Calibrated microplate assays for total antioxidant capacity using DPPH and comparisons with the FRAP assay

Ng Hau Ying, Jo B00672470

BSc (Hons) Human Nutrition School of Biomedical Sciences

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• Oxidative stress: an imbalance in metabolism of controlling and detoxifying reactive oxygen species – cancer, cardiovascular disease, cognitive disease

• Antioxidant: substances that inhibit oxidation of biological molecules

• USDA has established a database for flavonoid content of 506 food items since 2013 – Potential beneficial health effects of antioxidants – A great demand on total antioxidants capacity (TAC) testing

• An advanced testing system is needed for comparisons and quality assurance

• Using more than one assay for objective antioxidants capacity

quantitation

(USDA, 2013)

(Davies, 1995)

(Halliwell, 1995)

(Fogarasi et al., 2015)

Introduction

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hjh Aims

• The possibility of incorporation and agreement on pathlength

determinations between microplate and conventional methods without using expensive equipment was indicated.

• Ulster University: Universal molar absorptivity (ɛ) standard for FRAP assay determined and successfully applied on Manuka honey.

(Bolanos de la Torre et al., 2015)

(Smith et al, 2001)

1) Determine the molar absorptivity and calibration parameters for five antioxidants: ascorbic acid, gallic acid, resveratrol, caffeic acid and trolox using microplate- FRAP Assay.

2) Determine similar parameters for microplate based DPPH assay 3) Establish pathlength corrections for the FRAP and DPPH assays

using 1-cm conventional spectrophotometric

FRAP + DPPH microplate-based assay comparisons

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Methodology 1. FRAP and DPPH working solution preparation

2. Standard phenolic compounds for calibration preparation - 100ml 1000µM stock solution of ascorbic acid , gallic acid, resveratrol,

caffeic acid and trolox - 2-fold serial dilutions using distilled water to form

1000,500,250,125,62.5, 0µM solutions.

FRAP DPPH1 75µl 2-fold diluted solutions + 1425µl FRAP/DPPH working solution

DPPH2 20µl 2-fold diluted solutions + 280µl DPPH working solution

Store in the dark for 30 minutes Warm in 37 ℃ for 30minutes 4

Solution were transferred to 1-cm cuvette. Absorbance was then recorded at 593nm (FRAP) and 515nm (DPPH).

200µl solution were transferred from cuvette to 96-well microplate (replicated for 4 times). Absorbance was then recorded at 593nm (FRAP) and 515nm (DPPH).

Absorbance was then recorded at 515nm.

Statistical analysis Molar absorptivity (sensitivity) calculation (ɛ) from Beer’s law (A=ɛLC)

determined from a slope (m) of calibration graph, y = mx + c

Pathlength calculation (cm) ɛ’ (microplate)/ ɛ (cuvette) Excel Graphs & tables production SPSS one way ANOVA analysis

*All tests replicated (≧2) 5

Results

Figure 1 Calibration graphs for microplate FRAP assays for the five antioxidants in descending sensitivity: gallic acid > caffeic acid > ascorbic acid > trolox > resveratrol. The linear range of gallic acid is 0-25µM while the others are 0-50µM. Solutions (200µl) of mixture (75µl sample and 1425µl warmed in Eppendorf tube) were transferred in 96-well microplate. Readings in A593nm were recorded. 6

Figure 2 Calibration graphs for microplate DPPH1 assays for the five antioxidants in descending sensitivity: gallic acid > caffeic acid > ascorbic acid > trolox > resveratrol. The linear range of gallic acid is 0-6.3µM while caffeic acid and ascorbic acid are 0-16.6µM; trolox and resveratrol are 0-33.3µM. Solutions (200µl) of mixture (75µl sample and 1425µl warmed in Eppendorf tube) were transferred in 96-well microplate. Readings in A515nm were recorded. 7

Results (con’t) Figure 3 Molar absorptivity (ɛ) of the five antioxidants using mFRAP, mDPPH1 and mDPPH2. Letters denote significant difference in FRAP assay only, P<0.05.

Table 1 Antioxidant activity of phenolic compounds determined using FRAP.

Sensitivity is equal to the molar absorptivity (ɛ) in M-1cm-1.MDC= minimum detectable concentration. L’ = optical pathlength of a microplate in cm. TE= Trolox equivalent. GAE= Gallic acid equivalent.

