Purification, characterization and antioxidant properties of C-Phycocyanin from Spirulina platensis

Transcription

1 SIRJ-APBBP Volume 2 Issue 1 (2015) ISSN Scrutiny International Research Journal of Agriculture, Plant Biotechnology and Bio Products (SIRJ-APBBP) Purification, characterization and antioxidant properties of C-Phycocyanin from Spirulina platensis A. Avila Jerley 1 and D. Magesh Prabu 2 1 Assistant Professor, PG & Research Department of Zoology, Holy Cross College, Trichy, Tamilnadu, India 2 Microbiological Laboratories, R.S Puram, Coimbatore, Tamilnadu, India Article history: Submitted 3 September 2014; Accepted 8 November 2014; Published 28 February 2015 Abstract C- Phycocyanin (C-Pc) a blue coloured, water soluble fluorescent protein was isolated from spirulina platensis is of great importance because of its various medical and pharmacological properties. Phycocyanin is also a powerful tonic agent and has anticancer, antioxidant, antiviral and anti-inflammatory activities. In the present study, the culture was maintained in modified Zarrouk s medium and growth was determined. The crude phycocyanin was extracted and purified by ammonium sulphate precipitation and dialysis. The UV Visible absorbtion spectrum of the eluted fractions showed a broad peak range at 280,615 and 652nm. Finally the purification steps were ministered using DEAE column. The purified phycocyanin was further confirmed by structural elucidation by FT-IR and this confirms the presence of the functional groups of phycocyanin. Further in this research the antioxidant activity of the phycocyanin was carried out by free radical scavenging assay and this blue pigment showed the presence of antioxidant potency which is one of the important health component for functional foods. As there is an enormous increase in the population day by day, the alternative therapy in the market is getting its glimpse. Hence, microbial pigments have therapeutic ability and can provide a novel material for human health concern. Key words: Phycocyanin, Spirulina platensis, antioxidant, FT-IR, DEAE column Corresponding author Introduction Dr. A. Avila Jerley, Assistant Professor, PG & Research Department of Zoology, Holy Cross College, Trichy , Tamilnadu, India In algae and cyanobacteria, pigments are responsible compounds for capturing the energy from sunlight for photosynthesis. In addition to their role in photosynthesis, natural

2 pigments carry a variety of important biological functions. There are three basic classes of light harvesting pigments: chlorophylls, carotenoids and phycobiliproteins plays important role on human health benefits. Phycobiliproteins are proteins with linear tetrapyrrole prosthetic groups called bilins, found not only in cyanobacteria but also in red algae and crytomonads. Phycobiliproteins are divided into three main classes according to their structure: Phycoerythrins, phycocyanin and allophycocyanins (Bermejo et al., 2005).The other pigments like fucoxanthin and peridinin belong to carotenoid group of photosynthetic pigment. The Phycocyanin has been mainly used as food pigment and small quantities are included as biochemical tracers in immunoassay due to their fluorescent properties and it has been observed as good anti-inflammatory, anti-cancer, anti-viral, neuroprotective and hepatoprotective properties (Reddy et al., 2003). Spirulina is one of the several algal genera that have attracted special attention due to importance as human food stuff with their in vitro and in vivo functional properties. Among these genera, Spirulina platensis has been extensively grown to obtain a protein rich material of nutritional and industrial use (blue pigment). They contain phycobilisomes as light harvesting protein pigment complexes. Nearly 80-85% of phycobilisomes are composed of colored polypeptides namely phycobiliproteins and it is the excellent source of blue pigment phycocyanin. Spirulina has 62% amino acid content and is the world richest natural source of vitamin B12 and contain a whole spectrum of natural phytopigments. Several studies show that bioactive compounds of Spirulina prevent or inhibit cancer in human and animals. It is well known that microbial pigments are highly responsible for the health benefits and plays a key role as antioxidants due to the presence of hydroxyl substituent and their aromatic structures which enables them to scavenge free radicals. Antioxidants are substances that protect the cells from damage caused by unstable molecules known as free radicals. Antioxidants include carotenoids: β carotene, phenolic compound: gallic acid, flavanoids, quercetin, alkanoids, capsaicin, hydroxytoulene, etc., (Bhat et al., 2005). Phycocyanin (PC) is one of the major blue pigments from Spirulina, a microalga has a mass of evidence in favor of antioxidant properties. PC can scavenge alkyl, hydroxyl and peroxyl radicals. Many disease are formed due to excessive formation of reactive oxygen species (ROS) and natural antioxidants like PC is useful for the treatment of some neurodegenerative disorders such as alzheimers, Parkinsons and Huntingtons diseases (Romay et al., 2003). The aim of this research work is to explore the cyanobacteria Spirulina platensis for the production of natural biopigments with special emphasis upon in vitro experiments for screening and analyzing the therapeutic efficacy as antioxidants. Materials and Methods Collection of algal strain The algae Spirulina platensis was obtained from S.S Biotech, Madurai. Growth and cultural maintenance of Spirulina platensis The Cyanobacteria Spirulina platensis was cultivated in modified Zarrouk s medium, ph 9 in 1 liter conical flask. 20ml of inoculam was added to 500ml of medium and the culture flasks were kept in sunlight at day time and at night these were exposed to total darkness. This was maintained for 20 days. After 20 days, it was viewed for its morphological structure under microscope and the biomass was collected, filtered, dried and analysed at 40 o C for 48 hours. Then it is homogenized, sieved and used for further investigations. 8

