Bulletin of the Chemists and Technologists 2017

of Bosnia and Herzegovina

48
Print ISSN: 0367-4444 UDC: __________________________
Online ISSN: 2232-7266
9-14
Original Scientific Paper

Inhibitory effects of selected phenolic acids on the oscillations of

the Briggs-Rauscher reaction

Džomba, E., Gojak-Salimović, S.*

University of Sarajevo, Faculty of Science, Department of Chemistry, Zmaja od Bosne 33-35, 71000 Sarajevo, B&H

Abstract: Phenolic acids are secondary metabolites of aromatic plant that possess prominent
Article info
Received: 13/04/2017 antioxidant activity. When an antioxidant is added to an active oscillating Briggs-Rauscher
Accepted: 14/06/2017
reaction mixture, there is an immediate cessation of the oscillations, an inhibition time that
linearly depends on the concentration of the antioxidant added, and a subsequent regeneration
Keywords:
Briggs-Rauscher reaction of oscillations. In this study, the effects of concentration of the ethanol solutions of selected
Phenolic acids
Inhibition time phenolic acids (gallic, caffeic, chlorogenic, rosmarinic, p-coumaric and m-coumaric acids) on
Oscillation the oscillatory system Briggs-Rauscher reaction were investigated. The reaction was
performed in a constantly stirred reactor, with accurately defined concentrations of reactants,
at constant temperature of 25°C. Flow oscillations in the Briggs-Rauscher reaction mixture
were monitored as a change in potential between the platinum electrode and silver/silver
chloride reference electrode. Relative antioxidant activities of phenolic acids were determined
in three ways on the basis of inhibition times. The obtained results showed that the gallic and
p-coumaric acids have much less antioxidant activity than the caffeic, chlorogenic and
*Corresponding author: rosmarinic acids. The ability to inhibit oscillations of the Briggs-Rauscher reaction mixture is
E-mail: sgojak@pmf.unsa.ba
Phone: 00-387-33-279-907 not showed for m-coumaric acid.
Fax: 00-387-33-649-359

INTRODUCTION

Phenolic acids are widespread plant secondary Although the role of phenolic acids as secondary
metabolites. They belong to the subclass of polyphenols metabolites in plants is not fully clarified, it is considered
with more than 8000 of natural compoundsthat possess to participate in many processes such as nutrient, protein
one common structural feature, a phenol (an aromatic ring synthesis, enzyme activity, photosynthesis and others.
bearing at least one hydroxyl group). According to the Phenolic acids have prominent antioxidant activity.
basic structure they are divided into hydroxybenzoic and Hydroxycinnamic acids has better antioxidant properties
hydroxycinnamic acids. than most hydroxybenzoic acids (Rice-Evans et al.,
Caffeic, vanillic, ferulic and p-coumaric acids are found 1996;Robards et al., 1997).
in almost all plants. Other acids are found in selected Gallic acid and its derivatives have potential for
plants or foods (Robbins, 2003). The hydroxycinnamic combating oxidative damages, cancer manifestations and
acid class, which includes p-coumaric, caffeic and ferulic microbial infestations. Large number of research studies
acids, occur most frequently as simple esters with are available to show its ability for the treatment of
hydroxy carboxylic acids or D-glucose. In contrast, the diabetes, ischemic heart diseases, ulcer and other ailments
hydroxybenzoic acid class, such as p-hydroxybenzoic, (Nayeemet al., 2016).
gallic and ellagic acids, is present mainly in the form of
glucosides (Ota et al., 2011).
10 Gojak-Salimović et al.

