Accepted Manuscript Analytical Methods A comparative study of the antioxidant scavenging activity of green tea, black tea and coffee extracts: a kinetic approach Anissi Jaouad, El Hassouni Mohammed, Ouardaoui Abdelkrim, Sendide Khalid PII: DOI: Reference:
S0308-8146(13)01632-4 http://dx.doi.org/10.1016/j.foodchem.2013.11.009 FOCH 14959
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
16 April 2012 27 October 2013 2 November 2013
Please cite this article as: Jaouad, A., Mohammed, E.H., Abdelkrim, O., Khalid, S., A comparative study of the antioxidant scavenging activity of green tea, black tea and coffee extracts: a kinetic approach, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.11.009
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1 2
A comparative study of the antioxidant scavenging activity of green tea, black tea and coffee extracts: a kinetic approach.
3 4
Anissi Jaouad1, 2, El Hassouni Mohammed,2 Ouardaoui Abdelkrim1, Sendide Khalid1*.
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1:
6 7 8 9 10
Al Akhawayn university, School of Science and Engineering, Laboratoty of Biotechnology,
Av. Hassan II, P. O Box 104-Ifrane. 2:
Université Sidi Mohamed Ben Abdellah, Faculté des Sciences Dhar el Mehrez, Laboratoire
de Biotechnologie, Unité de Génétique Moléculaire des Microorganismes, Fés-Morocco. *:
Corresponding author: Tel: 00212661673006, Fax: 00212535862030, e-mail:
[email protected]
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Abstract:
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The antioxidant activities of three beverages, coffee, black tea and green tea, along with their
14
major components, were investigated in terms of their reaction with the stable radical 2,2-
15
diphenyl-2-picrylhydrazyl (DPPH•). We used a kinetic approach in parallel with quantification
16
methods based on a fixed end-point to determine the scavenging efficiency of compounds
17
abundant in these beverages during their reaction with DPPH• using a stopped-flow
18
spectrophotometer-based method. Ascorbic acid, (+)-catechin, (-)-epigallocatechin, tannic acid,
19
and caffeic acid were selected as model antioxidants to study in coffee, black tea and green tea.
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We applied a second-order model to demonstrate similarities in the kinetics behavior of
21
beverages and related compounds. Our findings showed the slopes k’2 ((mol/l)-1.s-1) and k’2max
22
((mol/l)1.s-1)
23
time is more informative about antioxidant properties than reaction with DPPH• alone. We also
24
used IC100 to test the reliability of the relative stoichiometry using a new comparative parameter
25
“n,” which was calculated as:
exhibited similar and correlated values; we suggest the variation in k’2 as a function of
(mol/l. (mol/l)-1, (mol/l).ml.mg-1 or mol.g-1.
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Keywords: DPPH• free-radical, kinetic behavior, second-order model, green tea, black tea, coffee, DPPH• scavenging efficiency comparison
1. Introduction
2 30
The human body relies on antioxidants to limit the damaging effects of reactive oxygen
31
species (ROS). Exogenous antioxidants include phenolic compounds (Croft, 1999; Cai et al.,
32
2006), alkaloids (Racková et al., 2004) and steroids (Reyes et al., 2006). Many methods have
33
been used to study the antioxidant activities of different compounds and foods in vitro. These
34
include phosphomolybdenum method (Prieto et al., 1999), ferric reducing antioxidant power
35
(FRAP) (Benzie and Strain, 1999), oxygen radical absorbance capacity (ORAC) (Cao et al.,
36
1993), electron spin resonance (ESR) (Wasek et al., 2001), hydroxyl radical scavenging activity
37
(Yoshioka et al., 2001) and the ABTS•+ (2,2'-azino-bis3-ethylbenzthiazoline-6-sulphonic acid)
38
method (Yu and Ong, 1999). The stable radical DPPH• (2,2-diphenyl-1-picrylhydrazyl) was
39
initially discovered by Goldschmidt and Renn (1922), and was later introduced by Blois (1958).
40
Today, DPPH remains the most popular assay to measure antioxidant activity in biological
41
samples, and to evaluate the scavenging activity of processed and unprocessed food (Peng et al.,
42
2000; Hirano et al., 2001). However, researchers such as Sharma and Bhat (2009) and Andrzej et
43
al. (2012) have suggested that DPPH• scavenging activity needs to be standardized in order to
44
compare results. In general, studies express the „strength‟ of antioxidants using IC50, defined as
45
the amount of antioxidant necessary to decrease the initial DPPH• concentration by half. In 2009,
46
Sherer and Godoy (2009) presented a new antioxidant activity index (AAI), calculated as:
47
, which considers the both concentration of DPPH• and IC50 to determine a
48
constant for each antioxidant. The antioxidant activity unit (AAU), suggested by Deng et al.
49
(2011), is defined as “1 mol of DPPH• scavenged to consume an amount (in moles) of the
50
scavenger”:
51
“B” is the fitting equation slope of the free radical scavenging ratio; “C” is the initial
52
concentration of DPPH• (g/ml); and “Mr” stands for the molecular weight of the sample.
, where “R” is the ratio of the sample volume to DPPH• volume;
53
Thus far, the majority of kinetic investigations in to the reactions between DPPH• and
54
potential antioxidants have been mechanistic, i.e. focus on the steps by which reactants are
55
converted into products. Some research papers reported DPPH• scavenging reactions using
56
pseudo-first order or second-order kinetic models (Pap et al., 2005). However, no deterministic
57
presentations exist comparing multiple samples activities. In 1995, Halliwell defined an
58
antioxidant as "any substance that when present at low concentrations compared to those of an
3 59
oxidized substrate significantly delays or prevents its oxidation" and, this definition is still used
60
to determine the strength of antioxidants.
61
Tea (Camellia sinensis), is rich in polyphenolic compounds known as tea flavonoids (mainly
62
catechins), which have strong antioxidant properties (Paquay et al., 2000). Green tea has been
63
shown to exhibit a potent antioxidant activity by means of its flavonoids (Suganuma et al., 1999).
64
Black tea, which is an oxidized product of green tea, has approximately 20–30% polyphenols
65
(Sanderson et al., 1972), including bisflavonols, aflavins and arubigins (Gupta et al., 2002).
66
Comparisons of the antioxidant activity of green and black teas have been described in the past
67
and shown green tea to exhibit greater antioxidant activity (Van Gadow et al., 1997).
68
Coffea arabica (arabica) has been suggested to contribute up to 64% of total antioxidant
69
intake in the human diet (Svilaas et al., 2004), but its antioxidant activity has been shown to be
70
considerably lower than that of tea.
71
In this paper, we examined the kinetic behavior of some abundant (antioxidant) compounds
72
in selected teas and coffee to compare parameters describing the reaction kinetics for antioxidant
73
activity in a DPPH• system. In the past, a number of authors have presented models that overlap
74
with our experimental results. A major objective of this work was, therefore, to build on existing
75
work in order to assess the parameters for effective evaluation of DPPH• scavenging. This was
76
achieved through a comparison of kinetic parameters (k'2, slope calculated from the reciprocal of
77
DPPH• concentration as a function of time, k'2max: maximum value of the variation of k'2 as a
78
function of time ), IC100 (amount of antioxidant needed to scavenge 100 % of the initial DPPH•
79
concentration), and n (ratio of the initial DPPH• concentration to IC100) and, thus, this study
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provided both quantitative and qualitative measures of the reactions of antioxidants with DPPH•.
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2. Material and methods 2.1.
Chemicals and samples preparation
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DPPH• and 2,2-diphenyl-1-picrylhydrazine (DPPH-H) were purchased from Sigma (Lyon,
84
France). HPLC grade methanol was purchased from Panreac (Barcelona, Spain). L-ascorbic acid
85
(98%), gallic acid (98%), tannic acid (98%), (+)-catechin (98%), (-)-epigallocatechin (98%) and
86
caffeic acid (98%) were acquired from Fluka (Lyon, France).
4 87
2.2.
Preparation of plants extracts
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Green and black teas and coffee (Arabica) were bought from a local specialty teashop.
89
Samples (10 g) were ground to a powder, and extracts prepared by heating samples in double-
90
distilled water (100 ml) for 15 min at 80 °C with stirring. This process was repeated three times.
91
Extracts from each sample were combined, filtered through a Whathman paper N. 5, centrifuged
92
(4500 rpm, 15 minutes) and freeze-dried.
93
2.3.
Apparatus
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Spectrophotometric data were acquired using a Jasco J-815 Series spectrophotometer (Jasco,
95
USA) equipped with a JASCO SFS-492 Series Stopped-flow systems apparatus. The mixing
96
ratio was fixed to 1:1 with a flow speed of approximately 4 ml/s. Results were displayed using
97
Spectra Manager II software (Jasco, USA). A Jasco V-530 spectrophotometer and disposable
98
cuvettes (1 cm × l cm x 4.5 cm) from Muller Ratiolab (Germany) were used for routine
99
absorbance measurements. All experiments were performed at 25 °C.
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2.4.
Kinetic analysis
101
A typical reaction mixture contains 0.2 mmol/l DPPH• radicals and either 0.5 mmol/l, 0.20
102
mmol/l, 0.125 mmol/l, 62.5 µmol/l, or 31.25 µmol/l of the pure compound. When plant extracts
103
were used, the extract concentration ranged from 10 to 300 µg/ml. The kinetic measurements
104
were performed at 25 °C for 40 minutes; negative controls (i. e. methanol only) were also run.
105
The decrease in the absorbance for the DPPH• radicals was followed at 515 nm (ε515 nm = 10870
106
(mol/l)-1.cm-1 in methanol) (Fotti et al., 2004). Experimental DPPH• concentrations were
107
determined using a standard curve with DPPH• concentrations ranging from 0 mol/l to 0.3
108
mmol/l, with a correlation coefficient of R2=0.999. The amount of antioxidant needed to
109
scavenge 100% of the initial DPPH• concentration (IC100) was determined graphically. The value
110
n was calculated from the formula:
111
2.5.
Data analysis
112
Data were analyzed using STATISTICA® 10. Values were presented as arithmetic means at
113
95% confidence intervals. Means were compared using F-test. P values < 0.05 were considered
114
statistically significant.
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3. Results and discussion
3.1.
