Methods for Extraction , Isolation and Purification of C-phycocyanin : 50 years of Research in Review

Context: Spirulina (Arthrospira) exerts a wide spectrum of pharmacological activities that are largely attributed to its phycobiliprotein content, mainly to C-phycocyanin. The extraction, isolation and purification of C-phycocyanin have been studied for many years, resulting in diverse methodologies with a range of yields and grades of purity. Objective: We performed a systematic review of the literature, consulting all the available years in TOXNET, PubMed/MEDLINE and Science Direct-Scopus. Search criteria included the separation, isolation, and purification methods for C-phycocyanin from different microorganisms. Search words were: extraction, separation, isolation and purification of C-phycocyanin. Results: The combination of aqueous two-phase systems for extraction and ultrafiltration for purification results in the best yields and highest purity of the desired nutraceuticals. It is also essential to consider the freshness and species of the primary biomass, as these factors heavily influence the concentration and viability of the phycobiliproteins and therefore affect the yield and purity. Conclusion: In order to preserve the valuable properties and health benefits of nutraceuticals, such as C-phycocyanin, it is essential to seek innovative methods for isolating and purifying these bioactive substances from natural sources. The information herein gathered indicates the best methods currently available. *Corresponding author: German Chamorro-Cevallos, Department of Pharmacy, National School of Biological Sciences, National Polytechnic Institute , Avenida Wilfrido Massieu s / n. Unit Adolfo Lopez Mateos, Del. Gustavo A. Madero, 07738 Mexico, DF, Mexico, Tel: + (52) (55) 5729 6000/ ext. 52398; E-mail: gchamcev@yahoo.com.mx Received Date: May 25, 2016 Accepted Date: June 10, 2016 Published Date: June 15, 2016


Obtention of C-phycocyanin: brief historical perspective
Different methods of extraction, isolation and purification (summarized in Figure 1) have been assayed in order to obtain these phycobiliproteins (especially C-phycocyanin) from Spirulina spp (Glazer, Lundell, Yamanaka, & Williams, [15] ; Khan et al., [14] ). The first attempts involved simple chromatography by using precipitations previously obtained with ammonium sulphate. Subsequently, phycobiliproteins were isolated by crystallization (Carra, [16] ). However, these methods lacked specificity as they extracted the whole phycobilisome without separating each pigment.

Extraction, Isolation and Purification of C-phycocyanin
Years later, a thermal shock-based separation technique allowed pigment separation via density-gradient centrifugation with sucrose (Bekasova, Muslimov, & Krasnovskii, [17] ). Although these processes represented an advance, the overall purity and yield of the process was still low.
With the use of reversed phase high-performance liquid chromatography (RP-HPLC), it was possible to achieve an isolation of up to 85% of C-phycocyanin and allophycocyanin. (Swanson & Glazer,[18] ) Nevertheless, other compounds apart from the phycobilisome were present in the final products due to the sample pre-treatments needed to perform HPLC. In order to optimize phycobiliprotein isolation, the chromatographic method was modified by adapting resins used in the solid phase, varying the polarity and pH of the eluent solution (Moreno et al., [19] ), and using magnesium chloride precipitation with further diffusion in polyethylene glycol gel. By enhancing overall specificity, the isolation of pure C-phycocyanin and allophycocyanin was obtained, but the yielding mass was significantly decreased.
Towards the end of the 20 th century, electrophoresis-based techniques were tested with the addition of laser-induced fluorescence (LIF) detectors. This novel achieved separation with a fairly good yield (≈90 -100%). Nevertheless, such extractions corresponded to a mixture of both C-phycocyanin and allophycocyanin (Viskari & Colyer, [20] ). Yet another type of electrophoresis was used with polyacrylamide/dodecyl sulphate gel, pre-treating the samples by precipitation with ammonium sulphate followed by further separation in chromatographic columns by Sephadex (Minkova et al., 2003). This technique achieved an isolation of pure C-phycocyanin with a yield of ≈ 45%.
Afterwards, different combinations of methods were tested in order to increase the ease of separation and isolation as well as the grade of purity and final yield. One such method, HPLC coupled with a flame ionization detector (Zolla & Bianchetti, [21] ), was able to separate C-phycocyanin from allophycocyanin, yet it destroyed the original sample. In order to resolve this problem, an integral procedure was devised that extracted the phycobiliproteins with sodium phosphate neutral buffer, and then further purified them via dialysis and gel filtration chromatography (Bhaskar, Gopalaswamy, & Raghu, [22] ). This procedure yielded C-phycocyanin with a purity of 4.98. Another method, pre-treated the sample in the same manner, but the purification process consisted of ion-exchange chromatography (Patel, Mishra, Pawar, & Ghosh, [23] ), yielding C-phycocyanin with a purity of 4.42.
Another widely studied method for isolating C-phycocyanin from Spirulina platensis combined chromatography with expanded bed adsorption, anion interchange, and hydroxyapatite columns (Niu, Wang, Lin, & Zhou, [24] ). These techniques yielded 4.45 mg of C-phycocyanin per gram of dried S. platensis with a purity of 3.2. This method offers several advantages, such as the possibility of using different resins with special charge characteristics (i.e., anionic or cationic). For instance, by using Q-Sepharose (Silveira, de Menezes Quines, Burkert, & Kalil, 2008) it was possible to isolate C-phycocyanin from Spirulina platensis with ~75% yield and a purity of 3.4. The problem with these kinds of techniques is that they are often strongly dependent on the pH and temperature of the eluent solutions.
On the other hand, hydrophobic interaction chromatography with ammonium sulphate and liquid nitrogen precipitation pretreatments (Soni, Trivedi, & Madamwar, [25] ) are capable of isolating C-phycocyanin with a purity of 4.5 but with poor yields. This may be due to the original cyanobacterium (Phormidium fragile) from which the phycobilin was isolated. To improve the yield, C-phycocyanin was extracted from Spirulina platensis with high-speed counter-current chromatography (HSCCC) (Yin et al., [26] ), obtaining 78.7 mg per 200 mg of crude extract with a purity of 4.25. Nowadays, one of the most widely used methods is ionic exchange chromatography, which involves pre treating vegetable samples of Spirulina platensis with two aqueous phases(Patil, Chethana, Sridevi, & Raghavarao, [27] ), leading to a purity of 6.69.
The aim of the present review was to describe different methods for C-phycocyanin extraction and purification and compare the results in order to determine the method with the best cost-benefit ratio.

