 h before GO displayed antiinflammatory properties (Romay et al., 1998a)(Table 4). GO reactswithendogenousglucosegeneratingH2O2 and •OH.Therefore the PC scavenging activity for these ROS was important in this protective effect (Romay et al., 1998a). PC was subsequently employed in other inflammatory models,including ear edema-induced by either arachidonic acid or 12-Otetradecanoylphorbol-13-acetate (TPA); carrageenan-induced paw edema and cotton pellet granuloma (Romay et al., 1998b). In these models the activation of cyclooxygenase (COX 1 and 2, Fig. 6A), lipoxygenase (LPX), protein kinase C and ROS production were important damage mediators. PC inhibited in a dose-dependent manner the inflammation in all models; however for the TPA model the inhibition was weak (Romay et al., 1998b).


7.2  Liver

The liver is the main organ where exogenous chemicals are metabolized and eventually excreted.As a consequence, liver is exposed to high concentrations of these chemicals, which mayresultinliverdysfunction,cellinjury,andevenorganfailure (Klaassen, 2007).One of these toxic agents is CCl4,which is metabolized by cytochrome P450 to trichloromethyl radical (•CCl3) to initiate lipid peroxidation. Interestingly, PC prevented the CCl4-induced hepatotoxicity (Fig. 6B, Table 4). A first study showedthattheantioxidantactivityofPCpreventsthedamage induced by free radical formation secondary to the metabolism of CCl4 by cytochrome P-450 (Vadiraja et al., 1998).Then, it was shown that PC prevents in a dose-dependent manner the CCl4-induced liver damage which was evident by the attenuation in the serum activities of aspartate transaminase (AST) and ALT (Ou et al., 2010). PC prevents the oxidative damage by measuring MDA and GSH level, and the activity of antioxidant enzymes SOD and glutathione peroxidase (GPx). Inaddition,PCsignificantlydecreasedthemRNAlevelsoftransforming growth factor-beta1 (TGF-β1) and hepatocyte growth factor (HGF). Histological analysis revealed that PC prevents vacuole formation, inflammatory infiltration, edema and necrosis of hepatocytes (Ou et al., 2010). Another study showed that PC prevents galactosamineinduced hepatic necrosis, leukocyte infiltration and serum increase of AST and ALT activities, as well as of hepatic levels of secondary oxidation products represented by MDA (Gonzαlez et al., 2003). PC protects against hepatic encephalopathy when rats are treated with thioacetamide (AAT) (Sathyasaikumar et al., 2007). In this model, PC increased the survival rate and reduced alterations in ammonia (serum,liver and brain) and serum levels of ALT, AST and albumin. Furthermore, PC improved the prothrombin time.The histological analysis revealed a reduction innecrosisandinfiltrationofneutrophilsandmononuclearcells compared withAAT group.PC attenuates the oxidative damage and the loss of CAT activity in brain. Besides, the transmission electron microscopy showed a reduction of cellular and mitochondrial edema (Sathyasaikumar et al., 2007). Finally, PC prevented in a dose-dependent manner, the effects of alloxan-induced increase in triglycerides and total cholesterollevelsandtheexpressionofglucokinase(GK)inliver homogenate (Ou et al., 2012).


7.3. Heart

The protective effect of PC has also been studied in the field of ather-ogenesis. Riss et al. (2007) fed hamsters with an ather-ogenic diet (without vitamins C and E and selenium) with or without a daily dose of PC and PC-Se for 84 days (Table 4). Results showed that PC reduced aortic O2• production,expression of p22phox subunit of NADPH oxidase and total cholesterol content,and increased the plasma scavenging capacity in comparison with the non-treated group.Also PC-Se reduced high density lipoprotein cholesterol (HDL-C) concentrations (Riss et al., 2007). On the other hand,PC attenuates heart ischemia reperfusion (IR)-induced damage by decreasing ROS formation, apoptotic death,Bax/Bcl-2 ratio,caspase-3 activity,and increase the extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and mitogen-activated protein kinase (p38 MAPK) activation compared with the control group (Khan et al., 2006a).


7.4. Lung

The lung is the main organ affected by paraquat (PQ) that induces the formation of free radicals associated with lipid peroxidation and inflammation (Blanco-Ayala, AndéricaRomero,&Pedraza-Chaverri,2014).Inthisregard,PChasshown to attenuate lung damage; it reduces NF-κB,TNF-α,IκB and secondary oxidation MDA formation, and increases the activity of SOD and GPx compared to the non-exposedgroup(Sunetal., 2011)(Fig. 6C, Table 4). The acute lung injury induced by lipopolysaccharide inhalation was prevented by PC treatment (Leung, Lee, Kung, Tsai, & Chou, 2013). Such protection was associated to the attenuation of apoptosis (evaluated by the expression of Bax, Bcl-2 and caspase-3), O2•− production and inflammation that was evaluated by TNF-α, interleukin-1 beta (IL-1β), IL-6, cytokine-induced neutrophil chemoattractant 3 (CINC-3) and NF-κB expression.