General
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Re-consideration of kinetic parameters has previously been proposed by Perez-Jimenez and
118
Saura-Calixto (2008) in order to provide more information about antioxidant activities. Also,
119
Goupy et al. (2003) suggested that reaction kinetics could be more informative than overall
120
antioxidant potential determined by conventional endpoint parameters. In this context, we used a
121
stopped-flow technique to follow rates of DPPH• scavenging and, thus, provide more reliable,
122
real-time data for comparison of antioxidant scavenging.
123
Data describing the reaction between DPPH• and other hydrogen donor molecules are
124
available in the literature. Our purpose was to better understand reactions with DPPH• and to
125
assess DPPH• scavenging activity of some popular beverages alongside established bioactive
126
compounds through mechanistic measurements. Preliminary observations showed most of the
127
scavengers reached a steady-state reaction after a period of time, which depended to the
128
DPPH•/antioxidant ratio and chemical structure. We assessed the reliability of several models to
129
obtain kinetic parameters capable of characterizing the behavior of bioactive compounds. Those
130
compounds were chosen from the chemical composition of the selected beverages.
131
Scavenging of DPPH• is achieved through hydrogen/electron transfer from a given
132
antioxidant to DPPH•. Although DPPH-H is the final product, other complexes form between
133
DPPH• and the oxidized intermediates of the antioxidant or two or more oxidized forms, which
134
generate high molecular weight polymers (Osman, 2011). This hydrogen/electron transfer can be
135
simulated by a reaction where the product does not participate in the reverse reaction and where
136
stoichiometry may vary as the reaction proceeds. In this context, species either regenerate
137
through interaction with the solvent or produce other active species that carry on the reaction
138
responsible for the formation of the final products.
139
In general, the first (rapid) step is attributed to the oxidation of antioxidants by DPPH•, and
140
the subsequent slow step(s) is/are due to secondary reaction(s) of the oxidized antioxidants. In
141
addition, we observed the following features:
142
(1) Stoichiometric coefficients varied throughout the evolution of the reaction;
143
(2) Initial rates were not affected by the presence of the product, and
144
(3) Initial reaction rates were not necessarily the most important during scavenging reactions.
6 145
We also noticed three major kinetic behaviors that varied in speed (fast, moderate and slow),
146
as previously discussed by Brand-Williams et al. (1997). DPPH• scavenging reactions can be
147
simulated by a chain reaction (Denga et al., 2011) and is commonly used during kinetic analysis.
148
Therefore, scavenging of DPPH• by a scavenger (AH) can be presented as:
149 150 151 152
The rates of DPPH• loss during the initial step can follow a pseudo-first or a second-order
153
reaction, depending on the antioxidants chemical structure. Our kinetic results show that ascorbic
154
acid and gallic acid follow a pseudo-first-order.
155
During our analysis of other compounds (e. g. (+)-catechin, tannic acid, (-)-epigallocatechin,
156
and caffeic acid), we observed these reactions appeared to follow a second-order model. By
157
applying such a model, we supposed the antioxidant (AH) concentration decreased with
158
decreasing DPPH• concentration according to the equation
159
variable that maintains this equilibrium throughout the reaction. Therefore, the equation for the
160
reaction rate can be presented as:
161
variables and integration, eqn. 2 can be written as:
162
written as
163
nor as function of DPPH• concentration, the integral
164
function and, thus, included within the rate constant value of the second-order model. The
165
second-order integrated rate equation can be presented as:
166
where
167
168
, where α is a
(eqn. 2). After separation of (eqn. 3). Thus, eqn. 3 can be
(eqn. 4). As α can neither be integrated as a function of time can be considered as a primitive
.
3.2.
Kinetic analysis of the pure compounds in reaction with DPPH•
3.2.1. Ascorbic acid
(eqn. 5)
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Fig. 1 (A) shows how the natural logarithm of DPPH• concentration is dependent on time
170
and Fig. 1 (B) shows the dependence of the pseudo-first-order rate constant on the initial
171
concentration during the reaction with DPPH•. Experimental data showed a lack of fit with the
172
second-order model, in contrast with the results presented by Mishra et al., (2012). (See Table 1
173
for the rate constants.) Ascorbic acid exhibits a rapid reaction with DPPH• through a two-step
174
mechanism. The first step is rapid with rate constants dependent on the initial concentration of
175
ascorbic acid. The second step is slower and involves dehydroascorbic acid as downstream
176
intermediate, with an n value of ≈ 2: (Sawai, 2000). IC100 for ascorbic acid was 98.9000 ± 0.0091
177
µmol/l (mean ± SD, N = 4) and n of 2.222 ± 0.284.
178
3.2.2. Gallic acid
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Gallic acid is a phenolic compound occurring in different food plants that has been widely
180
used as a standard in the DPPH• assay. In our experiments, its reaction with DPPH• followed a
181
pseudo-first-order kinetic model. Fig. 2 (A) shows the linear evolution of the logarithm for
182
DPPH• concentration as a function of time. The calculated slopes (k’2) showed a linear
183
correlation with the initial concentration of gallic acid (Fig. 2 (B)). IC100 for gallic acid was
184
36.000 ± 0.004 µmol/l (mean ± SD, N = 4) and n of 5.555 ± 0.642. The segments slopes in Fig. 2
185
define the pseudo-first-order constant rate and are summarized in Table 1.
186
3.2.3. (+)-Catechin
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The reaction of (+)-catechin’ with DPPH• is relatively slow. The reaction time to reach the
188
steady state was relatively long and depended on the (+)-catetchin concentration. Inconsistencies
189
in the total stoichiometric values for (+)-catechin have been reported previously (Villano et al.,
190
2007) and were attributed to the incubation time, the nature of the used solvent and the ratio of
191
flavanol to DPPH• radicals (Dimitrios et al., 2006). We calculated (+)-catechin IC100 and n as
192
49.000 ± 0.005 µmol/l (mean ± SD, N = 4) and 4.081 ± 0.503 (mean ± SD, N = 4), respectively.
193
n was in agreement with previous reports on (+)-catechin stoichiometry (Osman, 2011). As
194
shown in Fig. 3 (A), the variation in the reciprocal for DPPH• concentration versus time
195
indicated second-order kinetics and showed a linear evolution of the rate constant (k’2) as a
196
function of the initial concentration of (+)-catechin, (Fig. 3B, and Table 1). When we plotted the
8 197
evolution of the second-order rate constant (k’2) (using eqn. 5) as a function of time (Fig. 3 (C)),
198
we observed that the rate constants reached a maximum value (k’2max), similar to those for slope
199
of the Fig. 3 linear segment (A-B) at a given concentration (see Table 1 for the corresponding
200
rate constants values), followed by a progressive but slow decrease, indicating the progress of the
201
reaction. Furthermore, the variation in the second-order rate constant as a function of the initial
202
concentration of (+)-catechin correlated with the dependence of k’2max on the initial concentration
203
of (+)-catechin (Fig. 3 (D)). Osman (2011) showed the reaction of (+)-catechin with DPPH• is a
204
multistep, mainly biphasic, reaction with the first step followed by a slow decrease, probably due
205
to the low reactivity of the downstream intermediates (Sang et al., 2003). We showed that the
206
evolution of the second-order rate constant can tell us more about the behavior of the reaction
207
than simply the reciprocal of DPPH• concentration as a function of time.
208
3.2.4. Tannic acid
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Tannic acid is a plant polyphenol found in several beverages including red wine, beer,
210
coffee, and black and green teas (Chung et al., 1998; King et al., 1999). Like other polyphenols,
211
tannic acid has been shown to possess antioxidant activities (Andrade et al., 2005). Tannic acid
212
exhibited second-order kinetics for its reaction with DPPH•. The slope of the reciprocal for the
213
DPPH• concentration as a function of time, extracted from the linear curve in Fig. 4 (A), showed
214
a linear dependence on the initial concentration of tannic acid (Fig. 4 (B) and Table 1). Plotting
215
k’2 values as a function of time showed the occurrence of two maxima (k’2max), as shown in Fig.
216
4 (C) and Table 1, and these showed a linear correlation when plotted as a function of the initial
217
tannic acid concentration, as presented in Fig. 4 (D). IC100 and n were 31.500 ± 0.005 µmol/l
218
(mean ± SD, N = 4) and 6.349 ± 0.353 (mean ± SD, N = 4), respectively.
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3.2.5. (-)-epigallocatechin
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(-)-epigallocatechin, one of the most important catechins found in tea, exhibited moderate
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kinetics in reaction with DPPH•. Fig. 5 (A-B) shows the dependence of the reciprocal for the
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DPPH• concentration as a function of time. Slopes in Fig. 5 (A) show a linear relation with the
223
initial concentration of (-)-epigallocatechin. Such a relationship suggests a second-order model in
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this reaction. Values for the second-order rate constants (Table 1) show the significant activity of
9 225
(-)-epigallocatechin toward DPPH• as compared with (+)-catechin. IC100 and n were 48.100 ±
226
0.002 µmol/l (mean ± SD, N = 4) and 4.154 ± 0.264 (mean ± SD, N = 4), respectively. When
227
plotting the evolution of the second-order rate constant (k’2) (calculated from eqn. 5) as a
228
function of time (Fig. 5 C), we observed the second-order rate constants reached a maximum
229
(k’2max) similar to that of the linear segment slope from Fig. 5 (A-B) at a given concentration,
230
(see Table 1 for the corresponding rate constants values); these rates remained constant with a
231
slow decrease indicating the progress of the reaction. Furthermore, the variation in the second-
232
order rate constant as a function of the initial of (-)-epigallocatechin concentration correlated
233
with the dependence of k’2max on the initial concentration of (-)-epigallocatechin (Fig. 5 (D)).
