COMPARISON OF DPPH, FRAP AND TAS ANTIOXIDANT TAIWAN. Luis Valenzuela, Yew-Hu Chien Department of Aquaculture National Taiwan Ocean University

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1 COMPARISON OF DPPH, FRAP AND TAS ANTIOXIDANT ACTIVITY ASSAYS AGAINST TPC TO DETERMINE THE ANTIOXIDANT ACTIVITY OF VARIOUS ALGAE FROM TAIWAN Luis Valenzuela, Yew-Hu Chien Department of Aquaculture National Taiwan Ocean University 1

2 2 INTRODUCTION 1/2 Why Algae? Superfood! Functional-food! Vitamins (A,B,C,D,K and E) Rich in PUFA Minerals, such as I, Ca, Mg, N, P, and K Protein source as high as 71% Rich in antioxidants (carotenoids, phenolic compounds, phycobilins, polysaccharides Antibacterial and antifungal properties

3 3 INTRODUCTION 2/2 What are antioxidants? Why Phenols? Antioxidants are nutrients that help to minimize free-radical damage to the body cells by: reducing their energy, stopping them from forming interrupting an oxidizing chain reaction to minimize the damage of ROS.

4 4 OBJECTIVE The aim of this study was to prepare methanol and water extracts and conduct in vitro tests to estimate the antioxidant potential (DPPH, FRAP and TAS), total phenolic content (TPC), and keep the records for further use of on in vivo trials in aquatic organisms.

5 5 MATERIAL AND METHODS 1/4 Macroalgae Collected from Keelung s offshore Ulva lactuca Sargassum wightii Microalgae Provided by Taiwan Gene Biothech Co. Chlorella sp. Spirulina sp. Nannochloropsis sp.

6 6 MATERIAL AND METHODS 2/4 Total Phenolic Content (TPC) Algae Samples Methanol extraction Water Extraction DPPH Total Antioxidant Status (TAS) Ferric Antioxidant Reducing Power (FRAP)

7 7 MATERIAL AND METHODS 3/4 Kirby Bauer antibiotic testing or disk diffusion method 4 gram-negative bacteria (Vibrio alginolyticus and parahaemolyticus, Edwardsiella tarda, Aeromonas hydrophila) 30µl of algae extract were used per each paper disk (6mm) The agar petri dishes were inoculated with the bacteria and 5 disks were placed in each petri dish The petri dishes were incubated for 24hr before reading

8 8 MATERIAL AND METHODS 4/4 Statistical Analysis All essays were carried out in triplicates and the average was used. The results are expressed as mean ± SD. The statistical analysis and significance were assessed using SAS software (SAS 9.4 Cary, NC). Two-way ANOVA was applied to evaluate significant differences (P<0.05). Pearson s correlation coefficients and Fisher s z transformation test was used to obtain the correlation of the antioxidant assays versus total phenolic content.

9 9 RESULTS 1/7 Total Phenolic Content Total phenolic content (TPC) on methanol and water extracts were evaluated, using Folin-Ciocalteu method, results are presented in the following table. Microalgae Strains Phenolic Content Water Fraction (mg GAE/g) Phenolic Content Methanol Fraction (mg GAE/g) Chlorella sp ± 0.003* ± 0.001* Spirulina sp ± 0.002* ± 0.003* Values represent means ± standard deviation of triplicate absorbance value readings. * Values P <0.05 were considered significant Nannochloropsis sp ± 0.001* ± 0.001* Ulva lactuca ± 0.002* ± 0.003* Sargassum wightii ± 0.003* ± 0.002*

10 10 Total Antioxidant Status Determination (ABTS) RESULTS 2/7 The antioxidant activity ranged from 1.31 mmol/l (Ulva lactuca) to 2.88 mmol/l (Nannochloropsis sp.), being the last one the highest reading 3.50 Total Antioxidant Status mmol/l Water Methanol * Values P <0.05 were considered significant 0.00 * Chlorella sp. * Spirulina sp. * Nannochloropsis * Ulva lactuca Sargassum wightii * sp.

11 11 RESULTS 3/7 Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay Samples with relatively high DPPH activity (<50%) belonged to Spirulina sp. (85.60%) and Nannochloropsis sp. (68.15%) DPPH radical scavenging % Water Methanol * Values P <0.05 were considered significant Chlorella sp. * Spirulina sp. Nannochloropsis sp. Ulva lactuca * * * * Sargassum wightii

12 12 Ferric Reducing Antioxidant Power (FRAP) RESULTS 4/7 The highest readings were obtained from Spirulina sp. (35.48 µm/fe2+) followed by Nannochloropsis sp. (34.19 µm/fe2+) and Ulva lactuca (21.79 µm/fe2+) FRAP antioxidat power μm/l Fe Water Methanol * Values P <0.05 were considered significant * Chlorella sp. * Spirulina sp. * Nannochloropsis * Ulva lactuca * Sargassum wightii sp.

