Total phenolic content - Folin-Ciocalteu reducing … and DPPH - COST 927...Singleton V.L., Rosi...

Total phenolic content - Folin-Ciocalteu reducing capacity(FCR assay)

Antiradical activity against DPPH radical

Ryszard Amarowicz

Institute of Animal Reproduction and Food Research

General chemistry of the F-C method

Na2WO4/Na2MnO4 → (phenol - MoW11O40)-4

Mo(VI) (yellow) + e- → Mo(V) (blue)

Folin O., Ciocalteu V.: Tyrosine and tryptophan determination on proteins. J. Biol. Chem., 1927, 73, 627-649.

Modyfication of the F-C method by Singleton & Rosi

Molybdotungstophosphoric hereopolyanion reagent:

3H2O – P2O5 – 13WO3 – 5 MoO3 – 10H2O

and

3H2O – P2O5 – 14WO3 – 4 MoO3 – 10H2O

Singleton V.L., Rosi J.A.: Colorimetri of total phenolics with phosphomolybdic-phosphotungstic acid reagent. Am. J. Enol. Vitic., 1965, 16, 144-158.

Absorption spectra produced by wine (W) and gallic acid (G) withFolin-Ciocalteu (C) and Folin-Denis (D) reagent

Singleton V.L., Orthofer R., Lamuela-Raventos R.M.: Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol., 1999, 299, 152-178.

The explicit conditions of the Singleton-Rossi method are as follows:Mix 1 mL of sample (properly diluted) with at least 60 mL of water and 5 mL of F-C reagent; after 30 s and before 8 min, add 15 mL of Na2Co3; mix and bring to 100 mL total volume with water; incubate for 2 h at 75 oF and measure absorbance.

The F-C method suffers from a number of interfering substances:

•Sugars•Aromaric amines•Sulfur dioxide•Ascorbic acid•Organic acid•Fe(II)

Nonphenolic organic substances that react with F-C- reagent includd:

Addenine Adenosine Alanine Anilin Aminobenzoic acidAscorbic acid Benzaldehyde Creatinine Cysteine CytosinDimethylaniline Diphenylamine EDTA Fructose GuanineGuanosine Glycine Histamine Histidine Indole ProteinsPyridoxine Sucrose Sulfanilic acid Thiourea Thymine ThymidineTrimethylamine Tryptophan Uracil Uric acid Xanthine

Some inorganic substances may also react with F-C reagent:

Hydrazine Hydroxyammonium chloride Iron ammonium sulfate Manganese sulfate Potassium nitrite Sodium cyanide Sodium metabissulfite Sodium phosphate Sodium sulfite Tin chloride

Molar absorptivity in F-C assay from selected phenols and potentialy interfering substances

Compound Molar absorbance

(1000÷÷÷÷)

Molar absortivityper reactive

group

Compound Molar absorbance

(÷÷÷÷)

Molar absortivityper reactive

group

Phenolp-Coumaric acidTyrosineCatecholChlorogenic acidDOPAFerulic acidVanillinPyrogallolGallic acidSinapic acidPhloroglucinolp-HydroquinoneResorcinol(+)-CatechinKaempferol

12.715.615.722.528.924.619.214.924.825.033.313.312.819.834.329.6

12.715.615.711.214.412.319.214.912.412.516.613.312.819.811.514.8

QuercetinQuercitrinMalvino-Aminophenolp- Aminophenolp- MethylanilineFlavoneFlavanone3-Hydroxyflavone4-hydroxycoumarinAcetylsalicylic acidD-FructoseAscorbic acidFerrous sulfateSodium sulfite

48.344.840.531.121.811.60.11.93.50.10.20.017.53.417.1

16.114.920.215.610.911.60.11.93.50.10.20.017.53.417.1

Singleton V.L., Orthofer R., Lamuela-Raventos R.M.: Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol., 1999, 299, 152-178.