234
3.2.6. Caffeic acid
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Caffeic acid (3,4-dihydroxycinnamic acid) is found in a wide variety of plant-derived
236
products such as wine, coffee beans, fruits, vegetables, olive oil and tea (Shahidi & Naczk,
237
1995). It reacts with the DPPH• through moderate kinetics. Fig. 6 (A) shows the dependence of
238
the reciprocal for the DPPH• concentration as a function of time and indicates second-order
239
kinetics. The dependence of the second-order rate constant (k’2) shows a linear correlation, as a
240
function of the initial concentration of caffeic acid (Fig. 6 (B)). The second-order rate constant
241
k’2 was extracted from the slope for each caffeic acid concentration, and values are summarized
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in Table 1. The evolution of second-order rate constants (calculated from eqn. 5) as a function of
243
time shows the rate constants reached a maximum, similar to the corresponding k’2max, calculated
244
from the curves in Fig. 6 (C) (see Table 1 for values). Furthermore, the dependence of the
245
second-order rate constants on the initial concentrations of caffeic acid shows a linear correlation
246
with the dependence of the k’2max on the initial concentration of caffeic acid. This first step is
247
governed by the reaction of caffeic acid with DPPH•. A lower k’2max was also observed in a
248
second step. These observations suggest a multistep reaction; a moderate step followed by a slow
249
step, probably due to the low reactivity of the downstream intermediate(s). Also, the
250
corresponding values for k’2max showed a linear dependence on the initial concentration of the
251
caffeic acid. IC100 and n were 71.000 ± 0.005 µmol/l (mean ± SD, N = 4) and 2.814 ± 0.315
252
(mean ± SD, N = 4), respectively.
253
10
In summary, (-)-epigallocatechin showed the highest k’2max at IC100 913.7 ± 57.1 (nmol/l)-1.s-
254 255
1
, followed by caffeic acid (458.900 ± 0.053 (µmol/l)-1. s-1) then tannic acid with two k’2max
256
(0.2873 ± 0.0061(µmol/l)-1. s-1 and 0.0582 ± 0.0041 (µmol/l)-1. s-1) and finally (+)-catechin
257
(6.2.10-7 ± 0.6.10-7 (mol/l)-1. s-1). In general, k’2max corresponded to the antioxidant activity of
258
the initial molecule, which remained independent of the downstream reactions involving
259
intermediates. This involvement explains the different order in scavenging activity when the
260
antioxidants are compared according to IC100. This difference exists because IC100 is derived
261
from the action of the initial antioxidant as well as intermediate forms.
262
We demonstrates that not all antioxidants in a DPPH• system behave according to the same
263
kinetic model, suggesting the choice of kinetic model has to be determined independently for
264
each sample, particularly if its chemical composition is not fully determined, in order to
265
understand fully its antioxidant activity. Our results show most significant antioxidant
266
compounds present in the selected beverages (coffee, and green and black teas) exhibited
267
second-order kinetics. The variation of k’2 as a function of time is, therefore, a valuable
268
parameter for describing kinetic behavior of an antioxidant. This provides a clear description of
269
the extent of DPPH• scavenging. Furthermore, the profiles of such plots were characteristic for
270
each compound.
271
During the course of the reaction, reactive intermediate(s) may be generated and such
272
intermediate(s) may exhibit higher or lower activity compared to the initial compound. The
273
evolution of k’2 over time could, therefore, be used to predict complexity of the mechanistic
274
model. Indeed, values obtained for k’2 and the k’2max correlate significantly with a Pearson values
275
of 0.9894, 0.9928, 0.9969, and 0.9981 for (+)-catechin, (-)-epigallocatechin, caffeic acid and
276
tannic acid, respectively. In order to apply a second-order reaction kinetic model, the
277
concentration of DPPH• has to be maintained in proportion to the concentration of the
278
antioxidant through the arbitrary α factor.
279
3.3.
Determination of the IC100 and n values
280
We performed a kinetic analysis of six pure compounds (ascorbic acid, gallic acid, (+)-
281
catechin, (-)-epigallocatechin, tannic acid and caffeic acid), each present in at least one of the
282
three beverages studied. Each compound was tested at different concentrations. The IC100 value,
283
defined as the amount of antioxidant necessary to decrease the concentration of DPPH• by 100
11 284
%, was used as a comparative parameter to define the strength and the stoichiometry of an
285
antioxidant in reaction with DPPH•. An example illustrating the calculation of the IC100 of tannic
286
acid is shown in Fig. 7. In general, the higher the IC100, the weaker the antioxidant. Thus, we can
287
conclude from the IC100 values (Table 1) that tannic acid exhibits the highest antioxidant activity,
288
followed by caffeic acid, (-)-epigallocatechin, (+)-catechin, ascorbic acid, and then gallic acid.
289
Also, the relative stoichiometry (n) was shown to be relevant for the comparison of the
290
antioxidant strength between beverages, as well as between pure compounds, especially since we
291
did not note any significant difference in this parameter when observed at different
292
DPPH•/antioxidant ratios.
293
3.4.
Kinetic analysis of the extracts from green tea, black tea and coffee
294 295
We investigated the antioxidant effects of green and black teas and coffee on DPPH•. Table 2
296
presents the kinetic parameters extracted from the analysis of the three beverages. As shown in
297
Fig. 8 (A-B), green tea extract showed rapid kinetics, fitting a second-order model. Fig. 8 (C-D)
298
shows the reaction is characterized by a rapid increase in k’2 as a function of time reaching a
299
maximum value in a short period of time followed by a decrease to a minimal. Similarities in
300
k’2max and k’2 support our suggestion the reaction kinetic sequence should be presented as the
301
change in k’2 as a function of time and elucidate scavenging properties of food/ food extract
302
when using DPPH•. IC100 was calculated and values are presented in Table 2.
303
Black tea showed a lower antioxidant activity than green tea. The kinetic behavior of black
304
tea (extract) followed a second-order kinetic model, and its rate constants and IC100 were
305
calculated accordingly (Table 2). IC100 was largely higher (≈ 5 fold) than that for green tea. In
306
addition, analysis of the evolution of k’2 for black tea (Fig. 9 (C)) indicated the occurrence of low
307
kinetic behavior antioxidant compounds compared with green tea extracts, which exhibits a rapid
308
kinetics and high second-order rate constants (Fig. 8 (C)). Values for the slopes extracted from
309
Fig. 9 (A) and presented in Fig. 9 (B) are in agreement with those obtained for k’2max. The change
310
in green tea composition during fermentation to produce black tea is probably the main causes of
311
the change in its antioxidant activity (Rietveld and Wiseman, 2003). This change in composition
312
would certainly explain the relatively high antioxidant potential of green tea found in our study.
12 313
Coffee (extract) showed DPPH• scavenging activity somewhere between those exhibited by
314
green and black teas, in contrast to what was previously reported by Svilaas et al. (2004). The
315
kinetic behavior showed its dependence on a second-order kinetic model. The values for second-
316
order model rate constants and IC100 were calculated and are presented in Table 2. Interestingly,
317
IC100 for coffee was similar to that for green tea extract, but the second-order rate constants were
318
significantly different. We observed the evolution of k’2 from coffee extract (Fig. 10 (C)) and
319
black tea (Fig. 9 (C)) was similar to that for tannic acid, which would correlate with the
320
occurrence of high concentrations of tannins accumulated in both beverages during processing.
321
Similarly, the same correlation was observed when comparing the evolution of k’2 for green tea
322
with those of (+)-catechin and (-)-epigallocatechin. All these beverages demonstrate antioxidant
323
activity as a result of the concentration of naturally occurring bioactive compounds.
324
4.
Conclusion
325
We attempted to shed light on kinetic behavior as a tool for evaluation and comparison of
326
different antioxidants in reaction with DPPH•. Although used extensively, the DPPH• assay
327
remains a crude tool for comparing the efficiency of natural compounds against free radicals.
328
Indeed, data about parameters such as IC50 or IC100 lack information about the kinetic
329
performance of antioxidant. This study provides more reliable kinetic tools for comparing
330
antioxidants. Kinetic analysis of the reactions of the selected pure compounds with DPPH•
331
allowed us to evaluate progress of the reactions as a function of time through changes in rate
332
constant. This analysis reflects real-time monitoring of the reaction. Based on the kinetic and an
333
endpoint analyses, our results revealed different rankings for scavenging for selected antioxidant
334
compounds. These differences suggest a combination of both approaches would allow
335
researchers to perform a better comparison between antioxidant or food samples. Expression of
336
the results in kinetic terms not only takes into account antioxidant activity, but also provides
337
valuable information on the general behavior (i.e. speed, reversibility, etc.). Thus, we
338
recommend use of both kinetic and fixed endpoints be combined so as to provide comprehensive
339
information about antioxidant potential in a given sample. The proposed approach, based on a
340
second-order model, as compared with suggested kinetic models previously, was more
341
discriminative and consistent with regard to the DPPH•/antioxidant reaction behavior, especially
342
when considering a multistep mechanism that includes intermediate(s) exhibiting different
13 343
efficiencies toward DPPH•. Parameters extracted from this model provide a complementary
344
method and additional understanding of potential antioxidants in food or pharmaceutical
345
applications. Based on the analysis of the kinetic behavior in the present paper, we recommend:
346
High concentrations of DPPH• to maintain the proportion of DPPH• to antioxidant.
347
Prolonged reaction times for compounds exhibiting low kinetic reaction.
348
Pure compounds should be used as standards and selected from the same family of
349 350
351
compounds in the food source.
Actual kinetic model be applied in analyses should be selected based on preliminary data.
Acknowledgments
352
The authors are thankful to Dr. Siân Astley and Dr. Jack Kalpakian for their thorough review of
353
the manuscript. The project has been funded by Al Akhawayn University in Ifrane University‟s
354
seed Research Fund.
