Bulletin of the Chemists and Technologists 2017

of Bosnia and Herzegovina

49
Print ISSN: 0367-4444 UDC: __________________________
Online ISSN: 2232-7266 Original Scientific Paper
35-38

Evaluation of the antioxidant activity of ferulic, homovanillic and

vanillic acids using the Briggs-Rauscher oscillating reaction method
Aljović, I., Gojak-Salimović, S.*
University of Sarajevo, Faculty of Science, Department of Chemistry, Zmaja od Bosne 33-35, 71000 Sarajevo, B&H

Article info Abstract: In this study, the antioxidant activity of aqueous and ethanolic solutions of ferulic,
Received: 06/12/2017
Accepted: 19/12/2017 homovanillic and vanillic acids were evaluated using Briggs-Rauscher oscillating reaction
method. This method is based on the inhibitory effects of antioxidants on the oscillations of the
Keywords: Briggs-Rauscher reaction mixture. The inhibitory effect consists of an immediate quenching of
Antioxidants
Phenolic acids
oscillations, an inhibition time that depends on the amount and type of the antioxidant added,
Briggs-Rauscher reaction and a subsequent regeneration of oscillations. Flow oscillations in the Briggs-Rauscher reaction
Inhibition time
Oscillations mixture were followed potentiometrically. In all samples, the inhibition times increased with
increasing concentration and linearity was found in a wide concentration range of phenolic acid
added. The antioxidant activity decreased in following order: ferulic acid > homovanillic acid
> vanillic acid. It was also investigated the antioxidant activity for two-component and three-
*Corresponding author:
E-mail: [email protected] component mixtures of aqueous solutions examined phenolic acids.
Phone: 00-387-33-279-907
Fax: 00-387-33-649-359

INTRODUCTION Homovanillic acid (HVA) is a major catecholamine

metabolite and is produced by the consecutive action of
Phenolic acids are widely distributed in the plant world and monoamine oxidase and catechol-O-methyl transferase on
have prominent antioxidant activity. They are ubiquitous dopamine. It is used as reagent for the fluorimetric
in all plant organs and are therefore an integral part of the determination of glucose oxidase and other oxidative
human diet (Ota et al., 2011). It is estimated that humans enzymes (Curzon et al., 1970). In psychiatry and
consumed from 25 mg to 1 g phenolic acids a day neuroscience, brain and cerebrospinal fluid levels of
depending on diet (Robbins, 2003). According to the basic homovanillic acid are measured as a marker of metabolic
structure, phenolic acids are divided into two classes: stress caused by 2-deoxy-D-glucose (Marcelis et al.,
derivates of benzoic acid such as vanillic acid and derivates 2006). Homovanillic acid presence supports a diagnosis of
of cinnamic acid as ferulic acid (Rice-Evans et al., 1996). neuroblastomas and malignant pheochromocytoma
Ferulic acid (FA) is a ubiquitous plant constituent that (Candido et al., 2002).
occurs primarily in seeds. Ferulic acid is found in wheat, Vanillic acid (VA) is an oxidized form of vanillin produced
maize, rye, barley, oats, spinach, sugar beet, and water during the conversion of vanillin to ferulic acid. The
chestnuts, generally esterified, and rarely as free form, highest quantity of vanillic acid in plants has been found in
such as in barley. Due to its phenolic nucleus and an the roots of Angelica sinensis. Vanillic acid is used as a
extended side chain conjugation, it readily forms a flavoring agent. Various studies have provided evidence of
resonance stabilized phenoxy radical that accounts for its the effectiveness of vanillic acid in the management of
potent antioxidant activity. Ferulic acid has been shown to immune or inflammatory responses (Kim et al., 2010).
be protective against oxidative stress in vitro. It is absorbed Vanillic acid showed antigenic and genotoxic effects
and excreted by humans. The low lipophilicity impairs its depending on the dose on human lymphocytes (Bival
in vivo efficiency, bioavailability and stability (Fraga, Štefan, 2015).
2010).
36 Aljović & Gojak-Salimović