Callibration curves for F-C method

Compound Regression r Range Compound Regression r Rangesinapic acid y = 2.6715x + 0.0101 0.9996 0.0-0.3 chlorogenic y = 3.2033x + 0.0352 0.9971 0.0-0.31gallic y = 6.2295x + 0.017 0.9997 0.0-0.214 protocatechuic y = 5.8417x + 0.0177 0.9989 0.0-0.15caffeic y = 7.1463x + 0.0075 0.9995 0.0-0.1 egcg y = 4.5783x + 0.0134 0.9995 0.0-0.2p-cumaric y = 3.0353x + 0.0227 0.9981 0.0-0.25 baicalin y = 4.0829x + 0.0251 0.9981 0-0.2o -coumaric y = 3.2019x + 0.0342 0.9975 0.0-0.3 naringenin y = 4.0863x + 0.0314 0.9970 0.0-0.2vanillic y = 3.2699x + 0.0253 0.9979 0.0-0.2575 naringin y = 0.669x + 0.0516 0.9886 0-1homovanillic y = 3.6029x + 0.0373 0.9957 0.0-0.22 arbutin y = 1.9775x + 0.0199 0.9989 0.0-0.43salicilic y = 0.0678x + 0.0436 0.9739 0-5 rutin y = 2.2528x + 0.0316 0.9975 0.0-0.38syringic y = 2.0896x + 0.0103 0.9996 0.0-0.416 chryzin y = 2.9379x + 0.0313 0.9964 0.0-0.255ferulic y = 3.1078x + 0.0213 0.9978 0.0-0.25 quercitin y = 6.264x + 0.0044 0.9994 0.0-0.12isoferulic y = 3.3648x + 0.0441 0.9962 0.0-0.3 catechine y = 4.6179x +0.0127 0.9987 0.0-0.1tannic y = 4.4556x + 0.0088 0.9994 0.0-0.15 morin y = 3.8719x + 0.019 0.9989 0.0-0.22homogentisin y = 3.7893x + 0.0073 0.9990 0.0-0.22 coniferyl aldehyde y = 3.4441x + 0.0186 0.9977 0.0-0.22gentisin y = 6.3517x + 0.0169 0.9993 0.0-0.15 metferulald y = 2.1977x + 0.021 0.9978 0.0-0.4ellagic y = 4.4621x + 0.0175 0.9988 0.0-0.206 3,5dimetoxy cinald y = 3.4992x + 0.0199 0.9991 0.0-0.25

Converting factors for individual phenolic compounds determining using F-C method

sinapic acid gallic acid catechinesinapic acid 1.000 0.418 0.573gallic 2.358 1.000 1.358caffeic 2.665 1.132 1.536p -cumaric 1.183 0.496 0.679o -cumaric 1.289 0.542 0.740vanillic 1.281 0.538 0.735homovanillic 1.450 0.611 0.833salicilic 0.151 0.054 0.082syringic 0.783 0.325 0.447ferulic 1.205 0.506 0.692isoferulic 1.387 0.584 0.797tannic 1.663 0.702 0.956homogentisic 1.408 0.593 0.809gentisic 2.403 1.019 1.385ellagic 1.698 0.717 0.977chlorogenic 1.293 0.543 0.742egcg 2.215 0.939 1.276baicalin 1.726 0.729 0.993naringenin 1.584 0.668 0.911naringin 1.609 0.679 0.925arbutin 0.406 0.163 0.229rutin 0.777 0.322 0.444chryzin 0.924 0.385 0.529protocatechuic 1.179 0.495 0.676quercitin 2.323 0.985 1.338coniferyl aldehyde 1.738 0.734 1.000metferulald 1.483 0.625 0.852morin 1.321 0.555 0.7593,5dimetoxy cinald 0.863 0.359 0.494catechine 1.347 0.566 0.773

Chemical structure of DPPH• and DPPH - H

DPPH• + AH → DPPH-H + A•

Kinetic behaviour of ascorbic acid

Brand-Williams W., Cuvelier M.E., Berset C.: Use of free radical method to evaluate antioxidant activity. LWT –Food Sci. Technol., 1995, 28, 25-30.