3 Extraction of blue pigment phycocyanin Phycocyanin were extracted by taking approximately 2gm of dried Spirulina 200ml of 0.1 M Sodium phosphate buffer PH 7.0 containing 100μg/ml lysozyme and 10mm EDTA. This mixture was placed in rotary shaker at 30 o C for 24 hours, to disintegrate the cell walls of the algae by enzymatic digestion method. After 24 hours of incubation the mixture is centrifuged for 1 hour at 8000 rpm in 4 o C. This gives a clear blue colour supernatant which is further purified and the cell debris is discarded. Separation of phycocyanin by ammonium sulphate precipitation Solid ammonium sulfate was gradually added into the beaker containing crude extracts of phycocyanin to obtain 50% saturation with continuous stirring for 1 hr. This solution was kept overnight in dark condition and the precipitation was collected by centrifugation at 10,000 rpm for 10min at 4 0 C. The colourless, clear supernatant was discarded and blue precipitate was dissolved in small volume of 0.025M sodium phosphate buffer ph 7 and stored at 4 o C in brown bottle for further purification. Partial Purification of phycocyanin using dialysis The pellets obtained from ammonium sulfate precipitation were further purified by dialysis. The dialysis membrane has to be pretreated to remove the chemicals and other compounds present in the membrane. Purification by Ion exchange chromatography The protein fractions of phycocyanin were further purified using ion exchange chromatography which is a type of column chromatography. Diethyl amino ethyl cellulose (DEAE-C) is a positively charged resin used as adsordent in protein and nucleic acid purification/separation. This DEAE-C matrix was mixed with 50mM Phosphate buffer ph 7 in ratio 1:1. The saturated protein pigments were eluted with phosphate buffer. Step wise elution was done with phosphate buffers of increasing ionic strength at ph 7.0. Fractions are collected for further analysis. Estimation of phycocyanin The Phycocyanin concentration in the supernatant was calculated spectrophotometrically by measuring the absorbance at 615 and 652 nm using the following calculation (Baussiba and Richmond, 1979.) Where, A615 absorbance at 615 nm A652 absorbance at 652 nm 5.34 constant factor C phycocyanin mg ml = A 651 A