Many analytical methods have been developed for the Preparation of the solutions of phenolic acids
determination the activity of antioxidants (Shalaby and Solutions of acids where in the concentration range of 8-
Shanab, 2013). The Briggs-Rauscher oscillating reaction 40 mg/L, 8-80 mg/L and 160-320 mg/L for ferulic,
method is very useful and applicable method as a test for homovanillic and vanillic acids, respectively; all of
the activity of antioxidants because it is works at pH about phenolic acids are ethanol and water soluble.
2 and partially mimics the physiological conditions similar
to those of the fluids in the human stomach. When Apparatus
antioxidants are added to an active oscillating Briggs- The oscillating behavior of the Briggs-Rauscher reaction
Rauscher reaction mixture, there is an immediate was followed visually and potentiometrically by recording
quenching of the oscillations for a certain time, the potential of the reaction mixture using a platinum wire
denominated as inhibition time. An inhibition time is electrode and Ag/AgCl/KCl(sat) reference electrode (+197
linearly dependant on the concentration of the antioxidant mV vs. SHE). The electrodes were connected to a pH
added to the reaction mixture (Cervellati et al., 2001; multimeter (Phywe, Model 13702.93). The accuracy of the
Cervellati et al., 2002). The efficiency of the multimeter was ±1 mV. All measurements were conducted
corresponding antioxidant is expressed as inhibition time at temperature (25±0.5°C) using a suitable thermostating
before oscillations restart. A better antioxidant as well as system. The reaction mixture was stirred by a magnetic
higher concentration of it leads to a prolonged inhibiton stirrer (600 rpm).
time. Relative antioxidant activity with respect to a
substance chosen as standard can then be determined on The Briggs-Rauscher oscillating reaction method for
the basis of inhibition time (Hönor and Cervellati, 2002; the determination of antioxidant activity
Hönor et al., 2002). The Briggs-Rauscher oscillating The Briggs-Rauscher reaction mixture were prepared by
reaction method can give useful in vitro information on the mixing the appropriate amounts of stock solutions of
antioxidant activity at low pH values and has many reagents of A, B and C. For each measurement 10 mL of
advantages. Milos and Makota (2012) demonstrated some each solution A and B were mixed into the double-wall
new possibilities of this method for determine the thermostated beaker equipped with a magnetic stir bar and
synergistic and antagonistic effects in mixture of placed on a stirring plate. The 10 mL of solution C was
compounds, which often poses a problem when using used to initiate the oscillations. After the third oscillation,
standard methods. 1 mL solution of phenolic acid at corresponding
Oscillatory behaviour in the Briggs-Rauscher reaction concentration was added to 30 mL of an active Briggs-
system can be easily followed potenciometrically using a Rauscher reaction mixture. The inhibition times were then
bright platinum electrode and a suitable reference electrode measured from the recordings. Typical potentiometric
under thermostated and stirred conditions. recordings for a non-inhibited and an inhibited Briggs-
The aim of the present study is to evaluate the antioxidant Rauscher reaction mixture were shown in our previous
activity of aqueous and ethanolic solutions of ferulic acid, works (Dacić and Gojak-Salimović, 2016; Džomba and
homovanillic acid and vanillic acid using the Briggs- Gojak-Salimović, 2017).
Rauscher oscillating reaction method. The inhibition time defined as the time elapsed between
the end addition of the phenolic acid and first regenerated
EXPERIMENTAL oscillation. The addition of 1 mL of ethanol or water,
without phenolic acid does not interrupt the oscillations.
Reagents The pH value of a non-inhibited-Briggs-Raucher reaction
All used reagents were of analytical grade and were used mixture was 1.56.
without further purification. Potassium iodate, sulfuric
acid, hydrogen peroxide and ethanol were obtained from RESULTS AND DISCUSSION
Semikem (Sarajevo, BiH), malonic acid, manganese(II)
sulfate monohydrate and starch were obtained from Merck Our previous works showed the ability of some selected
(Darmastadt, Germany), ferulic acid, homovanillic acid phenolic acids (gallic, caffeic, chlorogenic, rosmarinic and
and vanillic acid were obtained from Sigma (St. Louis, p-coumaric acids) to inhibited oscillations of the Briggs-
USA). Rauscher reaction mixture (Džomba and Gojak-Salimović,
2017). In this study, the inhibitory effect of various
Preparation of the solutions for the Briggs-Rauscher concentrations of the ethanolic and aqueous solutions of
reaction ferulic acid, homovanillic acid and vanillic acid on the
Three colourless stock solutions were prepared daily. oscillatory system Briggs-Rauscher reaction were evaluted
Solution A: 43 g potassium iodate and 4.5 mL 96% sulfuric at 25°C.
acid were dissolved in distilled water and diluted to 1 L; Addition of a solution of ferulic, homovanillic or vanillic
Solution B: 15.6 g malonic acid, 3.4 g manganese(II) acids in the active Briggs-Rauscher reaction mixture
sulfate monohydrate and 3.0 g starch were dissolved in causes an immediate effect of quenching of oscillations
distilled water and diluted to 1 L; Solution C: 500 mL of and after period inhibition start again because the reaction
30% hydrogen peroxide was diluted to 1 L. produces hydroperoxyl radicals that are quenched by
Mixture of equal volumes of stock solutions (A, B and C) antioxidants. The total antioxidant activity is measured as
represents the Briggs-Rauscher reaction mixture, which is an inhibition time of Briggs-Rauscher oscillating reaction.
used for evaluation of antioxidant activity (Džomba and The obtained values of inhibition time of aqueous and
Gojak-Salimović, 2017). ethanolic solutions of ferulic, homovanillic and vanillic
acids are presented in Table 1.
Bulletin of the Chemists and Technologists of Bosnia and Herzegovina 2017, 49, 35-38 37