355
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Table 1: Summary of the kinetic parameter constant rate of the reaction of gallic acid, ascorbic acid, (+)-catechin, (-)-epigallocatechin, caffeic acid and tannic acid with DPPH• at 0.2 mmol/l. 0.5 mmol/l
0.20 mmol/l
0.125 mmol/l
0.0625 mmol/l
0.03125 mmol/l
k1
k1
k1
k1
k1
0.0170 ± 5.6.10-3 0.101 ± 0.093 k’2 k’2max
0.0092 ± 2.5.10-3 0.095 ± 0.063 k2 k’2max
0.0051 ± 4.1.10-4 0.076 ± 0.061 k2 k’2max
0.0030 ± 3.3.10-4 0.0101 ± 2.11-3 k2 k’2max
Constant rate (s-1) Gallic acid Ascorbic acid Constant rate ((mmol/l) -1.s-1) (+)-catechin
(-)-epigallocatechin
Caffeic acid Constant rate ((mmol/l) -1.s-1)
8.13.10-6 ± 1.3.10-6
15.4.10-6 ± 9.2.10-6
3.1.10-6 ± 0.2.10-3
7.1.10-6 ± 0.9.10-6
1.6.10-6 ± 0.8.10-3
2.7.10-6 ± 0.9.10-6
0.0010 ± 0.0005
1.6.10-6 ± 0.3.10-3
0.601.10-6 ± 0.012.10-3
0.62.10-6 ± 0.06.10-3
1.79.10-3 ± 0.18.10-3 1.94.10-3 ± 84.2.10-6
1.76.10-3 ± 0.43.10-3 3.21.10-3 ± 0.98.10-3
0.812.10-3 ± 0.035.10-3 0.85.10-3 ± 58.1.10-6
1.002.10-3 ± 0.204.10-3 1.23.10-3 ± 51.1.10-6
0.47.10-3 ± 58.1.10-6 0.46.10-3 ± 59.1.10-5
19.74.10-5 ± 6.3.10-6 0.32.10-3 ± 78.3.10-6
25.24.10-5 ± 16.4.10-6 0.42.10-3 ± 49.1.10-6
10.58.10-5 ± 51.6.10-6 0.11.10-3 ± 88.1.10-6
11.37.10-5 ± 38.1.10-6 0.24 .10-3 ± 94.1.10-6
k’2
k’2max1/k’2max2
k’2
k’2max1/k’2max2
k’2
0.538.10-3 ± 0.054.10-3 62.25.10-5 ± 58.1.10-6 k’2max1/ k’2max2 0.42.10-3 ± 81.5.10-5 0.18.10-3 ± 1.75.10-6
k’2
k’2max1/k’2max2
k’2
k’2max1/k’2max2
-3
-3
Tannic acid
0.0015 ± 5.1.10-4 0.0030 ± 3.110-3 k2 k’2max
1.79.10 ± 0.46. 10-3
0.75.10 ± 32.8.10-6 0.47.10-3 ± 22.1.10-6
-3
-3
0.75.10 ± 32.7.10-3
0.48.10 ± 1.6.10-6 0.25.10-3 ± 52.2.10-6
-3
0.47.10 ± 8.1.10-6
-3
-3
0.20.10 ± 44.6.10-6
0.33.10 ± 5.21.10-3 9.35.10-5 ± 4.3.10-6
IC100 (mol/l)
n (mmol/l.( mmol/l) -1
36.1.10-6 ± 4.10-6 98.9.10-6 ± 9.110-3
5.555 ± 0.642 2.222 ± 0.284
0.0491 ± 0.0057
4.081 ± 0.503
0.0481± 0.0021
4.154 ± 0.264
0.0712 ± 0.0053
2.81 ± 0.31
IC100 (mol/l)
n (mmol/l. (mmol/l )-1)
0.0315 ± 0.0051
6.349 ± 0.353
-3
-3
0.11.10 ± 8.1.10-6
0.29.10 ± 6.1.10-3 0.058.10-3 ± 0.004.1.10-6
Table 2: Summary of the kinetic parameters of the pseudo-first order and second order, and the IC100 and the stoichiometry of the reaction of green tea, black tea and coffee extracts with DPPH• at 0.2 mmol/l 0.210 mg/ml Constant rate (mg/ml-1.s-1) Green tea
Black tea
Coffee
k’2 3.2781 ± 0.9393 0.0421 ± 0.0328 0.2589 ± 0.0832
k’2max 3.2124 ± 0.5810 0.1795 ± 0.0511 0.2789 ± 0.0471
0.180 mg/ml k’2 2.7725 ± 0.7021 0.0314 ± 0.0062 0.2340 ± 0.0921
k’2max 2.6489 ± 0.531 0.1485 ± 0.0512 0.2540 ± 0.0571
0.150 mg/ml k’2 2.216 ± 0.371 0.0262 ± 0.0511 0.2067 ± 0.0441
k’2max 2.2577 ± 0.6429 0.1082 ± 0.0113 0.2053 ± 0.0381
0.11 mg/ml k’2 1.6912 ± 0.0536 0.0164 ± 0.0022 0.1837 ± 0.0552
k’2max 1.5955 ± 0.3812 0.0940 ± 0.0081 0.1633 ± 0.0121
0.050 mg/ml k’2 0.7630 ± 0.0381 0.0070 ± 0.0001 0.0712 ± 0.0021
k’2max 0.5632 ± 0.0562 0.0382 ± 0.0051 0.0866 ± 0.0011
0.025 mg/ml k’2 0.2545 ± 0.0516 0.0036 ± 0.00074 0.0451 ± 0.0045
k’2max 0.2475 ± 0.0231 0.0213 ± 0.0013 0.0555 ± 0.0081
IC100 (mg/ml)
Stoichiometry (n, (mmol/l). ml/mg)
0.0318 ± 0.0024
6.289 ± 0.8724
0.1531 ± 0.0431
1.300 ± 0.424
0.0365 ± 0.0024
5.479 ± 0.524
(A') 2.0
(B)
(A) 0.15
5.10-4 mol/l -5 25.10 mol/ l 62.5.10 -6 mol/ l 31.25.10 -6 mol/ l -6 11.5625.10 mol/ l
0.4 0.3 0.2
5.10-4 mol/l 25.10-5 mol/l 62.5.10-6 mol/l 31.25.10-6 mol/l 11.5625.10-6 mol/l
0.1 0.0
1.5
0
1
2
3
4
5
Time (s)
1.0
k 1 (s-1 )
ln([DP PH . ] t/[DPP H .] 0 )
ln([DPPH .] t /[DPPH .] 0 )
2.5
0.10
0.05
0.5 0.0 0
5
10
15
20
0.00 0.0
25
Time (s)
0.1
0.2
0.3
c0 ascorbic acid (.10 -3 mol/l)
Figure 1: (A), variation of the logarithm of the DPPH• concentration on the time in reaction with different concentrations of ascorbic acid at 25 °C. (A’) show the magnification from t=0 to t=4 (s), and (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of ascorbic acid.
(A)
(B) 0.020
-4
5.10 mol/l 25.10-5 mol/l
0.015
5
62.50.10-6 mol/l 4
100
200
Time (s)
0.010
31.25.10-6 mol/l 11.56.10-6 mol/l
0.005
300
0.000 0.0
3 0
k 1 ( s -1 )
ln ([D P PH .] t /[D PPH .] 0 )
6
0.2
0.4
0.6
c0 gallic acid (.10 -3 mol/l)
Figure 2: (A), variation of the logarithm of the DPPH• concentrations on the time in the reaction with gallic acid at 25 °C at the indicated concentrations, and (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of gallic acid.
4 -5
25.10 mol/l 62.50.10-6 mol/l 31.25.10-6 mol/l 11.56.10-6 mol/l
0 0
200
400
600
800
0.006 0.004 0.002 0.000 0.0
1000
Time (s)
(C) 12.5 10-5 mol/l
0.002
0.001 -5
62.510 mol/l 31.25 10-6 mol/l
0.000 0
500
1000
Time (s)
0.2
0.4
0.6
c0 (+)-catechin (.10 -3 mol/l)
0.003
k'2 (.10-3 mol/l -1.s -1)
slope (10 -3 (mol/l) -1.s -1)
6
1500
k'2max (10-3 (mol/l)-1.s-1)
1 /[ D P P H .] t -1 /[ D P P H .] 0 ( 1 0 - 3 (m ol/l) - 1 )
5.10-4 mol/l
2
(B)
0.008
(A)
8
(D)
0.015
0.010
0.005
0.000 0.0
0.2
0.4
0.6
-3
c0 (+)-catechin (.10 mol/l)
Figure 3: (A), dependence of the reciprocals of the DPPH• concentrations on the reaction time with (+)catechin at the indicated concentrations at 25 °C, (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of (+)-catechin. (C), variations of k’2 as function of time. (D), dependence of k’2max on the initial concentration of (+)-catechin.
40
25.10-5 mol/l
20
62.50.10-6 mol/l 31.25.10-6 mol/l 11.56.10-6 mol/l
0 0
50
100
150
200
slope (.10 -3 (mol/l) -1.s -1)
5.10-4 M
0.20 0.15 0.10 0.05 0.00 0.00
250
Time (s)
(C) 5.10 mol/l 25.10-5 mol/l 62.50.10-6 mol/l 31.25.10-6 mol/l 11.56.10-6 mol/l
0.6 0.4 0.2 0.0 100
200
0.10
0.15
0.20
0.25
300
Time (s)
(D)
0.8 -4
0
0.05
c0 tannic acid (.10 -3 mol/l)
400
500
k'2max (.10-3 (mol/l)-1.s -1)
1/[DPPH.] t -1/[ DPPH.] 0 (.10-3 (mol/l) -1)
60
0.8
k'2 (.10-3 (mol/l)-1.s-1)
(B)
0.25
(A)
First peak Second peak
0.6 0.4 0.2 0.0 0.0
0.2
0.4
0.6
c0 tannic acid (.10 -3 mol/l)
Figure 4: (A), dependence of the reciprocals of the DPPH• concentrations on the time in the reaction with tannic acid at a temperature of 25 °C at the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l, (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of tannic acid, (C), variations of k’2 versus time, (D), dependence of k’2max on the initial concentration of tannic acid.
40
62.5.10-6 mol/l 20 -6
31.25.10 mol/l
slope (.10 -3 (mol/l) -1.s -1)
25.10-5 mol/l 12.5.10-5 mol/l
60
0
200
400
600
800
1.5 1.0 0.5 0.0 0.0
0 1000
0.2
0.4
0.6
c0 (-)-epigallocatechin (.10 -3 mol/l)
Time (s)
(D)
(C) 2.0
k'2max (.10-3 (mol/l)-1.s-1)
1/[DPPH.] t -1/[ DPPH .] 0 (.10-3 (mol/l) -1)
2.0
5.10-4 mol/l
2.0
k'2 (.10-3 (mol/l)-1.s-1)
(B)
(A)
80
5.10-4 mol/l 25.10-5 mol/l 12.5.10-5 mol/l 62.5.10-6 mol/l 31.25.10-6 mol/l
1.5 1.0 0.5 0.0 0
100
200
300
Time (s)
400
500
1.5 1.0 0.5 0.0 0.0
0.2
0.4
0.6
c0 (-)-epigallocatechin (.10 -3 mol/l)
Figure 5: (A), Dependence of the reciprocals of the DPPH• concentrations on the time in the reaction with (-)epigallocatechin at a temperature of 25 °C at the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l, (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of of (-)epigallocatechin, (C), variations of k’2 versus time, (D), dependence of k’2max on the initial concentration of (-)epigallocatechin.