Kinetic behaviour of quaiacol

Brand-Williams W., Cuvelier M.E., Berset C.: Use of free radical method to evaluate antioxidant activity. LWT –Food Sci. Technol., 1995, 28, 25-30.

Disappearance of DPPH radical as a function of the moles of zingerone/mole DPPH radical

Brand-Williams W., Cuvelier M.E., Berset C.: Use of free radical method to evaluate antioxidant activity. LWT – Food Sci. Technol., 1995, 28, 25-30.

Classification of antiradical efficiacies and stechiometry, according to kinetic behavior

Kinetic behavior

Compound ARP Stoichiometricvalue

Number of reduced DPPH••••

Rapid

Intermediate

Slow

IsoeugenolAscorbic acidIsoascorbic acid

δ-TocopherolRosmarinic acid

PhenolCoumaric acidVanillinVanillic acidΓ-resorcylic acidFerulic acidEugenolZingeroneGuaiacolBHABHTProtocatechuic acidCaffeic acidGentisic acidGallic acid

1.943.703.70

46.90

0.0020.020.050.170.362.333.73.74

4.174.207.149.111.112.5

1.030.540.54

0.500.30

2709844

11.85.60.860.540.540.50.380.380.280.220.180.16

0.971.851.85

23.33

<1<1<1<1<1

1.161.851.85

22.632.633.64.545.66.25

Brand-Williams W., Cuvelier M.E., Berset C.: Use of free radical method to evaluate antioxidant activity. LWT –Food Sci. Technol., 1995, 28, 25-30.

Antiradical activity of selected phenolic acids against DPPH radical

Phenolic acid EC50(µmol/assay)

ARP Relative ARP(%)

GallicGentisicSyringicCaffeicProtocatechuicSinapicFerulicIsoferulicVanillicp-Coumarico-Coumaricm-CoumaricSalicylicp-Hydroxybenzoic

0.02370.02920.04270.04780.05740.07240.09275.6814.3766.29130.05> 300> 800> 800

42.1934.2523.4220.9217.4213.8110.790.1760.0690.0150.008

<0.003<0.001<0.001

10081.8155.5149.5841.3132.7325.570.420.160.040.02

<0.007<0.002<0.002

KaramaćM., Kosińska A., Pegg R.B.: Comparison of radical-scavenging activities for selected phenolic acids. Pol. J. Food Nutr. Sci., 2005, 55, 165-170.

Stoichiometry n = 1

DPPH• + AH → DPPH2 + A•

A• + A• → A-A

--------------------------------------------------------

2 DPPH• + 2AH → 2 DPPH2 + A-A

Stoichiometry n = 2

DPPH• + AH → DPPH2 + A•

DPPH• + A• → DPPH-A

-----------------------------------------------------

2 DPPH• + AH → DPPH2 + DPPH-A

Stoichiometry n = 3

DPPH• + AH → DPPH2 + A•

DPPH• + A• → DPPH-A

DPPH• + DPPH-A → DPPH2-Q

--------------------------------------------------------------

3 DPPH• + AH → DPPH2 + DPPH-Q

DPPH• + AH → DPPH2 + A•

DPPH• + A• → DPPH-A (P1)

DPPH• + P1 → DPPH2 + P2

DPPH• + P2 → DPPH2 + P3

----------------------------------------------

4 DPPH• + AH → 3 DPPH2 + P3

Stoichiometry n = 4

Vessela D. Kancheva: Natural antioxidants for the preservation of oils – kinetic studies and mechanisms . Eur. J. Lipid Sci. Technol. – in press.

Antiradical activity against DPPH radical

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.004 0.008 0.012 0.016 0.02

Content (mg/assay)

Abs

orba

nce

at 5

17 n

m

Extract

Fraction II0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.02 0.04 0.06 0.08 0.1

Content (mg/assay)

Abs

orba

nce

at 5

17 n

m

Fraction I