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Calibrants SensitivitymFRAP1 *

1-cm cuvette* MDC mFRAP1(mol/L)

Pathlenth (L')(cm)*

TE (ratio ofsensitivity)

GAE (ratio ofsensitivity)

Ascorbic acid 26086 ± 741a 52629 ± 992a 6.3 x 10-7 0.496 ± 0.0081a 1.1 1.1Gallic acid 83770 ± 1971b 167173 ± 4663b 1.7 x 10-7 0.493 ± 0.0032a 3.6 3.6Resveratrol 19287 ± 260d 35344 ± 600d 6.7 x 10-7 0.546 ± 0.0166ab 0.8 0.8Caffeic acid 44708 ± 1987c 79660 ± 3459c 1.9 x 10-7 0.561 ± 0.0128b 1.9 1.9Trolox 23240 ± 252a 45030 ± 557 a 4.1 x 10-7 0.516 ± 0.0008ab 1.0 1.0

Figure 4 Calibration graph of trolox using microplate-based and spectrophotometric FRAP assay. The molar absorptivity of mFRAP (ɛ’) and colorimetric assay (ɛ) were 23240 M−1 cm−1 and 45030 M−1 cm−1 respectively. L’ (optical pathlength) = 23240 L’/45030 x 1 = 0.516cm.

*Parameters with significant different between antioxidants. Values in a column with different superscript letters are significantly different, P<0.05

Table 2 Antioxidant activity of phenolic compounds determined using mDPPH1.

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CalibrantSensitivitymDPPH1* 1-cm cuvette*

MDCmDPPH1(mol/L)

Pathlenth (L')EC50 (µmol/L)*

TE (ratio ofsensitivity)

GAE (ratio ofsensitivity)

Ascorbic acid 6858 ± 1199a 13759 ± 2092a 4.0 x 10-6 0.497 ± 0.017 27.77 ± 0.66a 1.1 1.1Gallic acid 22493 ± 1734b 43908 ± 2083b 1.0 x 10-6 0.512 ± 0.015 7.17 ± 0.44b 3.7 3.7Resveratrol 5912 ± 517a 11394 ± 639c 9.4 x 10-6 0.518 ± 0.020 34.41 ± 2.36d 1.0 1.0Caffeic acid 10950 ± 237c 20640 ± 57b 2.5 x 10-6 0.531 ± 0.013 16.19 ± 0.76c 1.8 1.8Trolox 6113 ± 98a 11976 ± 404a 6.6 x 10-6 0.511 ± 0.009 29.64 ± 0.93a 1.0 1.0

• Sensitivity and linear range of antioxidants were higher using FRAP

than DPPH • Total antioxidant capacity (TAC) : gallic acid > caffeic acid >

ascorbic acid > trolox > resveratrol – Number of –OH group in phenolic compound increases with higher TAC

• Trolox has relatively low sensitivity and long linear range for both FRAP and DPPH assays – Better to be a reference compound than ascorbic acid or gallic acid

Discussions

Figure 5 Pathlength of the five antioxidants using FRAP and DPPH. Letters denote significant difference in FRAP assay only, P<0.05.

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Conclusions • The molar absorptivity for five antioxidants: ascorbic acid, gallic acid,

resveratrol, caffeic acid and trolox of mFRAP are 26086, 83770, 19287, 44708 and 23240 M-1cm-1 respectively .

• The molar absorptivity for five antioxidants: ascorbic acid, gallic acid, resveratrol, caffeic acid and trolox of mDPPH1 are 6858, 22493, 5912, 10950 and 6113 M-1cm-1 respectively .

• TAC: gallic acid > caffeic acid > ascorbic acid > trolox > resveratrol

• Significant difference of pathlength correction was only shown in FRAP assay.

• Pathlength difference may due to different composition, viscosity, solvability of antioxidants in different solvent/ with FRAP/DPPH solution

• Limitation: absorbance of DPPH working solution was needed to be regularly monitored throughout tests

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• Bolanos de la Torre, A. A. S., Henderson, T., Nigam, S. & Owusu-Apenten, R. K. (2015) A universally calibrated microplate ferric reducing antioxidant power (FRAP) assay for foods and applications to Manuka honey, Food Chemistry, 174, 119-123.

• Davies, K. J. A. (1995) Oxidative stress: the paradox of aerobic life. Biochemical Society symposium, 61, 1-31. • Fogarasi, A. L., Kun, S., Tankó, G., Stefanovits-Bányai, E & Hegyesné-Vecseri B (2015) A comparative assessment of

antioxidant properties, total phenolic content of einkorn, wheat, barley and their malts. Food Chemistry, 167, 1-6. • Halliwell B (1995) How to characterize an antioxidant: an update. Biochemical Society symposium, 61, 73-101. • Smith AD, Morris VC & Levander OA (2001) Rapid Determination of Glutathione Peroxidase and Thioredoxin

Reductase Activities Using a 96-well Microplate Format: Comparison to Standard Cuvette-based Assays. International journal for vitamin and nutrition research. Supplement, 71(1), 87-92.

• USDA (2013) USDA Database for the Flavonoid Content of Selected Foods: Release 3.1. [Online] Available from: http://www.ars.usda.gov/SP2UserFiles/Place/80400525/Data/Flav/Flav_R03-1.pdf. [Assessed: 25th November 2014]

References

Acknowledgement I would like to express my gratitude to my supervisor, Dr. Richard Owusu-Apenten for all technical advice and guidance throughout this project, and thank you to the other students who contribute to the related studies. 12