20
0
50
100
150
2.0 1.5 1.0 0.5 0.0 0.0
0 200
Time (s)
0.4
0.2
0.0 100
200
Time (s)
0.4
0.6
300
(D)
4
10-4 mol/l 8.10-5 mol/l 6.10-5 mol/l 4.10-5 mol/l 2.10-5 mol/l
0
0.2
c0 caffeic acid (.10 -3 mol/l)
(C)
0.6
k'2 (.10-3 (mol/l) -1.s-1)
slope (.10 -3 (mol/l) -1.s -1)
5.10-4 mol/l 25.10-5 mol/l 12.5.10-5 mol/l 62.5.10-6 mol/l 31.25.10-6 mol/l
40
(B)
2.5
400
k'2m ax (.10-3 (mol/l) -1.s -1)
1/[DPPH.]t-1/[ DPPH.]0 (10-3 (mol/l)-1)
(A)
3 2 1 0 0.0
0.2
0.4
0.6
-3
c0 caffeic acid (.10 mol/l)
Figure 6: (A), Dependence of the reciprocals of the DPPH• concentrations on the time in the reaction caffeic acid at a temperature of 25 °Cat the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l, (B), ), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of caffeic acid, (C), variations of k’2 versus time during the reaction of 0.2 mmol/l, (D), dependence of k’2max on the initial concentration of caffeic acid.
DPPH.scavenging percentage (%)
100 80 60 40 20 0 0.00
0.02
0.04
0.06
0.08
0.10
c0 tannic acid (.10 -3 mol/l)
Figure 7: graphical determination, of IC100 value of tannic acid during its reaction with DPPH•. The scavenging • • percentage (%) was calculated as at a given concentration of an antioxidant. •
0.66 mg/ml 0.25 mg/ml 0.21 mg/ml 0.18 mg/ml 0.15 mg/ml 0.11 mg/ml
60 40 20 0 0
5
10
15
(B)
4
slope ((mg/ml)-1.s -1)
1/[DPPH.] t-1/[ DPPH.] 0 (mg/ml) -1
(A) 80
3 2 1 0 0.00
20
0.05
Time (s)
(C)
3 2 1 0 0
100
200
0.15
0.20
0.25
300
Time (s)
400
(D)
4
0.2 mg/ml 0.75 mg/ml 0.65 mg/ml 0.55 mg/ml 0.45 mg/ml 0.35 mg/ml
k'2max ((mg/ml)-1.s-1)
-1 -1
k'2 ((mg/ml) .s )
4
0.10
c0 green tea (mg/ml)
500
3 2 1 0 0.00
0.05
0.10
0.15
0.20
0.25
c0 green tea (mg/ml)
Figure 8: (A), dependence of the reciprocals of the DPPH• concentrations on the time in the reaction with green tea extract at a temperature of 25 °C at the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l, (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of green tea extract, (C), variations of k’2 versus time, (D), dependence of k’2max on the initial concentration of green tea.
40 30 20 10
50
100
0.04 0.03 0.02 0.01 0.00 0.00
0 0
(B)
0.05
0.75 mg/ml 0.45 mg/ml 0.35 mg/ml 0.25 mg/ml 0.1 mg/ml
slope ((mg/ml)-1.s -1)
1/[DPPH.] t -1/[ DPPH.] 0 (mg/ml) -1
(A)
150
0.05
0.10
0.15
(C)
0.25
(D) 0.20
0.75 0.65 0.55 0.45 0.35
0.4
mg/ml mg/ml mg/ml mg/ml mg/ml
0.2
0.0 0
100
200
300
Time (s)
400
500
k'2max ((mg/ml)-1.s -1)
0.6
k'2 ((mg/ml)-1.s-1)
0.20
c0 black tea (mg/ml)
Time (s)
0.15 0.10 0.05 0.00 0.00
0.05
0.10
0.15
0.20
0.25
c0 black tea (mg/ml)
Figure 9: (A), dependence of the reciprocals of the DPPH• concentrations on the time in the reaction with black tea extract at a temperature of 25 °C at the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l. (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of black tea, (C), variations of k’2 versus time, (D), dependence of k’2max on the initial concentration of black tea extract.
0.3
0.5 mg/ml 0.4 mg/ml 0.38 mg/ml 0.333 mg/ml 0.25 mg/ml
40
20
slope ((mg/ml)-1.s -1)
1/[DPPH.] t -1/[ DPPH .] 0 (mg/ml)-1
(B)
(A)
60
0 0
100
200
300
400
0.2
0.1
0.0 0.00
500
0.05
0.0 200
Time (s)
300
400
k'2max ((mg/ml)-1.s -1)
k'2max ((mg/ml)-1.s-1)
0.5 mg/ml 0.4 mg/ml 0.35 mg/ml 0.3 mg/ml 0.25 mg/ml
0.1
100
0.20
0.25
0.3
0.2
0
0.15
(D)
(C)
0.3
0.10
c0 coffee (mg/ml)
Time (s)
0.2
0.1
0.0 0.00
0.05
0.10
0.15
0.20
0.25
c0 coffee (mg/ml)
Figure 10: (A), dependence of the reciprocals of the DPPH• concentrations on the time in the reaction with coffee extract at a temperature of 25 °C at the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l. (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of coffee extract, (C), variations of k’2 versus time, (D), dependence of k’2max on the initial concentration of coffee extract.
Research highlights:
Kinetic and endpoint analysis were combined for reliable comparison of antioxidants activities
Parameter k’2 offers a better understanding of the antioxidant’s kinetic behavior
Parameters k ’2max, IC100 and n can be used to compare antioxidants potential
The kinetic analysis was applied to compare antioxidant activity of 3 natural beverages
S0308-8146(13)01632-4 http://dx.doi.org/10.1016/j.foodchem.2013.11.009 FOCH 14959
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
16 April 2012 27 October 2013 2 November 2013
Please cite this article as: Jaouad, A., Mohammed, E.H., Abdelkrim, O., Khalid, S., A comparative study of the antioxidant scavenging activity of green tea, black tea and coffee extracts: a kinetic approach, Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.11.009
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1
1 2
A comparative study of the antioxidant scavenging activity of green tea, black tea and coffee extracts: a kinetic approach.
3 4
Anissi Jaouad1, 2, El Hassouni Mohammed,2 Ouardaoui Abdelkrim1, Sendide Khalid1*.
5
1:
6 7 8 9 10
Al Akhawayn university, School of Science and Engineering, Laboratoty of Biotechnology,
Av. Hassan II, P. O Box 104-Ifrane. 2:
Université Sidi Mohamed Ben Abdellah, Faculté des Sciences Dhar el Mehrez, Laboratoire
de Biotechnologie, Unité de Génétique Moléculaire des Microorganismes, Fés-Morocco. *:
Corresponding author: Tel: 00212661673006, Fax: 00212535862030, e-mail:
[email protected]
11 12
Abstract:
13
The antioxidant activities of three beverages, coffee, black tea and green tea, along with their
14
major components, were investigated in terms of their reaction with the stable radical 2,2-
15
diphenyl-2-picrylhydrazyl (DPPH•). We used a kinetic approach in parallel with quantification
16
methods based on a fixed end-point to determine the scavenging efficiency of compounds
17
abundant in these beverages during their reaction with DPPH• using a stopped-flow
18
spectrophotometer-based method. Ascorbic acid, (+)-catechin, (-)-epigallocatechin, tannic acid,
19
and caffeic acid were selected as model antioxidants to study in coffee, black tea and green tea.
20
We applied a second-order model to demonstrate similarities in the kinetics behavior of
21
beverages and related compounds. Our findings showed the slopes k’2 ((mol/l)-1.s-1) and k’2max
22
((mol/l)1.s-1)
23
time is more informative about antioxidant properties than reaction with DPPH• alone. We also
24
used IC100 to test the reliability of the relative stoichiometry using a new comparative parameter
25
“n,” which was calculated as:
exhibited similar and correlated values; we suggest the variation in k’2 as a function of
(mol/l. (mol/l)-1, (mol/l).ml.mg-1 or mol.g-1.
26 27 28
29
Keywords: DPPH• free-radical, kinetic behavior, second-order model, green tea, black tea, coffee, DPPH• scavenging efficiency comparison
1. Introduction
2 30
The human body relies on antioxidants to limit the damaging effects of reactive oxygen
31
species (ROS). Exogenous antioxidants include phenolic compounds (Croft, 1999; Cai et al.,
32
2006), alkaloids (Racková et al., 2004) and steroids (Reyes et al., 2006). Many methods have
33
been used to study the antioxidant activities of different compounds and foods in vitro. These
34
include phosphomolybdenum method (Prieto et al., 1999), ferric reducing antioxidant power
35
(FRAP) (Benzie and Strain, 1999), oxygen radical absorbance capacity (ORAC) (Cao et al.,
36
1993), electron spin resonance (ESR) (Wasek et al., 2001), hydroxyl radical scavenging activity
37
(Yoshioka et al., 2001) and the ABTS•+ (2,2'-azino-bis3-ethylbenzthiazoline-6-sulphonic acid)
38
method (Yu and Ong, 1999). The stable radical DPPH• (2,2-diphenyl-1-picrylhydrazyl) was
39
initially discovered by Goldschmidt and Renn (1922), and was later introduced by Blois (1958).
40
Today, DPPH remains the most popular assay to measure antioxidant activity in biological
41
samples, and to evaluate the scavenging activity of processed and unprocessed food (Peng et al.,
42
2000; Hirano et al., 2001). However, researchers such as Sharma and Bhat (2009) and Andrzej et
43
al. (2012) have suggested that DPPH• scavenging activity needs to be standardized in order to
44
compare results. In general, studies express the „strength‟ of antioxidants using IC50, defined as
45
the amount of antioxidant necessary to decrease the initial DPPH• concentration by half. In 2009,
46
Sherer and Godoy (2009) presented a new antioxidant activity index (AAI), calculated as:
47
, which considers the both concentration of DPPH• and IC50 to determine a
48
constant for each antioxidant. The antioxidant activity unit (AAU), suggested by Deng et al.