Methods
We performed an exhaustive search (using Scopus and PubMed databases) to find methods for the separation, isolation and purification of C-phycocyanin from different microorganisms. Search words were: extraction, separation, isolation and purification of C-phycocyanin.

Results
Summaries and general characteristics are herein presented (see Table) for the 86 reports found.

Purification method Observations Reference
Thermal treatment Density gradient by centrifugation The phycobilisomes of N. muscorum were separated into two subunits containing C-phycocyanin and allophycocyanin. However, they had traces of phycoerythrocyanin, thus presenting low purity and low yields (data not shown).
Bekasova et al. [17] Reverse phase chromatography using dicarboxylic acids and methanol-butanol washes Traces of C-phycocyanin and phycoerythrocyanin were identified by mass spectrometry chromatography.
Fu, Friedman, and Siegelman [28] www.ommegaonline.org Patil et al. [29] Polyethylene glycol 4000 and potassium phosphate saturation C-phycocyaninwas obtained from Spirulina platensis in a single extraction step. With multiple extractions, the purity of the isolates increases from 3.23 to 4.02.
Rito-Palomares, Nuñez, and Amador [30] Hexane extraction SDS-PAGE electrophoresis C-phycocyanin was obtained from Spirulina spp at a yield of 10.2% and a purity of 1.
Seo et al. [31] Stirring-centrifugation Expanded bed anion exchange with 80% ammonium sulfate 25.7 mg g -1 dm of C-phycocyanin was obtained from fresh Spirulina platensis at a purity of 4.8.
Moraes, Mazutti, Maugeri, and Kalil [32] Precipitation with ammonium sulfate Fast flow chromatography DEAE-Sepharose and hydroxyapatite columns C-phycocyanin was isolated from Spirulina platensis with a yield of 30 mg g -1 dm and a purity of 3.94.
Cruz de Jesús [34] Ion exchange chromatography C-phycocyanin was obtained from Spirulina maxima with a yield of 37.5% and a purity of 3.5 (determined by DEAE-Cellulose).

Ultra filtration
In this last stage, C-phycocyanin was isolated from fresh Spirulina maxima with a yield of 57% and a purity of 3.9.
Benavides and Rito-Palomares [35] Organic solvents and buffer extraction Centrifugation and filtration C-phycocyanin obtained from Spirulina platensis had low yield and low purity (0.46).
Maurya, Maurya, and Pandey [38] Ultra filtration Ion exchange chromatography C-phycocyanin was isolated from P. ceylanicum with a yield of 63.50% anda purity of 4.15.
Singh, Parmar, and Madamwar (2009) A phycobiliprotein was obtained fromthe fresh biomass of Spirulina spp witha yield of 82.9 to 88.6% and a purity of 1.0.
Cian, López-Posadas, Drago, Medina, and Martínez-Augustin [40] Cell disruption by pressure / agitation and centrifugation Purification by hydroxyapatite column chromatography and anion exchange / ultra filtration / electrophoresis SDS-PAGE C-phycocyanin was extracted from the cyano bacteria Anabaena spp with a yield of 10% and a purity of 2.7.

