Evaluation of antioxidant property of purified phycobiliproteins and phenolic compounds containing extracts from bangia atropurpurea

Rajalakshmi, Punampalam (2018) Evaluation of antioxidant property of purified phycobiliproteins and phenolic compounds containing extracts from bangia atropurpurea. Master dissertation/thesis, UTAR.

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Abstract

Bangia atropurpurea is a freshwater red filamentous alga. It is one of the fastgrowing algae with survival capacity. B. atropurpurea has high adaptation to a broad range of salinities over time and is able to tolerate desiccation and osmotic stress where other filamentous algae do not typically grow. The antioxidant property of this red alga was compared with Chlorella vulgaris, a freshwater green microalga. C. vulgaris is similar to most phototrophs as it absorbs light via the chloroplast to synthesise organic compounds for nutrition. In this study, the recovery yield of purified phycobiliproteins extracted from B. atropurpurea was evaluated and compared the antioxidant capacity with ascorbic acid, butylated hydroxytoluene (BHT) and phenolics extracted from B. atropurpurea and C. vulgaris. The crude extract of phycobiliproteins from B. atropurpurea iii was purified by (NH4)2SO4 saturation before further fractionation of the phycobiliproteins extract to R-phycoerythrin (R-PE), R-phycocyanin (R-PC) and allophycocyanin (APC) by gel filtration with Sephadex G-200. The separated R-PE (bright pinkish) and R-PC (purplish blue) proteins were identified by RP-HPLC and SDS-PAGE while APC was untraceable after gel filtration. The percentage of recovery yield of R-PE and R-PC from total protein extracted from B. atropurpurea increased proportionally with the purity index after each subsequent purification process. The recovery yields (%) of R-PE and R-PC after RP-HPLC were 94.4% at purity index (A562/A280) of 5.42 and 86.1% at purity index (A615/A280) of 3.95, respectively. A total of 85.9 mg of R-PE and 44.2 mg of R-PC were separated by RP-HPLC from 50 g of B. atropurpurea while from the total phycobiliproteins recovered, 66% was R-PE and 34% was R-PC. Therefore, R-PE is the predominant phycobiliprotein in B. atropurpurea. Phenolic compounds were extracted with solvents of different polarity. The Folin-Ciocalteu method was used to determine the TPC while 1,1-Diphenyl-2- picrylhydrazyl (DPPH) radical scavenging and ferric-reducing antioxidant power (FRAP) assays were used to determine the antioxidant capacity of the extracted phycobiliproteins and phenolic compounds. The phenolic compounds have high solubility in methanol solvent compared to other solvents used for extraction. The TPC in the methanol extract from B. atropurpurea (80.97 ± 0.53 mg GAE/g dry weight) was higher than C. vulgaris (62.13 ± 1.28 mg GAE/g dry weight). Similarly, the phenolics extracted from B. atropurpurea and C. vulgaris using methanol exhibited effective DPPH radical scavenging with the lowest IC50 (30.82 ± 0.92 μg/mL and 34.28 ± 0.79 μg/mL) and the highest FRAP (37.81 ± 0.04 mg GAE/g dry weight and 23.97 ± 0.61 mg GAE/g dry weight), iv respectively. Analysis of the correlations between TPC and the antioxidant property measured by DPPH radical scavenging and FRAP assays showed good correlations with higher regression coefficient, R2 = 0.898 and R2 = 0.925, respectively. These data suggest that phenolic compounds are powerful free radical scavengers and effective metal ion reducing agents, however, this study has justified that the phenolic compounds were not the only contributor to the antioxidant capacity of this red alga. The purified R-PE and R-PC have contributed significantly in DPPH radical scavenging and metal ion reduction activity. The purified R-PE (IC50, 7.66 ± 0.81 μg/mL) exhibited better radical scavenging activity compared to R-PC (IC50, 9.42 ± 1.73 μg/mL), phenolic compounds in methanol extract (IC50, 30.82 ± 0.92 μg/mL) and the synthetic antioxidant BHT (IC50, 35.06 ± 1.15 μg/mL) while lower radical scavenging activity compared to ascorbic acid (IC50, 6.78 ± 0.28 μg/mL). R-PE also exhibited higher FRAP (54.81 ± 0.31 mg GAE/g dry weight) compared to RPC (42.18 ± 0.70 mg GAE/g dry weight), phenolic compounds in methanol extract (37.81 ± 0.04 mg GAE/g dry weight) and BHT (30.37 ± 0.12 mg GAE/g) while lower FRAP compared to ascorbic acid (65.77 ± 0.12 mg GAE/g). SDSPAGE of purified R-PE and R-PC proteins showed single narrow bands at molecular weight of 20.5 kDa and 17.6 kDa, respectively. The findings of this study supported that B. atropurpurea could be a promising new source of potential antioxidants to replace the synthetic antioxidants used in food and pharmaceutical products due to its natural, non-toxic antioxidant capacity contributed by R-PE, R-PC and phenolics extract.

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