49
(2011), is defined as “1 mol of DPPH• scavenged to consume an amount (in moles) of the
50
scavenger”:
51
“B” is the fitting equation slope of the free radical scavenging ratio; “C” is the initial
52
concentration of DPPH• (g/ml); and “Mr” stands for the molecular weight of the sample.
, where “R” is the ratio of the sample volume to DPPH• volume;
53
Thus far, the majority of kinetic investigations in to the reactions between DPPH• and
54
potential antioxidants have been mechanistic, i.e. focus on the steps by which reactants are
55
converted into products. Some research papers reported DPPH• scavenging reactions using
56
pseudo-first order or second-order kinetic models (Pap et al., 2005). However, no deterministic
57
presentations exist comparing multiple samples activities. In 1995, Halliwell defined an
58
antioxidant as "any substance that when present at low concentrations compared to those of an
3 59
oxidized substrate significantly delays or prevents its oxidation" and, this definition is still used
60
to determine the strength of antioxidants.
61
Tea (Camellia sinensis), is rich in polyphenolic compounds known as tea flavonoids (mainly
62
catechins), which have strong antioxidant properties (Paquay et al., 2000). Green tea has been
63
shown to exhibit a potent antioxidant activity by means of its flavonoids (Suganuma et al., 1999).
64
Black tea, which is an oxidized product of green tea, has approximately 20–30% polyphenols
65
(Sanderson et al., 1972), including bisflavonols, aflavins and arubigins (Gupta et al., 2002).
66
Comparisons of the antioxidant activity of green and black teas have been described in the past
67
and shown green tea to exhibit greater antioxidant activity (Van Gadow et al., 1997).
68
Coffea arabica (arabica) has been suggested to contribute up to 64% of total antioxidant
69
intake in the human diet (Svilaas et al., 2004), but its antioxidant activity has been shown to be
70
considerably lower than that of tea.
71
In this paper, we examined the kinetic behavior of some abundant (antioxidant) compounds
72
in selected teas and coffee to compare parameters describing the reaction kinetics for antioxidant
73
activity in a DPPH• system. In the past, a number of authors have presented models that overlap
74
with our experimental results. A major objective of this work was, therefore, to build on existing
75
work in order to assess the parameters for effective evaluation of DPPH• scavenging. This was
76
achieved through a comparison of kinetic parameters (k'2, slope calculated from the reciprocal of
77
DPPH• concentration as a function of time, k'2max: maximum value of the variation of k'2 as a
78
function of time ), IC100 (amount of antioxidant needed to scavenge 100 % of the initial DPPH•
79
concentration), and n (ratio of the initial DPPH• concentration to IC100) and, thus, this study
80
provided both quantitative and qualitative measures of the reactions of antioxidants with DPPH•.
81 82
2. Material and methods 2.1.
Chemicals and samples preparation
83
DPPH• and 2,2-diphenyl-1-picrylhydrazine (DPPH-H) were purchased from Sigma (Lyon,
84
France). HPLC grade methanol was purchased from Panreac (Barcelona, Spain). L-ascorbic acid
85
(98%), gallic acid (98%), tannic acid (98%), (+)-catechin (98%), (-)-epigallocatechin (98%) and
86
caffeic acid (98%) were acquired from Fluka (Lyon, France).
4 87
2.2.
Preparation of plants extracts
88
Green and black teas and coffee (Arabica) were bought from a local specialty teashop.
89
Samples (10 g) were ground to a powder, and extracts prepared by heating samples in double-
90
distilled water (100 ml) for 15 min at 80 °C with stirring. This process was repeated three times.
91
Extracts from each sample were combined, filtered through a Whathman paper N. 5, centrifuged
92
(4500 rpm, 15 minutes) and freeze-dried.
93
2.3.
Apparatus
94
Spectrophotometric data were acquired using a Jasco J-815 Series spectrophotometer (Jasco,
95
USA) equipped with a JASCO SFS-492 Series Stopped-flow systems apparatus. The mixing
96
ratio was fixed to 1:1 with a flow speed of approximately 4 ml/s. Results were displayed using
97
Spectra Manager II software (Jasco, USA). A Jasco V-530 spectrophotometer and disposable
98
cuvettes (1 cm × l cm x 4.5 cm) from Muller Ratiolab (Germany) were used for routine
99
absorbance measurements. All experiments were performed at 25 °C.
100
2.4.
Kinetic analysis
101
A typical reaction mixture contains 0.2 mmol/l DPPH• radicals and either 0.5 mmol/l, 0.20
102
mmol/l, 0.125 mmol/l, 62.5 µmol/l, or 31.25 µmol/l of the pure compound. When plant extracts
103
were used, the extract concentration ranged from 10 to 300 µg/ml. The kinetic measurements
104
were performed at 25 °C for 40 minutes; negative controls (i. e. methanol only) were also run.
105
The decrease in the absorbance for the DPPH• radicals was followed at 515 nm (ε515 nm = 10870
106
(mol/l)-1.cm-1 in methanol) (Fotti et al., 2004). Experimental DPPH• concentrations were
107
determined using a standard curve with DPPH• concentrations ranging from 0 mol/l to 0.3
108
mmol/l, with a correlation coefficient of R2=0.999. The amount of antioxidant needed to
109
scavenge 100% of the initial DPPH• concentration (IC100) was determined graphically. The value
110
n was calculated from the formula:
111
2.5.
Data analysis
112
Data were analyzed using STATISTICA® 10. Values were presented as arithmetic means at
113
95% confidence intervals. Means were compared using F-test. P values < 0.05 were considered
114
statistically significant.
5 115
116
3. Results and discussion
3.1.
General
117
Re-consideration of kinetic parameters has previously been proposed by Perez-Jimenez and
118
Saura-Calixto (2008) in order to provide more information about antioxidant activities. Also,
119
Goupy et al. (2003) suggested that reaction kinetics could be more informative than overall
120
antioxidant potential determined by conventional endpoint parameters. In this context, we used a
121
stopped-flow technique to follow rates of DPPH• scavenging and, thus, provide more reliable,
122
real-time data for comparison of antioxidant scavenging.
123
Data describing the reaction between DPPH• and other hydrogen donor molecules are
124
available in the literature. Our purpose was to better understand reactions with DPPH• and to
125
assess DPPH• scavenging activity of some popular beverages alongside established bioactive
126
compounds through mechanistic measurements. Preliminary observations showed most of the
127
scavengers reached a steady-state reaction after a period of time, which depended to the
128
DPPH•/antioxidant ratio and chemical structure. We assessed the reliability of several models to
129
obtain kinetic parameters capable of characterizing the behavior of bioactive compounds. Those
130
compounds were chosen from the chemical composition of the selected beverages.
131
Scavenging of DPPH• is achieved through hydrogen/electron transfer from a given
132
antioxidant to DPPH•. Although DPPH-H is the final product, other complexes form between
133
DPPH• and the oxidized intermediates of the antioxidant or two or more oxidized forms, which
134
generate high molecular weight polymers (Osman, 2011). This hydrogen/electron transfer can be
135
simulated by a reaction where the product does not participate in the reverse reaction and where
136
stoichiometry may vary as the reaction proceeds. In this context, species either regenerate
137
through interaction with the solvent or produce other active species that carry on the reaction
138
responsible for the formation of the final products.
139
In general, the first (rapid) step is attributed to the oxidation of antioxidants by DPPH•, and
140
the subsequent slow step(s) is/are due to secondary reaction(s) of the oxidized antioxidants. In
141
addition, we observed the following features:
142
(1) Stoichiometric coefficients varied throughout the evolution of the reaction;
143
(2) Initial rates were not affected by the presence of the product, and
144
(3) Initial reaction rates were not necessarily the most important during scavenging reactions.
6 145
We also noticed three major kinetic behaviors that varied in speed (fast, moderate and slow),
146
as previously discussed by Brand-Williams et al. (1997). DPPH• scavenging reactions can be
147
simulated by a chain reaction (Denga et al., 2011) and is commonly used during kinetic analysis.
148
Therefore, scavenging of DPPH• by a scavenger (AH) can be presented as:
149 150 151 152
The rates of DPPH• loss during the initial step can follow a pseudo-first or a second-order
153
reaction, depending on the antioxidants chemical structure. Our kinetic results show that ascorbic
154
acid and gallic acid follow a pseudo-first-order.
155
During our analysis of other compounds (e. g. (+)-catechin, tannic acid, (-)-epigallocatechin,
156
and caffeic acid), we observed these reactions appeared to follow a second-order model. By
157
applying such a model, we supposed the antioxidant (AH) concentration decreased with
158
decreasing DPPH• concentration according to the equation
159
variable that maintains this equilibrium throughout the reaction. Therefore, the equation for the
160
reaction rate can be presented as:
161
variables and integration, eqn. 2 can be written as:
162
written as
163
nor as function of DPPH• concentration, the integral
164
function and, thus, included within the rate constant value of the second-order model. The
165
second-order integrated rate equation can be presented as:
166
where
167
168
, where α is a
(eqn. 2). After separation of (eqn. 3). Thus, eqn. 3 can be
(eqn. 4). As α can neither be integrated as a function of time can be considered as a primitive
.
3.2.
Kinetic analysis of the pure compounds in reaction with DPPH•
3.2.1. Ascorbic acid
(eqn. 5)
7 169
Fig. 1 (A) shows how the natural logarithm of DPPH• concentration is dependent on time
170
and Fig. 1 (B) shows the dependence of the pseudo-first-order rate constant on the initial
171
concentration during the reaction with DPPH•. Experimental data showed a lack of fit with the
172
second-order model, in contrast with the results presented by Mishra et al., (2012). (See Table 1
173
for the rate constants.) Ascorbic acid exhibits a rapid reaction with DPPH• through a two-step
174
mechanism. The first step is rapid with rate constants dependent on the initial concentration of
175
ascorbic acid. The second step is slower and involves dehydroascorbic acid as downstream
176
intermediate, with an n value of ≈ 2: (Sawai, 2000). IC100 for ascorbic acid was 98.9000 ± 0.0091
177
µmol/l (mean ± SD, N = 4) and n of 2.222 ± 0.284.