Caffeic acid, one of the most prominent naturally EXPERIMENTAL

occurring cinnamic acids, is known to selectively block
the biosynthesis of leukotrienes, components involved in Reagents
immunoregulation diseases, asthma and allergic reactions All used reagents were of analytical grade. Potassium
(Robbins, 2003). iodate, sulfuric acid, hydrogen peroxide and ethanol were
Chlorogenic acid is an ester formed between caffeic acid obtained from Semikem (Sarajevo, BiH), malonic acid,
and quinic acid, and is one of major polyphenol manganese(II) sulfate monohydrate and starch were
compounds found in numerous plant species, including obtained from Merck (Darmastadt, Germany),
coffee beans, apples, and blueberries. Chlorogenic acid chlorogenic acid was obtained from Acros Organics,
protects cells from oxidative stress induced by UVB (Geel, Belgium), gallic acid, caffeic acid, rosmarinic acid,
radiation (Cha et al., 2014). Moreover, chlorogenic acid p-coumaric acid and m-coumaric acid were obtained from
protects mesenchymal stem cells against oxidative stress Sigma (St. Louis, USA).
(Li et al., 2012).
Rosmarinic acid has antioxidant and anti-inflammatory Preparation of the solutions for the Briggs-Rauscher
effects and is used for the treatment of asthma and reaction
reactive airway diseases, allergic disorders such as For the Briggs-Rauscher oscillating reaction (Marković
allergic rhinitis, otitis media, chemical sensitivity and and Talić, 2013; Dacić and Gojak-Salimović, 2016) the
multiple allergen reactivity (Stansbury, 2014). following mixture should be prepared: 0.067 mol/L
The p-coumaric acid has beneficial effects on human potassium iodate, 1.5 mol/L hydrogen peroxide, 0.0267
health through their prevention of degenerative mol/L sulfuric acid, 0.050 mol/L malonic acid, 0.0067
pathologies, such as cardiovascular disease and cancer mol/L manganese(II) sulfate monohydrate and 0.01%
(Ota et al., 2011). fresh starch. In our study, for achieving the Briggs-
The m-coumaric acid is a polyphenol metabolite from Rauscher oscillating reaction, three stock solutions were
caffeic acid, formed by the gut microflora and the amount prepared daily. Solution A: 43 g potassium iodate and 4.5
in human biofluids is diet-dependant (Konish and mL 96% sulfuric acid were dissolved in distilled water
Kobayashi, 2004; Mennen et al., 2016). and diluted to 1 L; Solution B: 15.6 g malonic acid, 3.4 g
All the methods for measurements of the antioxidant manganese(II) sulfate monohydrate and 3 g starch were
activity are based on the generation of free radicals in a dissolved in distilled water and diluted to 1 L; Solution
reaction mixture and the effects of added antioxidants on C: 500 mL of 30% hydrogen peroxide was diluted to 1 L.
some properties of the radical or of the mixture:
absorbance, quenching of chemiluminescence, electric Preparation of the solutions of selected phenolic acids
potential, ect. These properties change depending on the In the concentration range used in this work all of
amount of antioxidants added with respect to those of a selected phenolic acids are ethanol soluble.
reference mixture (Cervellati et al., 2002; Shalaby and
Shanab, 2013). TheBriggs-Rauscher oscillating reaction Apparatus
method is relatively new and inexpensive method for Oscillations of the Briggs-Rauscher reaction were
meausuring antioxidant activity. This method is based on monitored potentiometrically by recording the potential
the inhibitory effects by antioxidants on the oscillations of the reaction mixture using a platinum electrode and
of the Briggs-Rauscher reaction mixture. When Ag/AgCl/KCl(sat) reference electrode (+197 mV vs. SHE).
antioxidantsare added to an active oscillating Briggs- The electrodes were connected to a pH multimeter
Rauscher reaction mixture, some of which cause an (Phywe, Model 13702.93). The accuracy of the
immediate cessation of the oscillations, an inhibition multimeter was 1 mV. All measurements were
timethat linearly depends on the concentration of the conducted at constant temperature (25±0.5°C) using a
antioxidant added in a wide range of concentration, and a thermostating system. The reaction mixture was stirred by
subsequent regeneration of the oscillations (Cervellati et a magnetic stirrer (600 rpm).
al., 2001; Cervellati et al., 2002; Furrow et al., 2004).
The inhibition time (tinhib)is defined as the time elapsed Procedure
between the end of the addition of the antioxidant and the The Briggs-Rauscher reaction mixture was prepared by
first regenerated oscillation. Relative antioxidant activity mixing the appropriate amounts of stock solutions of
with respect to a substance chosen as standard can then be reagents. For each measurement 10 mL of each solution
determined on the basis of inhibition time (Hönor and A and B were mixed into the double-wall thermostated
Cervellati, 2002; Hönor et al., 2002). beaker equipped with a magnetic stir bar and placed on a
In this study, the inhibitory effects of concentration of the stirring plate. The 10 mL of solution C was used to
ethanol solutions of selected phenolic acids (gallic, initiate the oscillations. After the third oscillation, 1 mL
caffeic, chlorogenic, rosmarinic, p-coumaric and m- of ethanol solution of phenolic acid at corresponding
coumaric acids) on the oscillatory system Briggs- concentration was added to an active Briggs-Rauscher
Rauscher reaction, which consisted of hydrogen peroxide, reaction mixture. Typical potentiometric recordings for a
malonic acid, manganese(II) sulfate monohydrate, non-inhibited and an inhibited Briggs-Rauscher reaction
potassium iodate, sulfuric acid and starch as indicator, mixture are shown in Figure 1 and Figure 2. The
were investigated. inhibition times were then measured from the recordings.
Bulletin of the Chemists and Technologists of Bosnia and Herzegovina 2017, 48, 9-14 11