Table 1: Inhibitory effects of individual ferulic acid, homovanillic The antioxidant activity decreased in the following order:
acid and vanillic acid at different concentrations on total antioxidant
activity measured
ferulic acid > homovanillic acid > vanillic acid, similar to
the results previously shown in studies of other authors
Ferulic acid using Briggs-Rauscher oscillating reaction, TEAC and
Concentration tinhib (s)
(mg/L)
DPPH methods (Cervellati et al., 2001; Cervellati et al.,
Water Ethanol
8 20 16 2002; Rice-Evans et al., 1996; Karamać et al., 2005).
16 177 133 The synergism between the antioxidants in the mixture
24 319 251 makes the antioxidant activity not only dependent on the
40 550 483 concentration, but also on the structure and the interaction
Homovanillic acid
Concentration tinhib (s)
between the antioxidants. In order to evaluate the impact
(mg/L) Water Ethanol of interactions among examined phenolic acids on their
8 100 30 antioxidant activity, the inhibition time of ferulic acid,
16 163 135 homovanillic acid and vanillic acid was compared with
32 235 290
values obtained by mixing them in different combinations.
48 406 360
64 800 510 The results obtained experimentally for nine two-
80 1144 670 component mixtures and one three-component mixture of
Vanillic acid aqueous solutions of phenolic acids were compared with
Concentration tinhib (s) the theoretical values by adding up the effects of two or
(mg/L) Water Ethanol
160 20 64
three individual phenolic acids analyzed separately. The
200 314 410 obtained results are presented in Table 3.
240 660 640
280 829 710 Table 3: Inhibitory effects of ferulic acid (16 mg/L), homovanillic acid
320 1250 950 (32 mg/L) and vanillic acid (200 mg/L) with various combinations on
total antioxidant activity measured
The results collected in Table 1 shown that the majority of Combination of compounds tinhib (s)
the ethanolic solutions of phenolic acids have much less homovanillic acid + vanillic acid (50%:50%) 498 (275)
antioxidant activity than the corresponding aqueous homovanillic acid + ferulic acid (50%:50%) 101 (206)
solutions. vanillic acid + ferulic acid (50%:50%) 300 (246)
In all samples, the inhibition times increased with homovanillic acid + vanillic acid (75%:25%) 560 (255)
increasing concentration and linearity was found in a wide homovanillic acid + ferulic acid (75%:25%) 504 (220)
concentration range of phenolic acid added. Therefore, the vanillic acid + ferulic acid (75%:25%) 1502 (279)
requirement for the possibility of using Briggs-Rauscher vanillic acid + homovanillic acid (75%:25%) 1335 (294)
reaction as a test for antioxidant activity was fulfilled. The ferulic acid + vanillic acid (75%:25%) 1250 (255)
linear behavior of the inhibition time versus concentration ferulic acid + homovanillic acid (75%:25) 580 (192)
of ethanolic solutions of ferulic, homovanillic and vanillic ferulic acid + homovanillic acid + vanillic acid
320 (242)
acids added shown in Figure 1. (33.3%:33.3%:33.3%)
*The values in parentheses are the summations of antioxidant activities of individual
compounds at corresponding concentrations
1000 Ferulic acid
Homovanillic acid Our results indicate that all combinations of phenolic acids
800
demonstrated some level of discrepancy in antioxidant
Inhibition time, s