Extraction, Isolation and Purification of C-phycocyanin
Precipitation with ammonium sulfate (25%) Column elution hydroxyapatite / Sephadex -DEAE ion exchange / Bio-Gel electrophoresis P C-phycocyanin was extracted from lyophilized Spirulina platensis with a purity of 4.0.
Bermejo-Bescós, Piñero-Estrada, and Villar del Fresno [43] C-phycocyanin was isolated from Synechococcus spp and Aphanocapsa cyano bacteria with a yield of 50 % and a purity of 6.1.
Glazer and Cohen-Bazire [44] C-phycocyaninwas extracted and purified from Porphyrayezoensis cyano bacterium with a yield of 20 % and a purity of 0.9 He, Hu, and Jiang [45] C-phycocyanin was isolated and purified from the fresh biomass of Spirulina platensis with a yield of 13.1 % and a purity of 4.71.
Li, Zhang, Gao, and Chu [46] C-phycocyanin was extracted and isolated from Spp Chroomonas cyano bacterium with a yield of 59 % and a purity of 0.92.
Piñero Estrada, Bermejo Bescós, and Villar del Fresno [48] Step chromatography with DEAE cellulose-11 The extract isolated from Spirulina platensis was identified as C-phycocyaninby SDS-PAGE, with a yield of 80% and a purity of 4.5.
Kumar, Dhar, Pabbi, Kumar, and Walia [49] C-phycocyanin was extracted and isolated from Anabaena variabilis cyano bacterium with a yield of 36 % and a purity of 2.75.
Moon et al. [51] C-phycocyanin was isolated and purified from Spirulina maxima with yield of 24% and a purity of 2.25.
Abd El-Baky and El-Baroty [52] C-phycocyaninwas extracted and isolated from cyanobacterium of the Nostoc spp genus, with a yield of 59% and a purity of 2.8.
Gray, Lipschultz, and Gantt [53] C-phycocyaninwas isolated and purified from Spirulina fusiformis with a yield of 60% and purity of 3.8.
Stec, Troxler, and Teeter [55] Activated carbon and chitosan , flow filtration High purity C-phycocyanin was obtained from Limnotrix spp with low ammonium sulfate concentrations.
Liao, Zhang, Wang, Yan, and Zhang [57] Anion exchange chromatography by hydrophobic interactions ( butyl-Sepharose column ) Ion exchange chromatography (Q-Sepharose column) / filtration SDS-PAGE gel C-phycocyaninwas obtained from Synechococcus spp with high purity and good yield.

Purification method Observations References
Liquid nitrogen

Precipitation/ crystallization
Ammonium sulfate A mixture of C-phycocyanin and R-phycocyaninphycoblasts were obtained from N. muscorum.
Carra [16] Ammonium-sulfate / hydrophobic interaction chromatography C-phycocyanin was isolated from Phormidium fragile, with low yield and a purity of 4.52.
Kobayashi, Siegelman, and Hirs [60] Stirring and precipitation with ammonium sulfate / SDS-PAGE electrophoresis C-phycocyanin was isolated from Oscillatona cyanobacterial agardhii with 70% yield and a purity of 4.35.
Torjesen and Sletten [61] High performance liquid chromatography (HPLC ) Reversed phase C-phycocyanin and allophycocyan in were isolated with a yield of 85 %.The impurities were traces of compounds outside the phycobilisome.
Swanson and Glazer [18] Flame ionization C-phycocyanin was separated from allophycocyanin(derived from Synechocystis). However, in the identification process the sample was lost.
Freezing and thawing cycles / dialysis / centrifugation / ammonium sulfate (20%) precipitation, and chromatography by filtration on DEAE -Sepharose gel C-phycocyanin was obtained from cyano bacterium Oscillatoria tenuis with a yield of 61.8 % and a purity of 4.88.
Thangam et al. [62] Chromatography Magnesium chloride / polyethylene glycol 6000 A mixture of C-phycocyanin and allophycocyanin was obtained from Spirulina platensis with low yield and high purity, analyzed by X-ray diffraction.
Moreno et al. [19] Gel filtration / ammonium sulfate / dialysis C-phycocyanin was obtained from Spirulina platensis at a purity of 4.98.
Bhaskar et al. [22] Ionic exchange C-phycocyanin was extracted from Spirulina with a purity of 4.42.
Patel et al. [23] C-phycocyanin was isolated from S. platensisusing Q-Sepharose, with a yield of 77.3 % and a purity of 3.4.
Benedetti et al. [64] C-phycocyanin was purified from Porphyrayezoensisby electrophoresis, with a good yield and high grade of purity.
Cai et al. [65] C-phycocyanin was isolated from Anabaena marine with a yield of 62%and a purity of 4.
Huang, Yang, Zheng, and Guo [67] High-speed counter current chromatography (HSCCC) / reversed phase 79 mg of C-phycocyanin was extracted from Spirulina platensis with a purity of 4.25, and was identified by SDS-PAGE.
Moraes, da Costa Ores, Costa, and Kalil [68] C-phycocyanin was isolated from cyanobacterium Synechocystisaquatilis with a yield of 74% and a purity of 4.0.

