Antioxidant potential of indigenous cyanobacterial strains in relation with their phenolic and flavonoid contents

Abstract Antioxidant activities of eight indigenous cyanobacterial strains belonging to the genera Oscillatoria, Chroococcidiopsis, Leptolyngbya, Calothrix, Nostoc and Phormidium were studied in relation with their phenolic and flavonoid contents, ranging 3.9–12.6 mg GAE g−1 and 1.7–3.44 mg RE g−1. The highest activities were shown by Leptolyngbya sp. SI-SM (EC50 = 63.45 and 67.49 μg mL−1) and Calothrix sp. SI-SV (EC50 = 65.79 and 69.38 μg mL−1) calculated with ABTS and DPPH assays. Significant negative correlations were seen between total phenolic and flavonoid contents and the antioxidant activities in terms of EC50 values. Furthermore, HPLC detected 15 phenolic compounds with total concentrations ranging from 277.3 to 829.7 μg g−1. The prevalent compounds in most of the strains were rutin, tannic acid, orcinol, phloroglucinol and protocatechuic acid. Cyanobacterial strains showed high potential as a good source of phenolic compounds with potent antioxidative potential which could be beneficial for food, cosmetic and pharmaceutical industries.

compounds that fight against Reactive Oxygen Species and oxidative stress, which in turn could also be used in treating chronic diseases related to oxidative stress such as, inflammation, diabetes, cardiovascular diseases, neurodegenerative diseases and premature aging (Saranya et al. 2014). These antioxidants include the phenolic or polyphenolic compounds that have numerous beneficial effects in humans and other mammals (Plaza et al. 2008). They are structurally diverse with hydroxyl group covalently attached to an aromatic hydrocarbon group. From various algal species, phenolic acids and their ester, derivatives of Phloroglucinol, halogenated phenols and derivatives of sulphated phenols have been identified (La Barre et al. 2010). Dietary antioxidants comprise vitamins, carotenoids and phenols among which phenolics have the highest in vitro antioxidant activity affirming their importance in food and pharmaceutical industries (Oueslatia et al. 2012). Natural tannins are also one of the phenolic compounds with very high antioxidant abilities. They include hydrolysable tannins, flavonoids and non-hydrolyzable tannins or flavolans which are becoming interesting these days because not only do they have potent antioxidant abilities, they can also bind to pigments, proteins, other macromolecules and metal ions (Okuda & Ito 2011).

Total phenolic and flavonoid content
The total phenolic and flavonoid contents are given in Table S1. Among the eight strains, Leptolyngbya sp. SI-SM showed the highest amount of total phenolic and flavonoid content (12.6 and 3.44 mg g −1 ) closely followed by Calothrix sp. SI-SV (12.4 and 3.23 mg g −1 ). Kumar et al. (2015) showed lesser phenolic contents from these cyanobacteria. The unicellular strain Chroococcidiopsis sp. SI-ST showed total phenolic and flavonoid content of 4.32 and 2.44 mg g −1 . To the best of our knowledge no study has been done on phenolic content of Chroococcidiopsis sp. and this is the first one done on this cyanobacteria. In the case of total flavonoid content a somewhat similar trend was seen as shown in Table S1. Moreover, despite the fact that the total phenolic content of Oscillatoria sp. SI-SF and Chroococcidiopsis sp. SI-ST was low, they showed high flavonoid content of 2.82 and 2.44 mg RE g −1 . Singh et al. (2014) also showed similar amount of total flavonoid content from Oscillatoria acuta.