4 Characterization of microbial pigments Analysis by thin layer chromatography (Harborne, 1973) The phycocyanin was subjected to thin layer chromatography (TLC) in order to separate the bioactive compounds present. The TLC plates were prepared using silica gel G and distilled water. Silica gel G (20g) was added to 40ml of distilled water and thick slurry was made. The plate was allowed to dry at room temperature. The dried plate was placed in the oven at 100 o C for 30 minutes to activate the silica gel. The plate was taken from the oven and kept at room temperature for 15 minutes. Using a microcapillary tube, a small drop of pigment extract was placed on the TLC plate, and kept in the TLC chamber which was saturated with Hexane-Acetone (70:30). When the solvent reached 2 cm below the top, the plates were taken out of the chamber and detected for the spots. The chromatogram was developed and Rf values of the spots were calculated. Chemical structural analysis of purified pigments The chemical structure of the purified pigment was analyzed by UV-Visible Spectrophotometer and Infrared Analysis. Spectral analysis by UV- spectroscopy A UV-Visible spectrophotometric analysis was done to detect the presence of UVabsorbing compounds in the pigments using shimadzu (Japan) model UV series Spectrophotometer and the spectral range for Phycocyanin is around 520 to 680nm. Spectral analysis by Fourier Transfer Infrared Spectral (FTIR) IR spectral analysis of isolated pigment was done with by keeping the samples in vacuum desiccators over solid KOH for 48 hours and then IR spectral Analysis was done with 1mg sample in a Fourier transfer Infrared spectrophotometer (Shimadzu, japan). Antioxidant activity by DPPH radical scavenging assay The free radical scavenging activity of the fraction was measured in vitro by 1-Diphenyl-2-Picrylhydrazyl (DPPH Assay).About 0.3mm solution of DPPH in 100% ethanol was prepared and 1ml was added to 3ml of the pigment fraction (Sample) dissolved in ethanol at different concentration. The mixture was shaken and allowed to stand at room temperature for 30 min and the absorbance was measured at 517 nm using UV- Visible Spectrometer (Shimadzu). The scavenging activity at different concentration was determined and the IC50 value of the fractions was compared with that of ascorbic acid (Vitamin C) which was used as the standard. The percentage of radical scavenging activity was calculated % RSA = Absorbance of Control Absorbance of sample Absorbance of control

5 Results Spirulina platensis was successfully cultured in Zarrouks medium medium for 9 days and their biomass calculations and microscopic observations were done. When the culture became older there was a change in the appearance of culture from light green to dark green proportionate to the increasing cell mass. The microscopic and visual observations revealed culture was grown healthy and under the light microscope Spirulina appeared as blue green filament composed of cylindrical cells arranged in unbranched helicoidal trichome. And the dried biomass obtained from the medium was found to be 280mg/litre. The extraction of intracellular substances was done by cell disruption, followed by centrifugation process. The lyzozyme enzyme was used to breakdown the cell walls of Spirulina, while, EDTA and Tris Buffer helps to release the blue pigment phycocyanin from the cell to the medium. This enzymatic degradation process helped to improve the protein extraction efficiency. In this study, a three step purification process was employed to get purified phycocyanin. In the first step the blue pigment was saturated by 50% of ammonium sulphate. Then the precipitate was partially purified by dialyzing the pigment against the phosphate buffer. Finally the recovered fractions was purified and separated by DEAE cellulose anionic chromatography. The purity of phycocyanin was increased after every stage of separation. The absorbance measurements at the end of each step are measured and the purity of the sample was calculated and summarized below (Table 1). When the measurement of A620/A280 was greater than 4, the phycocyanin was considered to be highly pure (Boussiba and Richmond, 1979). Therefore combination of three techniques such as Ammonium Sulphate Precipitation, Dialysis and Ion exchange Chromatography provides a simple and rapid way to obtain large amount of blue pigment Phycocyanin. Table No. 1: Purification data of phycocyanin obtained from blue green algae Spirulina platensis Purification Step Absorbance ratio (nm) 620/ /280 Crude Extract % (NH4)2SO4 Precipitation Dialysis DEAE cellulose anionic chromatography The Phycocyanin concentration in the purified sample was calculated Spectroscopically by measuring their absorbance at 615nm and 652nm. The amount of phycocyanin was 10.8 mg/g. Here in this work characterization studies reveals that silica gel was used as absorbants to separate the more polar substrates such as alcohols, carboxylic acid and amines. The plate developed in Hexane and acetone (70:30) showed spots of phycocyanin chromophore. The Rf values are 0.36 and 0.34 respectively. 11

6 The blue pigment from Spirulina platensis was extracted by enzymolysis method and this purified compound showed a maximum absorption at 652, 615 and 280nm respectively (Fig.1). The spectral range between indicates the presence of pigment phycocyanin (Hongsachart, et al., 2004). Figure No.1: Spectra of Algal Pigment Phycocyanin IR spectral analysis of phycocyanin was done and their spectrum has the following functional groups (Table 2 and Fig. 2). Table No. 2: Functional groups of blue pigment from Spirulina platensis Wavenumber Functional groups (cm -1 ) C-H bending in alkene groups C-O stretching in alcohol groups O-H bending in carboxylic groups N-H stretching in amine groups (carbonyl β- unsaturated keto amide) N-H stretching vibrations presence of amine (proteins) groups Figure No. 2: IR Spectra of algal pigment phycocyanin From their IR spectrum there is a sharp peak for carboxylic, amine and alkene groups which revealed that this blue pigment is phycocyanin (Venkatesan et al., 2012). 12