Ruperto Bermejo and
Ramos [70] C-phycocyanin was isolated from Spirulina platensis with pharmaceutical purity (4), and was identified by SDS-PAGE.
R. Bermejo, Felipe, Talavera, and Alvarez-Pez [71] Enzymatic digestionwith lysozyme Activated carbon and chitosan C-phycocyanin was extracted from Synechococcusspp with a yield of 80 % and a purity of 4.27.
Gupta and Sainis [72] Dialysis / centrifugation/ precipitation with ammonium sulfate / ultrafiltration C-phycocyanin was extracted and isolated from Coccochloris cyanobacterial elabens with a yield of 0.12 mg per gram of dry biomass and a purity of 2.5.
Song, Zhao, and Wang [75] Rivanol-ammonium sulfate (50%) precipitation Activated carbon and chitosan / purification by Sephadex column C-phycocyanin was isolated from two species of cyanobacteria (Spirulina maximum and Spirulina fusiformis) with a yield of 55% and a purity of 4.50 in both species, and was identified by SDS-PAGE.
Kaledona Minkova et al. [76] Sodium chloride precipitation/ Sephadex column purification C-phycocyanin was obtained from cyanobacterium Africanum Arthronema with a yield of 55 % and a purity of 4.52, and was identified by SDS-PAGE.
K. Minkova et al. [77] Ion exchange chromatography ( DEAE -Sepharose column ) / filtration C-phycocyanin was isolated from cyanobacterium Spirulina platensis, which was grown in medium enriched with selenium, achieving a purity of 5.12.
Chen, Wong, and Zheng [78] C-phycocyanin was isolated from Spirulina platensis after two purification processes with a yield of 67.04 % and a purity of 5.59.
Yan et al. [79] C-phycocyanin was isolated from Spirulina platensis cyano bacterium with a purity of 4.0.
Sørensen, Hantke, and Eriksen [82] EDTA precipitation / filtration / agitation / centrifugation SDS-PAGE electrophoresis C-phycocyanin was extracted and isolated from Acaryochloris marine cyanobacterium with a yield of 15 % and a purity of 2.0.

Discussion
Novel extraction, isolation, and purification processes for C-phycocyanin have been sought and developed since the 1980's. (Khan et al., [73] ) Throughout this process, it has been demonstrated that the original biomass is of critical importance in order to reach the best cost-benefit ratio when isolating phycobiliproteins. Another feature that must be considered is the freshness of the biomass and the subsequent pretreatment processes. In this series, C-phycocyanin was obtained from fresh biomass and dried at room temperature (as opposed to using lyophilized powders) (Chaiklahan et al., [15] 2011; Niu et al., [50] ).
Regarding extraction and purification methods, previous studies have shown that multiple cycles improved the purity grade of the C-phycocyanin extract, although yields significantly decreased. For instance, an aqueous two-phase system with polyethylene glycol 4000 (Patil et al., [4] ) managed to increase purity, but yields were importantly reduced. Similarly, a multiple extraction process for obtaining C-phycocyanin by using a Sephadex column (Minkova et al., 2003) achieved a yield of 46% with acceptable purity (established by Rito-Palomares et al., [5] ).
Based on the information herein gathered, a protocol was proposed with a one-step extraction processin order to obtain both a good yield and a high grade of purity. This was accomplished by using an aqueous two-phase system with a posterior ultrafiltration, giving C-phycocyaninin a yield of 57% and a purity of 3.9, thus surpassing the results of previous methodologies. It can be clearly seen that the preferred method should not be based on adsorption or elucidation, thus ruling out chromatography, because these processes diminish the yield of the extract (Cruz de Jesus, [74] ).

Conclusions
To maximize the health benefits that may be obtained from nutraceuticals, such as C-phycocyanin, it is essential to seek innovative methods for their isolation and purification, and thus preserve the valuable properties of these bioactive substances from natural sources. The current review makes it evident that to obtain nutraceuticals from extracts and achieve good yield and high purity; it is convenient to use aqueous two-phase systems for extraction together with ultrafiltration for purification. It is also essential to consider the freshness and species of the primary biomass, as these factors heavily influence the concentration and viability of the desired phycobiliproteins and therefore affect the yield and purity.