The addition of 1 mL of ethanol, without phenolic acid 2500

does not interrupt the oscillations. All samples were run Gallic acid
2000 Rosmarinic acid
at least in duplicate and results were expressed as mean

Inhibition time, s
Caffeic acid
values. 1500
p-Coumaric acid
1000 Chlorogenic acid

500

0
0 100 200 300 400

Concentration, mol/L

Figure 3: Straight lines of inhibition time versus concentration for

the phenolic acids studied

Figure 1: Recording of the potential versus time of non-inhibited As shown in Figure 3, the slopes of the straight lines are
Briggs-Rauscher reaction different, so the calculation of the relative antioxidant
activity will depend on the substance chosen as standard
and the concentration of the sample. The parameters of
the straight lines and R-squared values are reported in
Table 1.

Table 1: Parameters of straight-lines equations (tinhib= m(antioxidant) +

q) and R-squared values

Antioxidant m ( mol/L)-1 s q (s) R2

Caffeic acid 326.0 1055 0.978
Chlorogenic acid 228.7 419.4 0.955
Figure 2: Recording of the potential versus time when 1 mL of a Rosmarinic acid 230.8 480.1 0.970
solution of gallic acid (100 mg/L) was added to 30 mL of an active
Briggs-Rauscher reaction mixture after the third oscillation Gallic acid 1.156 148.6 0.995
p-Coumaric acid 0.551 4.125 0.997

RESULTS AND DISCUSSION The relative antioxidant activity were calculated as

relative activity with respect to concentrations (rac),
Our previous work showed the ability of chlorogenic acid relative activity with respect to slopes (ras) and relative
to inhibited oscillations of the Briggs-Rauscher reaction activity with respect to inhibition times (rat) (Cervellati et
mixture at room temperature (Dacić and Gojak- al., 2001). Caffeic acid was chosen as standard.
Salimović, 2016). In this study, the effects of various Relative antioxidant activity with respect to
concentration of the ethanol solutions of selected phenolic concentrations (rac) is the ration between concentrations
acids (gallic, caffeic, chlorogenic, rosmarinic, p-coumaric of the chosen standard and samples that give the same
and m-coumaric acids) on the oscillatory system Briggs- inhibition time:
Rauscher reaction were evaluated at 25 C.
In all samples except m-coumaric acid, the inhibition rac = [std]/[smp]
times increased with increased concentration, and
linearity was found in a wide concentration range of The concentration of standard that should give the same
phenolic acid added (Figure 3). Below a certain inhibition time of the sample was calculated from the
concentration of phenolic acid added (different for each straight-line equation of the chosen standard.
phenolic acid), the behavior deviates from linearity. At Relative antioxidant activity with respect to slopes (ras)
low concentrations of phenolic acids added, the inhibition is the ratio between the slope of the straight line of the
times become too low to be measured as well for some sample and that of the standard:
other antioxidants (Cervellati et al., 2000). There is a
threshold under which inhibition time cannot be detected ras = slope(smp)/slope(std)
and we believe that the straight-lines curve toward zero
under these lower limits. At high concentrations of Relative antioxidant activity with respect to inhibition
phenolic acids the amplitudes of resumed oscillations times (rat) is the ratio between the inhibition time of the
becomes too low, until up to a given concentration sample and that of the standard at the same concentration:
(different for each phenolic acid) oscillations do not
restart. rat = tinhib(smp)/tinhib(std)
12 Gojak-Salimović et al.