7 DPPH is a stable purple colour radical that turns to yellow or yellowish brown, when it reacts with antioxidant analytes and the degree of discoloration indicates the scavenging potentials of antioxidant pigment extracts. This activity depends on the hydrogen donating ability (Brand et al., 1995). The antioxidants activity of phycocyanin was compared with the standard Vitamin C and the blue pigment showed radical scavenging activity of 25.21%. Therefore this microbial pigment is potent free radical scavenger and inhibits lipid peroxidations. Discussion Spirulina platensis, an edible microorganism with rich sources of phycobiloproteins, which aid in production of blue pigment phycocyanin. The Spirulina platensis can grow profusely under suitable environmental conditions and with sufficient nutrients. After 10 days of cultivation, the biomass was calculated and the dried biomass was found to be 280mg/l (Bhaskar, et al., 2005). The extraction efficiency, purity and concentration of the phycocyanin depends mainly on the cell envelope disruption. The cell envelop is broken and the phycocyanin located on the thylakoid cell membrane are released outside by the lytic enzyme lysozyme. EDTA and Tris- buffer chelates the mg 2+ cations and destabilize the cell membrane and release the phycocyanin. The purification of phycocyanin was usually done by three steps processes. First the PC were aggregated by 50% ammonium sulphate precipitation, based on its surface charges the protein pigments are neutralized and precipitated outside. Then it is dialysed against phosphate buffer for the removal of small contaminants by diffusion of molecules from higher concentration to lower through semi permeable dialysis membrane. Finally PC was purified by anionic batch chromatograph of DEAE- cellulose, which locks negatively charged protein molecules into the matrix and released PC by increasing the salt concentration of the solvent. The step wise elution of PC is checked for its purity ratio. In this study the highest purity ratio was approximately and PC with purity ratio < 1 is considered a low purity protein that can used in food and cosmetic Industries, while purity ratio > 4 is of high purity grade and can used as pharmaceutical agent (Cisneros and Rito, 2004). The separation of these biopigments was based on the differential affinity of the compounds and their respective stationary and mobile phase. The chromatogram of the organic pigment indicates the presence of several colouring bands, blue, orange, yellow, and green corresponding to photosynthetic pigments. The UV Visible absorption pattern of PC from Spirulina platensis shows λ max at 652 and 615 nm, indicates that they strongly absorb the visible region of spectrum because they carry various covalently attached tetrapyrrole prosthetic groups (billins) confirming that PC is of pure grade (Madhyastha et al., 2006). TIR is one of the most widely used methods to identify the chemical constituents and elucidate the compounds structure to identify medicine in Pharmacopeia of many countries (Margarita et al., 2000). It is an accepted tool for the characterization of biomolecules in drug discovery research by development of high- throughput screening and combinatorial chemistry. In this present work FTIR spectroscopy operates in mid infrared region of cm -1 which has been proved to be powerful tool for quantitative analysis of microbial pigments. The IR spectrum of shows the presence of ketone amide C=N and ester C-H stretching indicates more characteristic features of phytochemical pigment PC, which are responsible for the medicinal property of Spirulina platensis and used in toxicological studies (Venkatesan et al., 2012) Free radicals, mainly reactive oxygen species (ROS) has a noxious effect on cells and induce oxidative damage of DNA and other cellular components leading to cancer related mutations (Valko et al., 2004). Antioxidants play a major role in protection 13