178
3.2.2. Gallic acid
179
Gallic acid is a phenolic compound occurring in different food plants that has been widely
180
used as a standard in the DPPH• assay. In our experiments, its reaction with DPPH• followed a
181
pseudo-first-order kinetic model. Fig. 2 (A) shows the linear evolution of the logarithm for
182
DPPH• concentration as a function of time. The calculated slopes (k’2) showed a linear
183
correlation with the initial concentration of gallic acid (Fig. 2 (B)). IC100 for gallic acid was
184
36.000 ± 0.004 µmol/l (mean ± SD, N = 4) and n of 5.555 ± 0.642. The segments slopes in Fig. 2
185
define the pseudo-first-order constant rate and are summarized in Table 1.
186
3.2.3. (+)-Catechin
187
The reaction of (+)-catechin’ with DPPH• is relatively slow. The reaction time to reach the
188
steady state was relatively long and depended on the (+)-catetchin concentration. Inconsistencies
189
in the total stoichiometric values for (+)-catechin have been reported previously (Villano et al.,
190
2007) and were attributed to the incubation time, the nature of the used solvent and the ratio of
191
flavanol to DPPH• radicals (Dimitrios et al., 2006). We calculated (+)-catechin IC100 and n as
192
49.000 ± 0.005 µmol/l (mean ± SD, N = 4) and 4.081 ± 0.503 (mean ± SD, N = 4), respectively.
193
n was in agreement with previous reports on (+)-catechin stoichiometry (Osman, 2011). As
194
shown in Fig. 3 (A), the variation in the reciprocal for DPPH• concentration versus time
195
indicated second-order kinetics and showed a linear evolution of the rate constant (k’2) as a
196
function of the initial concentration of (+)-catechin, (Fig. 3B, and Table 1). When we plotted the
8 197
evolution of the second-order rate constant (k’2) (using eqn. 5) as a function of time (Fig. 3 (C)),
198
we observed that the rate constants reached a maximum value (k’2max), similar to those for slope
199
of the Fig. 3 linear segment (A-B) at a given concentration (see Table 1 for the corresponding
200
rate constants values), followed by a progressive but slow decrease, indicating the progress of the
201
reaction. Furthermore, the variation in the second-order rate constant as a function of the initial
202
concentration of (+)-catechin correlated with the dependence of k’2max on the initial concentration
203
of (+)-catechin (Fig. 3 (D)). Osman (2011) showed the reaction of (+)-catechin with DPPH• is a
204
multistep, mainly biphasic, reaction with the first step followed by a slow decrease, probably due
205
to the low reactivity of the downstream intermediates (Sang et al., 2003). We showed that the
206
evolution of the second-order rate constant can tell us more about the behavior of the reaction
207
than simply the reciprocal of DPPH• concentration as a function of time.
208
3.2.4. Tannic acid
209
Tannic acid is a plant polyphenol found in several beverages including red wine, beer,
210
coffee, and black and green teas (Chung et al., 1998; King et al., 1999). Like other polyphenols,
211
tannic acid has been shown to possess antioxidant activities (Andrade et al., 2005). Tannic acid
212
exhibited second-order kinetics for its reaction with DPPH•. The slope of the reciprocal for the
213
DPPH• concentration as a function of time, extracted from the linear curve in Fig. 4 (A), showed
214
a linear dependence on the initial concentration of tannic acid (Fig. 4 (B) and Table 1). Plotting
215
k’2 values as a function of time showed the occurrence of two maxima (k’2max), as shown in Fig.
216
4 (C) and Table 1, and these showed a linear correlation when plotted as a function of the initial
217
tannic acid concentration, as presented in Fig. 4 (D). IC100 and n were 31.500 ± 0.005 µmol/l
218
(mean ± SD, N = 4) and 6.349 ± 0.353 (mean ± SD, N = 4), respectively.
219
3.2.5. (-)-epigallocatechin
220
(-)-epigallocatechin, one of the most important catechins found in tea, exhibited moderate
221
kinetics in reaction with DPPH•. Fig. 5 (A-B) shows the dependence of the reciprocal for the
222
DPPH• concentration as a function of time. Slopes in Fig. 5 (A) show a linear relation with the
223
initial concentration of (-)-epigallocatechin. Such a relationship suggests a second-order model in
224
this reaction. Values for the second-order rate constants (Table 1) show the significant activity of
9 225
(-)-epigallocatechin toward DPPH• as compared with (+)-catechin. IC100 and n were 48.100 ±
226
0.002 µmol/l (mean ± SD, N = 4) and 4.154 ± 0.264 (mean ± SD, N = 4), respectively. When
227
plotting the evolution of the second-order rate constant (k’2) (calculated from eqn. 5) as a
228
function of time (Fig. 5 C), we observed the second-order rate constants reached a maximum
229
(k’2max) similar to that of the linear segment slope from Fig. 5 (A-B) at a given concentration,
230
(see Table 1 for the corresponding rate constants values); these rates remained constant with a
231
slow decrease indicating the progress of the reaction. Furthermore, the variation in the second-
232
order rate constant as a function of the initial of (-)-epigallocatechin concentration correlated
233
with the dependence of k’2max on the initial concentration of (-)-epigallocatechin (Fig. 5 (D)).
234
3.2.6. Caffeic acid
235
Caffeic acid (3,4-dihydroxycinnamic acid) is found in a wide variety of plant-derived
236
products such as wine, coffee beans, fruits, vegetables, olive oil and tea (Shahidi & Naczk,
237
1995). It reacts with the DPPH• through moderate kinetics. Fig. 6 (A) shows the dependence of
238
the reciprocal for the DPPH• concentration as a function of time and indicates second-order
239
kinetics. The dependence of the second-order rate constant (k’2) shows a linear correlation, as a
240
function of the initial concentration of caffeic acid (Fig. 6 (B)). The second-order rate constant
241
k’2 was extracted from the slope for each caffeic acid concentration, and values are summarized
242
in Table 1. The evolution of second-order rate constants (calculated from eqn. 5) as a function of
243
time shows the rate constants reached a maximum, similar to the corresponding k’2max, calculated
244
from the curves in Fig. 6 (C) (see Table 1 for values). Furthermore, the dependence of the
245
second-order rate constants on the initial concentrations of caffeic acid shows a linear correlation
246
with the dependence of the k’2max on the initial concentration of caffeic acid. This first step is
247
governed by the reaction of caffeic acid with DPPH•. A lower k’2max was also observed in a
248
second step. These observations suggest a multistep reaction; a moderate step followed by a slow
249
step, probably due to the low reactivity of the downstream intermediate(s). Also, the
250
corresponding values for k’2max showed a linear dependence on the initial concentration of the
251
caffeic acid. IC100 and n were 71.000 ± 0.005 µmol/l (mean ± SD, N = 4) and 2.814 ± 0.315
252
(mean ± SD, N = 4), respectively.
253
10
In summary, (-)-epigallocatechin showed the highest k’2max at IC100 913.7 ± 57.1 (nmol/l)-1.s-
254 255
1
, followed by caffeic acid (458.900 ± 0.053 (µmol/l)-1. s-1) then tannic acid with two k’2max
256
(0.2873 ± 0.0061(µmol/l)-1. s-1 and 0.0582 ± 0.0041 (µmol/l)-1. s-1) and finally (+)-catechin
257
(6.2.10-7 ± 0.6.10-7 (mol/l)-1. s-1). In general, k’2max corresponded to the antioxidant activity of
258
the initial molecule, which remained independent of the downstream reactions involving
259
intermediates. This involvement explains the different order in scavenging activity when the
260
antioxidants are compared according to IC100. This difference exists because IC100 is derived
261
from the action of the initial antioxidant as well as intermediate forms.
262
We demonstrates that not all antioxidants in a DPPH• system behave according to the same
263
kinetic model, suggesting the choice of kinetic model has to be determined independently for
264
each sample, particularly if its chemical composition is not fully determined, in order to
265
understand fully its antioxidant activity. Our results show most significant antioxidant
266
compounds present in the selected beverages (coffee, and green and black teas) exhibited
267
second-order kinetics. The variation of k’2 as a function of time is, therefore, a valuable
268
parameter for describing kinetic behavior of an antioxidant. This provides a clear description of
269
the extent of DPPH• scavenging. Furthermore, the profiles of such plots were characteristic for
270
each compound.
271
During the course of the reaction, reactive intermediate(s) may be generated and such
272
intermediate(s) may exhibit higher or lower activity compared to the initial compound. The
273
evolution of k’2 over time could, therefore, be used to predict complexity of the mechanistic
274
model. Indeed, values obtained for k’2 and the k’2max correlate significantly with a Pearson values
275
of 0.9894, 0.9928, 0.9969, and 0.9981 for (+)-catechin, (-)-epigallocatechin, caffeic acid and
276
tannic acid, respectively. In order to apply a second-order reaction kinetic model, the
277
concentration of DPPH• has to be maintained in proportion to the concentration of the
278
antioxidant through the arbitrary α factor.
279
3.3.
Determination of the IC100 and n values
280
We performed a kinetic analysis of six pure compounds (ascorbic acid, gallic acid, (+)-
281
catechin, (-)-epigallocatechin, tannic acid and caffeic acid), each present in at least one of the
282
three beverages studied. Each compound was tested at different concentrations. The IC100 value,
283
defined as the amount of antioxidant necessary to decrease the concentration of DPPH• by 100
11 284
%, was used as a comparative parameter to define the strength and the stoichiometry of an
285
antioxidant in reaction with DPPH•. An example illustrating the calculation of the IC100 of tannic
286
acid is shown in Fig. 7. In general, the higher the IC100, the weaker the antioxidant. Thus, we can
287
conclude from the IC100 values (Table 1) that tannic acid exhibits the highest antioxidant activity,
288
followed by caffeic acid, (-)-epigallocatechin, (+)-catechin, ascorbic acid, and then gallic acid.
289
Also, the relative stoichiometry (n) was shown to be relevant for the comparison of the
290
antioxidant strength between beverages, as well as between pure compounds, especially since we
291
did not note any significant difference in this parameter when observed at different
292
DPPH•/antioxidant ratios.