The chosen concentration must be specified together with The oscillatory time (the duration of the oscillatory
the rat values and must be in the linear concentration regime) and numbers of oscillations depends on the
range of the standard and of all the examined substances. concentration of studied phenolic acids. Variation of
The obtained rac, ras and rat values are reported in Table oscillations parameters in the Briggs-Rauscher reaction
2. mixture with concentration of m-coumaric acid are
reported in Table 3.
Table 2: Relative antioxidant activities with respect to concentrations,
slopes and inhibition times Table 3: Variation of oscillations parameters in the Briggs-Rauscher
reaction mixture with concentration of m-coumaric acid
Concentration
Antioxidant rac ras rat
( mol/L) Concentration Oscillations
Caffeic acid 3.85 1 1 1 ( mol/L) Duration (s) Number
Chlorogenic acid 2.71 1.420 0.700 0.872 0 220 16
Rosmarinic acid 2.95 1.305 0.707 0.850 24.6 255 22
Gallic acid 44.5 0.086 0.035 0.085 49.1 420 31
p-Coumaric acid 355 0.011 0.017 0.005 73.7 460 40
122.8 490 53
The rac values were calculated at an inhibition time of 245.6 420 52
200 s. On the basis of rac values, the order of antioxidant
358.6 285 36
activity of the studied phenolic acids is: chlorogenic acid
491.2 210 28
rosmarinic acid caffeic acid gallic acid p-
coumaric acid. When possible, it is convenient to
calculate a mean value of rac in the linear concentration The largest number of oscillations (60) caused addition 1
range of the sample and the standard. The value (rac)m is mL of p-coumaric acid concentration of 500 mg/L to the
more significant than the rac value calculated at only one 30 mL active Briggs-Rauscher reaction mixture. The
inhibition time (Cervellatiet al., 2002). lowest number of oscillations (23) caused addition 1 mL
On the basis of ras values, the order of antioxidant of gallic acid concentration of 300 mg/L to the 30 mL
activity of the studied phenolic acids is: caffeic acid active Briggs-Rauscher reaction mixture.
rosmarinic acid chlorogenic acid gallic acid p- The ranking order of the antioxidant activity of
coumaric acid. This method of relative activity antioxidants components of secondary plant products
calculation is useful for comparison of the effect of differed from assay to assay. The Briggs-Rauscher
changes in sample concentration with the effect of reaction method can give useful in vitro information on
changes in the reference concentration, within the linear the antioxidant activity at low pH values and has many
ranges (Cervellati et al., 2001). advantages. Milos and Makota (2012) demonstrated some
The rat values were calculated at concentration 9 mol/L. new possibilities of this method for determine the
On the basis of rat values, the order of antioxidant synergistic or antagonistic effects in mixture of
compounds, which often poses a problem when using
activity of the studied phenolic acids is: caffeic acid
traditional methods.
chlorogenic acid rosmarinic acid gallic acid p-
coumaric acid. This method of relative activity
CONCLUSIONS
calculation has the same limitations asthe rac. The
advantage is that the activity is reffered to a given
The Briggs-Rauscher oscillating reaction is suitable as an
specified concentration (Cervellati et al., 2001).
analytical method to determine relative activity of
The ability to inhibit oscillations of the Briggs-Rauscher
antioxidants. In this study, the effects of concentration of
reaction mixture is not showed for m-coumaric acid.
the ethanol solutions of selected phenolic acids (gallic,
Recording of the potential versus time when 1 mL of a
caffeic, chlorogenic, rosmarinic, p-coumaric and m-
solution of m-coumaric acid (2500 mg/L) was added to 30
coumaric acids) on the oscillatory system Briggs-
mL of active Briggs-Rauscher reaction mixture after the
Rauscher reaction were investigated. In all samples
third oscillation are reported in Figure 4.
except m-coumaric acid, the inhibition time increased
with increased concentration, and linearity was found in a
wide concentration range of phenolic acid added. Relative
antioxidant activities with respect to concentrations,
slopes and inhibition times were calculated. The obtained
results showed that the gallic acid and p-coumaric acid
have much less antioxidant activity than the caffeic acid,
chlorogenic acid and rosmarinic acid. The ability to
inhibit oscillations of the Briggs-Rauscher reaction
mixture is not showed for m-coumaric acid. Our future
investigation will be focused on the antioxidant
Figure 4: The effect of the m-coumaric acid on the active Briggs- synergisms and antagonisms among selected phenolic
Rauscher reaction mixture acids.
Bulletin of the Chemists and Technologists of Bosnia and Herzegovina 2017, 48, 9-14 13