8 of human body against damage of ROS. Previous epidemiology studies have shown that intake of natural antioxidants can reduced the risk of cancer, diabetes and other disease associated with oxidative stress (Rietveld and Wiseman, 2003). The microbial pigments reacts with DPPH, which is nitrogen centered radical with characteristic absorption at 517nm and convert it to stable diamagnetic molecule 1,1, dipenyl-picryl hydrazine, due to its hydrogen donating ability at rapid rate. When this electron becomes paired off the absorption decreases stoichiometrically with respect to the number of electrons taken up. The reduction in Purple colour indicates the presence of antioxidant molecules (Abdille, et al., 2005). Findings of this study revealed that phycocyanin from Spirulina has 25% of RSA, which has a capacity to scavenge free radicals of hydroxyl and peroxyl groups and inhibits microsomal lipid peroxidation. Nowadays, complementary and alternative medicine is widely available throughout the world. Several microorganisms have the capacity to produce multifaceted secondary metabolites like pigments, which can play various roles as biocolourants, antioxidants, antimicrobial agents and potent pharmacological drug against diseases. From the above results summarized in this present study, phycocyanin can be used as ecofriendly therapeutic molecules in human health concern. Reference Abdille, M.D., Singh, R., Jayaprakash, G. and Jena, B., Antioxidant activity of extracts from Dillenia indica fruits. Food Chem., 90: Bermejo, R., Alien, F.G., Ibanez, M.J., Fernandez, J.M., Moline, E. and Alvarez- Pez, J.M., Preparative purification of phycoerythrin from microalgae Porphyridium creuentum by expand bed - adsorption Chromatography. J. chromatogrp. B, 790: Bhaskar, S.U., Gopalswamy, G. and Raghu, R., A simple method for efficient extraction and purification of C- Phycocyanin from Spirulina platensis Gietler. Ind. J. Exp. Biol., 43: Boussiba, S. and Richmond, A.E., Isolation and characterization of Phycocyanin from blue green algae Spirulina Platensis. Arch. Microbiol., 120: Brand-Williams, W., Cuvelier, M.E. and Berset, C., Use of a free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technol., 28: Cisneros, M. and Rito- palomares, M., A simplified strategy for the release and the primary recovery of Phycocyanin produced by Spirulina maxima. Chem. Biochem. Eng. Q., 18(4): Harborne, J.B., Methods of plant analysis. In Phytochemical Methods.pg London: Chapman and Hall. Hongsachart, P., Awakairt, S., Peerapornpisal, Y. and Phutrakul, S., Extraction and Partially Purification of Natural Pigments from Spirulina platensis. The 15 th Annual Meeting of the Thai Society for Biotechnology Sustainable Development of SMEs Through Biotechnology and the JSPS-NRCT Symposium on the Forefront of Bioinformatics Application, February 3th-6th, Chiang Mai, Thailand. 14

9 Madhystha, H.K., Radha, K.S., Sugiki, M., Omeera, S. and Maruyama, M., Purification of C- Phycocyanin from Spirulina fusiformis and its effect on induction of urokinase- type plasminogen activator from calf pulmonary endothelial cells. Phytomedicine., 13: Margarita, P. and Quinteiro, R., Fourier Transform Infrared (FTIR) Technology for identification of organisms. Clinical Microbiol. Newslett., 22: 8. Reddy, M.C., Subhashini, J., Mahipal, S.V.K., Bhat, V.D., Reddy, P.S., Kiranmani, G., Madyastha, K.M. and Reddanna, P., C-Phycocyanin, a selective cyclooxygenase- 2 inhibitor, induces apoptosis in lipopolysaccharide stimulated Raw macrophages. Biochem. Biophys.Res. Commun., 304: Rietveld, Wiseman Romay, C.H., gonzaley, R., Ledon, N., Remirez, D. and Rimbav, V., C- Phycocyanin: A biliprotein with antioxidant, anti inflammatory and neuroprotective effects. Cur. Protein. Peptide. Sci., 4: Romay, C.H., gonzaley, R., Ledon, N., Remirez, D. and Rimbav, V., C- Phycocyanin: A biliprotein with antioxidant, anti inflammatory and neuroprotective effects. Cur. Protein. Peptide. Sci., 4: Valko, M., Izakovic, M., Mazur, M., Rhodes, C.J. and Telser, J., Role of oxygen radicals in DNA damage and cancer incidence. Mol. Cell. Biochem., 266: Venkatesan, S., Pugazhendy, K., Sangeetha, D., Vasntaraja, C., Prabakaran., S. and Meenambal, M., Fourier Transform Infrared Spectroscopic Analysis of Spirulina. Int. J. Phar. Bio. Arch., 3(4):