Vanillic acid
600 activity when compared to individual values of their
400
constituents. Eight combinations of two and one
combination of three phenolic acids showed more or less
200
synergistic effect. The mixture of homovanillic acid and
0 ferulic acid (50%:50%) showed a loss of antioxidant
0 50 100 150 200 250 300 350 activity when compared to their individual values.
Stock concentration, mg/L

CONCLUSIONS
Figure 1: Straight lines of inhibition time versus concentration for the
ethanolic solutions of phenolic acids studied
The Briggs-Rauscher oscillating reaction is suitable as an
As shown in Figure 1, the slopes of the straight lines are analytical method to determine antioxidant activity of the
different. The parameters of the straight lines and R- ethanolic and aqueous solutions of ferulic, homovanillic
squared values are presented in Table 2. and vanillic acids. In all samples, the inhibition time of
Briggs-Rauscher oscillating reaction increased with
Table 2: Parameters of straight-lines equations (tinhib = m(antioxidant) + increasing concentration and linearity was found in a wide
q) and R2 values concentration range of phenolic acid added. The ethanolic
Water solution m (s L/mg ) q (s) R2 solutions of examined phenolic acids had much less
ferulic acid 16.42 −94.77 0.994 antioxidant activity than the corresponding aqueous
homovanillic acid 14.23 113.7 0.924 solutions. The antioxidant activity decreased in following
vanillic acid 7.482 −1170 0.987 order: ferulic acid > homovanillic acid > vanillic acid. All
Ethanol solution m (s L/mg) q (s) R2
combinations of phenolic acids demonstrated some level
ferulic acid 14.59 −100.4 1.00
homovanillic acid 8.409 −15.08 0.989 of discrepancy in antioxidant activity when compared to
vanillic acid 5.195 −692.6 0.955 individual values of their constituents.
38 Aljović & Gojak-Salimović