REFERENCES Li, S., Bian., H., Liu, Z., Wang, Y., Dai, J., He, W., Liao,
X., Liu, R., Luo, J. (2012). Chlorogenic acid protects
Cervellati, R., Crespi-Perellino, N., Furrow, S.D., MSCs against oxidative stress by altering FOXO
Minghetti, A. (2000). Inhibitory effects by soy family genes and activating intrinsic pathway.
antioxidants on the oscillations of the Briggs- Europaen Journal of Pharmacology, 674(2), 65-72.
Rauscher reaction. Helvetica Chimica Acta, 83(12), Marković, M., Talić, S. (2013). Antioksidacijska
3179-3190. aktivnost odabranih hercegovačkih vina. Kemija u
Cervellati, R., Höner, K., Furrow, S.D., Neddens, C., industriji, 62(1-2), 7-12.
Costa, S. (2001). The Briggs-Rauscher Reaction as a Mennen, L.I., Sapinho, D., Ito, H., Bertrais, S., Galan, P.,
Test to Measure the Activity of Antioxidants. Hercberg, S., Scalbert, A. (2006). Urinary flavonoids
Helvetica Chimica Acta, 84(12), 3533-3547. and phenolic acids as biomarkers of intake for
Cervellati, R., Renzulli, C., Guerra, M.C., Speroni, E. polyphenol-rich foods. British Journal of Nutrition,
(2002). Evaluation of Antioxidant Activity of Some 96(1), 191-198.
Natural Polyphenolic Compounds Using the Briggs- Milos, M., Makota, D. (2012). Investigation of atioxidant
Rauscher Reaction Method. Journal of Agricultural synergisms and antagonisms among thymol,
and Food Chemistry, 50(26), 7504-7509. carcacrol, thymoquinone and p-cymene in a model
Cha, J.W., Piao, M.J., Kim, K.C., Yao, C.W., Zheng, J., system using the Briggs-Rauscher oscillating reaction.
Kim, S.M., Hyan C.L., Ahn, Y.S., Hyun, J.W. (2014). Food Chemistry, 131(1), 296-299.
The Polyphenol Chlorogenic Acid Attenuates UVB- Nayeem, N., Asdaq, S.M.B., Salem, H., Ahel-Alfqy, S.
mediated Oxidative Stress in Human HaCaT (2016). Gallic Acid: A Promising Lead Molecule for
Keratinocytes. Biomolecules and Therapeutics, 22(2), Drug Development. Journal of Applied Pharmacy,
136-142. 8(2), 213-216.
Dacić, M., Gojak-Salimović, S. (2016). The effect of Ota, A., Abramović, H., Abram, V., Poklar-Ulrih, N.
chlorogenic acid on the Briggs-Rauscher oscillating (2011). Interactions of p-coumaric, caffeic and ferulic
reaction. Glasnik hemičara i tehnologa Bosne i acids and their styrenes with model lipid membranes.
Hercegovine/Bulletin of the Chemists and Food Chemistry, 24(4), 1256-1261.
Technologists of Bosnia and Herzegovina, 46, 51-54. Rice-Evans, C.A., Miller, N.J., Paganga, G. (1996).
Furrow, S.D., Höner, K., Cervellati, R. (2004). Inhibitory Structure-antioxidant activity relationship of
Effects by Ascorbic Acid on the Oscillations of the flavonoids and phenolic acid. Free Radical Biology
Briggs-Rauscher Reaction. Helvetica Chemica Acta, and Medicine, 20(7), 933-956.
87(3), 735-741. Robards, K., Prenzler, P.D., Tucker, G., Swatsitang, P.,
Höner, K., Cervellati, R., Neddens, C. (2002). Glover, W. (1999). Phenolic compounds and their role
Measurements of the in vitro antioxidant activity of in oxidative processes in fruits. Food Chemistry,
German white wines using a novel method. European 66(4), 401-436.
Food Research and Technology, 214(4), 356-360. Robbins, R.J. (2003). Phenolic Acids in Food: An
Höner, K., Cervellati, R. (2002). Measurements of the Overview of Analytical Methodology. Journal of
antioxidant capacity of fruits and vegetables using the Agricultural and Food Chemistry, 51(10), 2866-2887.
BR reaction method. European Food Research and Shalaby, E.A., Shanab, S.M.M. (2013). Antioxidant
Technolology, 215(5), 437-442. compounds, assays of determination and mode of
Konishi, Y., Kobayashi, S. (2004). Microbial metabolites action. African Journal of Pharmacy and
of ingested caffeic acid are absorbed by the Pharmacology, 7(10), 528-539.
monocarboxylic acid transporter (MCT) in intestinal Stansbury, J. (2014). Rosmarinic Acids a Novel Agent in
Caco-2 cell monolayers. Journal of Agricultural and the Treatment of Allergies and Asthma. Journal of
Food Chemistry, 52(21), 6418-6424. Restorative Medicine, 3(1), 121-126.
14 Gojak-Salimović et al.