Histological analysis showed that PC reduced edema, thickening of the alveolar walls and neutrophil infiltration (Leung et al., 2013).


Nutraceutical properties of the antioxidant Phycocyanin (PC) on in vivo models.jpg 


Nutraceutical properties of phycocyanin (PC) on in vivo studies.jpg 

7.5. Brain

Brain-IR brings severe sequels.Although the mechanism of ischemic neuronal death is not fully understood, it is known that oxidative stress is involved. Different doses of PC were used in two different treatments(pre-andpost-treatment) using two different administration routes (oral and i.p.) (Table 4).Encouraging results were found. PC increased the survival rate and reduced the infarct area and neurological defects such as posture and gait. Similar results were obtained in the pretreatment (200 mg/kg,orally) and post-treatment (75–100 mg/ kg i.p.).These results indicate that the oral dose of 200 mg/kg is similar to the i.p. injection of 75–100 mg/kg. The prevention model significantly reduced the serum and renal MDA concentration,peroxidation potential,and the ability to reduce ferric ion.In fact,it was proved that 75 mg/kg i.p.(purity = 30%) has similar result than the dose of 25 mg/kg i.p.(purity = 90%). This indicates that PC with low purity (30%) produces very favorableresultsandsuggeststhatitistheresponsiblecompound of the beneficial effects found (Pentón-Rol et al., 2011a). An interesting and unconventional research about mitochondrial from rat brain was reported by Marín-Pridaetal.(2012) (Table 4).The use of a micromolar range of PC reduced the Ca2+ overload and prevented mitochondrial pore opening, mitochondria swelling,ROS increase and the release of cytochrome c in a dose-dependent manner.Therefore,these results evinced another mechanism of PC protection (Marín-Prida et al., 2012). Encephalomyelitis (EAE) is a neurological complication caused by viral infections most often related to the respiratory tract. Pre-treatment and post-treatment with PC induce protection against the EAE-induced increase of MDA, oxidized proteins,peroxidation potentialand ferric reducing ability evaluated in plasma and in brain homogenates.However,only pre-treatmentwithPCpreventedthedevelopmentofEAE,while the post-treatment only reduced clinical signs of the disease probably induced by PC remyelination.Authors hypothesized that PC reach to the parenchyma in a required concentration is needed to influence microglia functions (Pentón-Rol et al., 2011b). In fact, Rimbau, Camins, Romay, Gonzαlez, and Pallàs (1999) showed that PC crosses the blood–brain barrier probably by its bilirubin mimetic function (Rimbau et al., 1999).

7.6. Kidney

The kidney damage induced by calcium oxalate crystals may progress to the formation of kidney stones (Farooq, Asokan, Sakthivel, Kalaiselvi, & Varalakshmi, 2004). This phenomenon implicates the induction of ROS, lipid peroxidation and decreased GSH content.As in other organs, the use of PC prevents these undesirable effects (Farooq et al., 2006)(Table 4). On the other hand,thepre- and post-treatment with PC prevented the mercuric chloride (HgCl2)-induced kidney damage in mice. To achieve these results, three repeated doses of PC were used to prevent the increase of blood urea nitrogen (BUN), creatinine and the red cell count.The authors concluded that one of the possible mechanisms involved was the inhibition of lipid peroxidation. However, lipid peroxidation was never measured (El-Ayouty, Baredy, & Salama, 2007). In contrast, another study revealed that PC prevents lipid peroxidation. In addition, PC prevented the ROS production and the reduction of glutathione disulphide (GSSG) in mice kidneys treated with HgCl2.The histological analysis showed that PC prevented cellular damage, edema, cellular atrophy of distal and proximal tubulesandnucleusloss.Thesebeneficialeffectswereachieved probably due to the antioxidant activity and the chelating property of PC for removal of mercury (Rodríguez-Sanchez, Ortiz-Butrón, Blas-Valdivia, Hernandez-García, & Cano-Europa, 2012). In another animal model, the pre-treatment with PC prevented the increase of MDA and total antioxidant capacity in kidney homogenate of mice treated with alloxan (Ou et al., 2012). Furthermore, PC prevented the cisplatin-induced renal damage. One of the pathological mechanisms of cisplatin in the kidney is the oxidative damage, therefore the use of antioxidants may be a strategy to counteract the cisplatininduced alterations, and perhaps it may contribute to reduce the prevalence of some kidney diseases. A single dose of PC prevented the renal damage (creatinine, BUN), the reduction of antioxidant enzymes such as GPx, CAT, glutathione reductase (GR),GST,and the increase in markers of oxidative damage as MDA and 4-hydroxy-2-nonenal (4-HNE) and GSH reduction (Fernαndez-Rojas et al., 2014).Evidences suggest that the antioxidant activity of PC is involved against the cisplatininduced ROS production (Figure 6E, Table 4). As mentioned before, PC is composed of an apoprotein and PCB with similar structure to biliverdin. In fact, it is believed that PC mimetics the biliverdin function (Fernαndez-Rojas et al., 2014; Marín-Prida et al., 2013; McCarty, 2011; Zheng et al., 2013). A study tested PC and PCB in db/db and db/+ mice. PC prevented albuminuria and mesangial expansion and increased MDA levels,NADPH oxidase expression, 8-hydroxy-2′-deoxyguanosine (8-OHdG), tumor growth factor-βand fibronectin expression.Interestingly,a dose of PC 300 mg/kg given orally for 10 weeks showed the same protective effect than the oral administration of PCB 15 mg/ kg for 2 weeks (Zheng et al., 2013). Therefore, this finding indicates that PCB may represent the main compound of PC that shows this beneficial effect and probably, it is also involved with other beneficial properties.