Antioxidation potential
The antioxidant activities of eight strains of cyanobacteria and the standards, obtained from ABTS and DPPH assays are given in Table S1. In the ABTS assay, the highest radical scavenging activity was shown by Leptolyngbya sp. SI-SM, closely followed by Calothrix sp. SI-SV and Nostoc sp. SI-SN with low EC 50 values of 63.45, 65.79 and 75.99 μg mL −1 , respectively ( Figure  S1(A)). The standard Trolox gave highest antioxidation with low EC 50 value of 25.48 ( Figure  S2(A)). These results are comparable with the study done by Shanab et al. (2012) however; they extracted the biomass in water instead of 70% methanol and also reported higher antioxidation of 75.6% at 100 μg mL −1 from Oscillatoria sp. as opposed to the current study. The DPPH assay gave slightly lower antioxidation activities than the ABTS assay with similar trend as shown in Figure S1(B). Ascorbic acid as a standard showed much higher antioxidation with a low EC 50 value of 28.16 ( Figure S2(B)). These results are in agreement with study done by El-Aty et al. (2014). Similarly, when comparing the antioxidation of Leptolyngbya sp. SI-SM at 50 μg mL −1 by DPPH assay (Figure S1(B)), Suhail et al. (2011) showed lower antioxidant activity from Plectonema boryanum, a taxonomic synonym of Leptolyngbya boryana.

Correlation between phenolic, flavonoid content and antioxidation potential
The correlation coefficient (R 2 ) was calculated between the antioxidation activities obtained from ABTS and DPPH assay as shown in Figure S3(A) and (B). The results showed significantly strong negative correlations between the total phenolic and flavonoid content and EC 50 values obtained from both ABTS (R 2 = 0.868, 0.620) and DPPH (R 2 = 0.813, 0.616) assays. It was also observed that despite the fact that Oscillatoria sp. SI-SF comparatively contained less total phenolic content but due to its high flavonoid content, it showed high antioxidation activity. This phenomenon was conversely present in Nostoc sp. SI-SN. This showed that the antioxidation activity greatly depended upon the phenolic and flavonoid contents equally (Figueroa et al. 2014).

HPLC UV/vis analysis of phenolic and flavonoid content
Fifteen phenolic compounds were detected in the 70% methanolic extracts as shown in Table S2. The highest concentration of phenolic compounds was found in Leptolyngbya sp. SI-SM with 829.7 μg g −1 . Rutin, tannic acid, orcinol, phloroglucinol and protocatechuic acid were the dominant phenolic compounds in most of the strains with concentrations ranging 96. 3-176.2, 13.4-75.0, 71.2-167.4, 12.0-28.3 and 35.6-94.0 μg g −1 , respectively. Higher values of protocatechuic acid were found by Shalaby and Shanab (2013) in water and methanolic extracts of Spirulina platensis. In Leptolyngbya sp. SI-SM, Gallic acid was seen in very high amounts of 205.4 μg g −1 followed by rutin and ferulic acid with 176.2 and 98.1 μg g −1 , respectively. These compounds are strong free radical scavengers and, therefore, could be involved in the high antioxidation activity of Leptolyngbya sp. SI-SM. Singh et al. (2014) showed lower values in P. boryanum under high salt stress. As far as other cyanobacterial strains were concerned, resorcinol, vanillic acid and syringic acid were detected in high amounts (81.3, 21.7 and 13.4 μg g −1 ) in Phormidium sp. SI-SC. Caffeic acid and benzoic acid (28.2 and 17.5 μg g −1 ) were detected in highest amount in Oscillatoria sp. SI-SF. Salicyclic acid and acetyl salicyclic acid (23.5 and 13.2 μg g −1 ) were present in Oscillatoria sp. SI-SA in higher amounts. Tannic acid was present in highest amount (75.0 μg g −1 ) in Nostoc sp. SI-SN. Due to the unavailability of flavonoid standards other than rutin, not much flavonoid varieties were detected in the extracts.

Conclusion
It is concluded from the study that out of eight cyanobacterial strains Leptolyngbya sp. SI-SM and Calothrix sp. SI-SV showed promising results as good sources of phenolic compounds with potent antioxidant activities which is directly related with the abundance and types of phenolic compounds extracted from the biomass. The antioxidant benefits of phenolic compounds from terrestrial plants have long been established and exploited but the phenolic compounds from cyanobacteria are still an emerging research field and deserve more scientific attention and interdisciplinary research. Since there is very scarce data published on the phenolic compounds of cyanobacteria particularly, this study would contribute greatly for this purpose and would be a boon for food and pharmaceutical sciences to explore unusual and potent phenolic compounds from these blue-green microbes.

Disclosure statement
No potential conflict of interest was reported by the authors.