Summary/Sažetak
Fenolske kiseline su aromatski sekundarni biljni metaboliti koji posjeduju značajnu antioksidacijsku aktivnost. Kada se
antioksidans doda u aktivnu Briggs-Rauscher reakcijsku smjesu dolazi do neposrednog gašenja oscilacija. Vrijeme
inhibicije oscilacija je u proporcionalnom odnosu s količinom i svojstvima dodanog antioksidansa. U ovom radu ispitivan
je uticaj koncentracije etanolnih rastvora odabranih fenolskih kiselina (galna, kafena, hlorogenska, ruzmarinska, p-
kumarinska i m-kumarinska kiselina) na oscilirajući sistem Briggs-Rauscher reakcije.Reakcija je izvođena u reakcionom
sudu, uz stalno miješanje tačno definisanih koncentracija reaktanata, pri konstantnoj temperaturi od 25 C. Tok oscilacija
Briggs-Rauscher reakcijske smjese praćen je potenciometrijskom metodom uz platinsku elektrodu i srebro/srebro hloridnu
referentnu elektrodu. Relativne antioksidacijske aktivnosti bazirane na vremenima inhibicije izračunate su na tri načina.
Rezultati ispitivanja su pokazali da galna i p-kumarinska kiselina imaju mnogo manju antioksidacijsku aktivnost od kafene,
hlorogenske i ruzmarinske kiseline. Sposobnost inhibicije oscilacija Briggs-Rauscher reakcijske smjese nije pokazala m-
kumarinska kiselina.