7.7. Eyes

A cataract is a clouding of lens inside the eye that decreases the vision; its development is often a gradual process of normal aging. Nevertheless, Taylor (1999) has described other factors: daylight, diet, diabetes, dehydration, among others.The latter catch-all category probably involves genetic influences in nuclear and cortical opacification in humans.The final common path way by which these factors exert their influence is predominantly due to oxidation of lens proteins and peroxidation of lipids (Kothadiaetal.,2011).Inthisregard,thecataractmodelinduced with selenite was employed in Wistar rats pups of 9 day old (Kumari&Anbarasu,2014).RatsthatreceivedPC(200mg/kgi.p.) and selenite at same time prevented the cataract process and the subsequent damage to the eye lens that was associated to the prevention of the activity of SOD and CAT and GSH content (Kumari & Anbarasu, 2014)(Figure 6F, Table 4).


7.8. Pancreas

Alloxan is a toxic compound analogous to glucose that destroys pancreatic beta cells inducing type 1 diabetes mellitus. In a recent study,some mice were exposed to alloxan and were treated with PC. As a consequence, PC counteracted the glycosylated proteins and glucose serum levels.Also,PC reduced the MDA content and attenuated the loss of total antioxidant capacity in a dose-dependent manner.In addition,PC reduced the p53 expression. Furthermore, the histological examination revealed that PC re-sets the number of β cells of insulin-secreting glands.Thus,the use of PC promoted there generation of pancreatic alloxan-induced damage beta cells (Ouetal.,2012).



Over the years,it has been shown that PC acts as a nutraceutical compound as described in this contribution. The physiopathology of many diseases comprises the imbalance between oxidant species and those with antioxid antactivity causing oxidative damage.In this way,the evidence supports the benefits of nutraceuticals in the treatment and prevention of diseases. In fact, some researchers have suggested using nutraceuticals as adjuvants in the clinical practice (Braithwaite et al., 2014; Fergurson, 2009). Among nutraceuticals, vitamin D is related to a reduction on atherosclerosis, insulin resistance, dyslipidemia and hypertension (Menezes, Lamb, Lavie, & DiNicolantonio, 2014). A dietary supplemental containing gamma linolenic acid,vitamin E,vitamin C,beta carotene and coenzyme Q10 may be considered in the treatment and prevention of dry skin associated with the use of oral isotretinoin (Fabbrocini et al., 2014).The administration of omega-3 can effectively improve endothelial function in adolescents with metabolic syndrome by reducing serum levels of vascular endothelial growth factor (VEGF), as a major index for atherosclerosis progression and endothelial destabilization (Ahmadi et al., 2014).The Mediterranean style diet improves the glucose and lipid profile in children and adolescents with obesity and metabolic syndrome (Velazquez-López et al., 2014). The co-administration of curcumin capsules with glyburide may be beneficial to improve glycemic control in patients. Thelipidlowering and antidiabetic properties of the curcumin show this antioxidant as a potential future drug molecule(Neerati,Devde, & Gangi, 2014).The use of PC has been shown to prevent the imbalance“antioxidants/oxidants”at least in in vitro and in vivo models (See Table 2, 3 and 4); however further studies should be performed to know whether this

nutraceutical might be used in the clinical practice.Therefore, it is essential to determine the LD50,the metabolites produced during PC metabolism when is administrated by different routes (oral and i.p.), determine whether whole PC or its derivatives are responsible of its biological effects. Besides, the mechanism by which PC crosses the plasma membrane has to be established.As mentioned in this review,PC has antioxidant effects in more than one organ and in different disease models(summarizedin Fig.6 and Table 4).Therefore, the knowledge of PC properties should be used by researchers to develop future studies and clinical trials to decrease the prevalence of human diseases.



This work was supported by PAPIIT No.IN210713andCONACYT No. 220046.


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