13 13 RESULTS 5/7 Statistical Correlation of Different Antioxidant Assays Against TPC TPCm highly correlated not only to FRAPm but also to DPPHm TPCw had a strong correlation with FRAPw The strongest correlation in both fractions water and methanol was TPC versus FRAP. Tests TPCm DPPHm TASm FRAPm TPCm 1 DPPHm 0.95* 1 TASm -0.55* -0.48* 1 FRAPm 0.98* 0.89* -0.56* 1 Tests TPCw DPPHw TASw FRAPw TPCw 1 DPPHw 0.57* 1 TASw 0.34* 0.87* 1 FRAPw 0.99* 0.55* 0.66* 1

14 14 RESULTS 6/7 Statistical Correlation of FRAP assay Against TPC FRAP antioxidat power μm/l Fe TPC mg GAE/g Water Methanol Water Methanol

15 15 RESULTS 7/7 Antibacterial assay Bacterium Algae V. Alginolyticus Nannochloropsis sp. Spirulina sp. Chlorella sp Sargassum wightii Ulva lactuca ± ±

16 16 DISCUSSION 1/6 The total phenolic content data showed in this paper was comparable with previous studies of Miranda et al. (1998), Go et al. (2010), Shanab et el. (2012) and Goiris et el. (2012). Source Species Extraction method Results Goiris et el. (2012) Chlorella sp. hot water 0.75 ± 0.03 mg GAE/g ± 0.26 mg GAE/g Current study Chlorella sp. Cold water ± mg GAE/g Chlorella sp. Methanol ± mg GAE/g

17 17 DISCUSSION (TAS) 2/6 The TAS assay kit used for this experiment has not been used before to quantify antioxidant activity of algae samples. Results may be subjected to re-evaluation by other scientists. Source Species Extraction method Results Shanab et el., 2012 Spirulina plantesis Methanol 99.55% Current Study Nannochloropsis sp. Water 2.88 mmol/l

18 18 DISCUSSION (DPPH) 3/6 Source Species Extraction method Results Li et el. (2005) Chlorella sp. Spirulina sp. Water Extract ± 0.65 µmol of ascorbic acid equivalent/g ± 1.06 µmol of ascorbic acid equivalent/g Current Study Chlorella sp. Spirulina sp. Water Extract Methanol Extract ±0.002 µmol of ascorbic acid equivalent/g ±0.002 µmol of ascorbic acid equivalent/g.

19 19 DISCUSSION (FRAP) 4/6 Source Species Extraction method Results Su et el. (2010) Nannochloropsis sp. Dichloromethane extract ± 0.59 µmol TE per gram of dried extract Current Study Spirulina sp ± μm/fe 2+. Nannochloropsis sp. Methanol extract ± μm/fe 2+.

20 20 DISCUSSION (Correlation) 5/6 According to Jimenez et al., (2001) the total phenolic content of a sample could be significantly related to the total antioxidant capacity. Tests TPCm DPPHm TASm FRAPm TPCm 1 DPPHm 0.95* 1 TASm -0.55* -0.48* 1 FRAPm 0.98* 0.89* -0.56* 1 Tests TPCw DPPHw TASw FRAPw TPCw 1 DPPHw 0.57* 1 TASw 0.34* 0.87* 1 FRAPw 0.99* 0.55* 0.66* 1 Hajimahmoodi et al,. (2009) reported a positive correlation between antioxidant capacity and phenolic content only in FRAP assay and not in DPPH.

21 21 DISCUSSION (Previous in vivo works?) 6/6 Administration of hot-water extract of Gracilaria tenuistipitata (Ye et al., 2006). Administration of hot-water extract of Spirulina plantesis (Tayang et al., 2010). Successful improvement and enhance the immune system, tolerance to stress factors and resistance against Vibrio alginolyticus in L. vannamei Administration of hot-water extract of Gelladium amansii (Fu et el. 2007) Administration of hot-water extract of Sargassum duplicatum (Ye et al. 2009) Previous in vivo works showed that algae extracts improve and enhance the immune system of shrimps. However, their work is based on measurements of immune parameters. The present work is the first part of a two part study, where unlike previous work we are documenting antioxidant activity of the algae, so later we can use exact doses and compare the findings to known strong antioxidants, such as astaxanthin.

22 22 CONCLUSIONS The tested algae in general displayed high antioxidant activities. Polyphenols could potentially be used as feed additive on in vivo trials and enhance the immune system of the receptors. The measurement of antioxidant activities, cannot be evaluated satisfactorily by a simple antioxidant test, without due regard to the many variables influencing the results. The correlation showed that FRAP assay is more accurate to measure the antioxidant activity of phenols. Although some inhibition was detected on the antibacterial analysis, further test, such as MIC and disk diffusion method should be carried on to make sure the effectivity of these extracts to neutralize bacteria.

23 23 Thank you for your attention! Questions and comments are now welcome.