REFERENCES German white wines using a novel method. European

Food Research and Technology, 214(4), 356-360.
Bival Štefan, M. (2015). Biološki učinci fenolnih kiselina Höner, K., Cervellati, R. (2002). Measurements of the
iz odabranih vrsta porodice Lamiaceae. Doktorski rad, antioxidant capacity of fruits and vegetables using the
Farmaceutsko-biokemijski fakultet, Sveučilište u BR reaction method. European Food Research and
Zagrebu. Technolology, 215(5), 437-442.
Candido, M., Billaud, E., Chauffert, M., Cottet-Ernard, Karamać, M., Kosińska, A., Pegg, R.B. (2005).
J.M., Desmoulin, D., Garnier, J.P., Greffe, J., Hirth, C., Comparison of radical-scavenging activities for
Jacob, N., Millot, F., Nignan, A., Patricot, M.C., selected phenolic acids. Polish Journal of Food and
Peyrin, L., Plouin, P.F. (2002). Biochemical diagnosis Nutrition Sciences, 14/55(2), 165-170.
of pheochromocytoma and neuroblastomas. Annales de Kim, S.J., Kim, M.C., Um, J.U., Hong, S.H. (2010). The
Biologie Clinigue, 60(1), 15-36. Beneficial Effect of Vanillic Acid on Ulcerative
Cervellati, R., Höner, K., Furrow, S.D., Neddens, C., Colitis. Molecules, 15, 7208-7217.
Costa, S. (2001). The Briggs-Rauscher Reaction as a Marcelis, M., Suckling. J., Hofman. P., Woodruff, P.,
Test to Measure the Activity of Antioxidants. Helvetica Bullmore, E., van Os, J. (2006). Evidence that brain
Chimica Acta, 84(12), 3533-3547. tissue volumes are associated with HVA reactivity to
Cervellati, R., Höner, K., Furrow, S.D., Mazzanti, F. metabolic stress in schizophrenia. Schizophrenia
(2002). Inhibitory Effects by Antioxidants on the Research, 86 (1-3), 45-53.
Oscillations of the Briggs-Rauscher Reaction in Mixed Milos, M., Makota, D. (2012). Investigation of antioxidant
EtOH/H2O Medium. Helvetica Chimica Acta, 85(8), synergisms and antagonisms among thymol, carvacrol,
2523-2537. thymoquinone and p-cymene in a model system using
Curzon, G., Godwin-Austen, R.B., Tomlinson, E.B., the Briggs-Rauscher oscillating reaction. Food
Kantamaneni, B.D. (1970). The cerebrospinal fluid Chemistry, 131(1), 296-299.
homovanillic acid concentration in patients with Ota, A., Abramović, H., Abram, V., Poklar-Ulrih, N.
Parkinsonism treated with L-dopa. Journal of (2011). Interactions of p-coumaric, caffeic and ferulic
Neurology, Neurosurgery and Psychiatry, 33, 1-6. acids and their styrenes with model lipid membranes.
Džomba, E., Gojak-Salimović, S. (2017). Inhibitory Food Chemistry, 24(4), 1256-1261.
effects of selected phenolic acids on the oscillations of Rice-Evans, C.A., Miller, N.J., Paganga, G. (1996).
the Briggs-Rauscher reaction. Bulletin of the Chemists Structure-antioxidant activity relationship of
and Technologists of Bosnia and Herzegovina, 48, 9- flavonoids and phenolic acid. Free Radical Biology
14. and Medicine, 20(7), 933-956.
Dacić, M., Gojak-Salimović, S. (2016). The effect of Robbins, R.J. (2003). Phenolic Acids in Food: An
chlorogenic acid on the Briggs-Rauscher oscillating Overview of Analytical Methodology. Journal of
reaction. Bulletin of the Chemists and Technologists of Agricultural and Food Chemistry, 51(10), 2866-2887.
Bosnia and Herzegovina, 46, 51-54. Shalaby, E.A., Shanab, S.M.M. (2013). Antioxidant
Fraga, C.G. (2010). Plant Phenolics and Human Health, compounds, assays of determination and mode of
Biochemistry, Nutrition and Pharmacology, Jonh action. African Journal of Pharmacy and
Wiley and Sons, Inc. Pharmacology, 7(10), 528-539.
Höner, K., Cervellati, R., Neddens, C. (2002).
Measurements of the in vitro antioxidant activity of

Summary/Sažetak
U ovom radu ispitivana je antioksidacijska aktivnost vodenih i etanolnih rastvora ferulinske, homovanilinske i vanilinske
kiseline primjenom metode Briggs-Rauscher oscilirajuće reakcije. Ova metoda se bazira na inhibitorskom efektu
antioksidanasa na oscilacije Briggs-Rauscher reakcijske smjese. Inhibitorski efekat se sastoji od trenutnog gašenja oscilacija,
vremena inhibicije koje zavisi od količine i vrste dodanog antioksidansa, i ponovne regeneracije oscilacija. Tok oscilacija
Briggs-Rauscher reakcijske smjese praćen je potenciometrijskom metodom. Sa porastom koncentracije fenolskih kiselina
linearno se povećavalo vrijeme inhibicije u širokom opsegu koncentracija. Antioksidacijska aktivnost je opadala prema
sljedećem nizu: ferulinska kiselina > homovanilinska kiselina > vanilinska kiselina. Također je ispitana i antioksidacijska
aktivnost dvokomponentnih i trokomponentnih smjesa vodenih rastvora fenolskih kiselina.