Elsevier

Food Chemistry

Volume 140, Issues 1–2, 1–15 September 2013, Pages 262-272
Food Chemistry

C-Phycocyanin from Oscillatoria tenuis exhibited an antioxidant and in vitro antiproliferative activity through induction of apoptosis and G0/G1 cell cycle arrest

https://doi.org/10.1016/j.foodchem.2013.02.060Get rights and content

Abstract

This study was undertaken to develop an efficient single step chromatographic method for purification of C-phycocyanin (CPC) from species of Oscillatoria tenuis. Purification of CPC involves a multistep treatment of the crude extract by precipitation with ammonium sulphate, followed by gel filtration chromatography. Pure CPC was finally obtained from O. tenuis with purity ratio (A620/A280) 4.88. SDS–PAGE of pure CPC yielded two bands corresponding to α and β subunits; the molecular weight of α subunit is 17.0 kDa, whereas the molecular weight of β subunit is 19.5 kDa. Fluorescence and phase contrast microscopy revealed characteristic apoptotic features like cell shrinkage, membrane blebbing, nuclear condensation and DNA fragmentation. CPC exhibited antioxidant and antiproliferative activity against human cancer cells through apoptosis; nuclear apoptosis induction was accompanied by G0/G1 phase arrest and DNA fragmentation. CPC is a natural pigment with potential as an anticancer agent.

Highlights

► Efficient and low-cost method developed for purification of C-phycocyanin (CPC) from Oscillatoria tenuis. ► The purity ratio of CPC was 4.88, which is analytical grade value for pure CPC. ► CPC from O. tenuis potentially scavenges free radicals. ► CPC from O. tenuis can also potentially inhibit cancer cell growth in vitro. ► CPC induces apoptotic characteristics of membrane blebs, apoptosis and G0/G1 cell cycle arrest.

Introduction

Chemoprevention is an effective way to reduce cancer risk. Natural products have been the mainstay of cancer chemotherapy for the past 30 years. Blue-green algae are the most primitive life forms on earth with nutrient-dense, edible forms like Nostoc, Spirulina, Aphanizomenon species, etc. The cyanobacterial phycocyanin (CPC) is the major phycobiliprotein in cyanobacteria. This blue colour red fluorescing biliprotein was first reported in 1928 by Lemberg (Patil et al., 2006). It consist of a protein component and a chromophore, and the protein moiety consists of α and β subunit (Patil et al., 2006). Phycocyanin is a natural blue colourant, has uses as a food colourant for chewing gum, sorbets, soft drinks, candies and cosmetics, including lipstick and eyeliners. Small quantities are also used as biochemical tracers in immunoassays due to its fluorescent properties (Silveira, Burket, Costa, Burket, & Kalil, 2007).

Phycocyanin has been proven to have therapeutic properties including antioxidant, anti-inflammatory and anti-cancer activities (Eriksen, 2008, Madhyastha et al., 2009). Recent studies have demonstrated antioxidant, anti-mutagenic, antiviral, anticancer, anti-allergic, immune enhancing, hepatoprotective, blood vessel-relaxing and blood lipid-lowering effects of Spirulina extracts. The biological and pharmacological properties of Spirulina were attributed mainly to calcium-spirulan and CPC (Subhashini et al., 2004). The cost of phycocyanin products varies widely and is dependent on the purity ratio, which is defined as the ratio of absorbance at 620 and 280 nm (A620/A280).

CPC has been purified using a number of combinations of chromatographic steps such as ion exchange chromatography, gel filtration chromatography, chromatography on hydroxyapatite and expanded bed adsorption chromatography (Bermejo et al., 2003, Santiago-Santos et al., 2004, Soni et al., 2006, Wang, 2002). In most cases, it has been purified by sequential application of either cation or anion exchange chromatography, followed by size exclusion chromatography as a final polishing step. Though purification was achieved in the above methods, high purity methods were very expensive while low-cost methods were time-consuming. Moreover, the combination of several steps decreases the yield considerably, requiring large culture volumes for purification (Jobby & Sharma, 2003). Hence the present study attempted to simplify the purification process as well as reduce the cost of process. Additionally we were interested to study the biological activities of CPC from Oscillatoria tenuis.

Section snippets

Chemicals and reagents

Seralose 6B gel filtration column, Griess reagent and ascorbic acid were purchased from SRL Chemicals, Mumbai, India. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and Dulbecco’s Modified Eagles Medium (DMEM) were purchased from Hi Media, Mumbai, India. Nitroblue tetrazolium (NBT), nicotinamide adenine dinucleotide (NADH), (4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid) (HEPES), penicillin, streptomycin, propidium iodide, ethylenediaminetetraacetic acid (EDTA), WST-8 kit, and dimethyl sulphoxide

Purification and characterisation of CPC from O. tenuis

The extraction of phycobiliproteins in O. tenuis revealed CPC as the major component with respect to other phycobiliproteins, such as APC and PE, which makes the purification process easy as well as efficient (Fig. 1 and Table 1). Because PE is present in very low amount, this molecule does not interfere in the purification process. This may be due to O. tenuis specificity in the phycobiliprotein content and the culture conditions.

During the process of extraction, cyanobacterial cells were

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1

These authors contributed equally to this work.

2

Director, KIPM & R, Chennai 600 032, TN, India.

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