293
3.4.
Kinetic analysis of the extracts from green tea, black tea and coffee
294 295
We investigated the antioxidant effects of green and black teas and coffee on DPPH•. Table 2
296
presents the kinetic parameters extracted from the analysis of the three beverages. As shown in
297
Fig. 8 (A-B), green tea extract showed rapid kinetics, fitting a second-order model. Fig. 8 (C-D)
298
shows the reaction is characterized by a rapid increase in k’2 as a function of time reaching a
299
maximum value in a short period of time followed by a decrease to a minimal. Similarities in
300
k’2max and k’2 support our suggestion the reaction kinetic sequence should be presented as the
301
change in k’2 as a function of time and elucidate scavenging properties of food/ food extract
302
when using DPPH•. IC100 was calculated and values are presented in Table 2.
303
Black tea showed a lower antioxidant activity than green tea. The kinetic behavior of black
304
tea (extract) followed a second-order kinetic model, and its rate constants and IC100 were
305
calculated accordingly (Table 2). IC100 was largely higher (≈ 5 fold) than that for green tea. In
306
addition, analysis of the evolution of k’2 for black tea (Fig. 9 (C)) indicated the occurrence of low
307
kinetic behavior antioxidant compounds compared with green tea extracts, which exhibits a rapid
308
kinetics and high second-order rate constants (Fig. 8 (C)). Values for the slopes extracted from
309
Fig. 9 (A) and presented in Fig. 9 (B) are in agreement with those obtained for k’2max. The change
310
in green tea composition during fermentation to produce black tea is probably the main causes of
311
the change in its antioxidant activity (Rietveld and Wiseman, 2003). This change in composition
312
would certainly explain the relatively high antioxidant potential of green tea found in our study.
12 313
Coffee (extract) showed DPPH• scavenging activity somewhere between those exhibited by
314
green and black teas, in contrast to what was previously reported by Svilaas et al. (2004). The
315
kinetic behavior showed its dependence on a second-order kinetic model. The values for second-
316
order model rate constants and IC100 were calculated and are presented in Table 2. Interestingly,
317
IC100 for coffee was similar to that for green tea extract, but the second-order rate constants were
318
significantly different. We observed the evolution of k’2 from coffee extract (Fig. 10 (C)) and
319
black tea (Fig. 9 (C)) was similar to that for tannic acid, which would correlate with the
320
occurrence of high concentrations of tannins accumulated in both beverages during processing.
321
Similarly, the same correlation was observed when comparing the evolution of k’2 for green tea
322
with those of (+)-catechin and (-)-epigallocatechin. All these beverages demonstrate antioxidant
323
activity as a result of the concentration of naturally occurring bioactive compounds.
324
4.
Conclusion
325
We attempted to shed light on kinetic behavior as a tool for evaluation and comparison of
326
different antioxidants in reaction with DPPH•. Although used extensively, the DPPH• assay
327
remains a crude tool for comparing the efficiency of natural compounds against free radicals.
328
Indeed, data about parameters such as IC50 or IC100 lack information about the kinetic
329
performance of antioxidant. This study provides more reliable kinetic tools for comparing
330
antioxidants. Kinetic analysis of the reactions of the selected pure compounds with DPPH•
331
allowed us to evaluate progress of the reactions as a function of time through changes in rate
332
constant. This analysis reflects real-time monitoring of the reaction. Based on the kinetic and an
333
endpoint analyses, our results revealed different rankings for scavenging for selected antioxidant
334
compounds. These differences suggest a combination of both approaches would allow
335
researchers to perform a better comparison between antioxidant or food samples. Expression of
336
the results in kinetic terms not only takes into account antioxidant activity, but also provides
337
valuable information on the general behavior (i.e. speed, reversibility, etc.). Thus, we
338
recommend use of both kinetic and fixed endpoints be combined so as to provide comprehensive
339
information about antioxidant potential in a given sample. The proposed approach, based on a
340
second-order model, as compared with suggested kinetic models previously, was more
341
discriminative and consistent with regard to the DPPH•/antioxidant reaction behavior, especially
342
when considering a multistep mechanism that includes intermediate(s) exhibiting different
13 343
efficiencies toward DPPH•. Parameters extracted from this model provide a complementary
344
method and additional understanding of potential antioxidants in food or pharmaceutical
345
applications. Based on the analysis of the kinetic behavior in the present paper, we recommend:
346
High concentrations of DPPH• to maintain the proportion of DPPH• to antioxidant.
347
Prolonged reaction times for compounds exhibiting low kinetic reaction.
348
Pure compounds should be used as standards and selected from the same family of
349 350
351
compounds in the food source.
Actual kinetic model be applied in analyses should be selected based on preliminary data.
Acknowledgments
352
The authors are thankful to Dr. Siân Astley and Dr. Jack Kalpakian for their thorough review of
353
the manuscript. The project has been funded by Al Akhawayn University in Ifrane University‟s
354
seed Research Fund.
355
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Table 1: Summary of the kinetic parameter constant rate of the reaction of gallic acid, ascorbic acid, (+)-catechin, (-)-epigallocatechin, caffeic acid and tannic acid with DPPH• at 0.2 mmol/l. 0.5 mmol/l
0.20 mmol/l
0.125 mmol/l
0.0625 mmol/l
0.03125 mmol/l
k1
k1
k1
k1
k1
0.0170 ± 5.6.10-3 0.101 ± 0.093 k’2 k’2max
0.0092 ± 2.5.10-3 0.095 ± 0.063 k2 k’2max
0.0051 ± 4.1.10-4 0.076 ± 0.061 k2 k’2max
0.0030 ± 3.3.10-4 0.0101 ± 2.11-3 k2 k’2max
Constant rate (s-1) Gallic acid Ascorbic acid Constant rate ((mmol/l) -1.s-1) (+)-catechin
(-)-epigallocatechin
Caffeic acid Constant rate ((mmol/l) -1.s-1)
8.13.10-6 ± 1.3.10-6
15.4.10-6 ± 9.2.10-6
3.1.10-6 ± 0.2.10-3
7.1.10-6 ± 0.9.10-6
1.6.10-6 ± 0.8.10-3
2.7.10-6 ± 0.9.10-6
0.0010 ± 0.0005
1.6.10-6 ± 0.3.10-3
0.601.10-6 ± 0.012.10-3
0.62.10-6 ± 0.06.10-3
1.79.10-3 ± 0.18.10-3 1.94.10-3 ± 84.2.10-6
1.76.10-3 ± 0.43.10-3 3.21.10-3 ± 0.98.10-3
0.812.10-3 ± 0.035.10-3 0.85.10-3 ± 58.1.10-6
1.002.10-3 ± 0.204.10-3 1.23.10-3 ± 51.1.10-6
0.47.10-3 ± 58.1.10-6 0.46.10-3 ± 59.1.10-5
19.74.10-5 ± 6.3.10-6 0.32.10-3 ± 78.3.10-6
25.24.10-5 ± 16.4.10-6 0.42.10-3 ± 49.1.10-6
10.58.10-5 ± 51.6.10-6 0.11.10-3 ± 88.1.10-6
11.37.10-5 ± 38.1.10-6 0.24 .10-3 ± 94.1.10-6
k’2
k’2max1/k’2max2
k’2
k’2max1/k’2max2
k’2
0.538.10-3 ± 0.054.10-3 62.25.10-5 ± 58.1.10-6 k’2max1/ k’2max2 0.42.10-3 ± 81.5.10-5 0.18.10-3 ± 1.75.10-6
k’2
k’2max1/k’2max2
k’2
k’2max1/k’2max2
-3
-3
Tannic acid
0.0015 ± 5.1.10-4 0.0030 ± 3.110-3 k2 k’2max
1.79.10 ± 0.46. 10-3
0.75.10 ± 32.8.10-6 0.47.10-3 ± 22.1.10-6
-3
-3
0.75.10 ± 32.7.10-3
0.48.10 ± 1.6.10-6 0.25.10-3 ± 52.2.10-6
-3
0.47.10 ± 8.1.10-6
-3
-3
0.20.10 ± 44.6.10-6
0.33.10 ± 5.21.10-3 9.35.10-5 ± 4.3.10-6
IC100 (mol/l)
n (mmol/l.( mmol/l) -1
36.1.10-6 ± 4.10-6 98.9.10-6 ± 9.110-3
5.555 ± 0.642 2.222 ± 0.284
0.0491 ± 0.0057
4.081 ± 0.503
0.0481± 0.0021
4.154 ± 0.264
0.0712 ± 0.0053
2.81 ± 0.31
IC100 (mol/l)
n (mmol/l. (mmol/l )-1)
0.0315 ± 0.0051
6.349 ± 0.353
-3
-3
0.11.10 ± 8.1.10-6
0.29.10 ± 6.1.10-3 0.058.10-3 ± 0.004.1.10-6
Table 2: Summary of the kinetic parameters of the pseudo-first order and second order, and the IC100 and the stoichiometry of the reaction of green tea, black tea and coffee extracts with DPPH• at 0.2 mmol/l 0.210 mg/ml Constant rate (mg/ml-1.s-1) Green tea
Black tea
Coffee
k’2 3.2781 ± 0.9393 0.0421 ± 0.0328 0.2589 ± 0.0832
k’2max 3.2124 ± 0.5810 0.1795 ± 0.0511 0.2789 ± 0.0471
0.180 mg/ml k’2 2.7725 ± 0.7021 0.0314 ± 0.0062 0.2340 ± 0.0921
k’2max 2.6489 ± 0.531 0.1485 ± 0.0512 0.2540 ± 0.0571
0.150 mg/ml k’2 2.216 ± 0.371 0.0262 ± 0.0511 0.2067 ± 0.0441
k’2max 2.2577 ± 0.6429 0.1082 ± 0.0113 0.2053 ± 0.0381
0.11 mg/ml k’2 1.6912 ± 0.0536 0.0164 ± 0.0022 0.1837 ± 0.0552
k’2max 1.5955 ± 0.3812 0.0940 ± 0.0081 0.1633 ± 0.0121
0.050 mg/ml k’2 0.7630 ± 0.0381 0.0070 ± 0.0001 0.0712 ± 0.0021
k’2max 0.5632 ± 0.0562 0.0382 ± 0.0051 0.0866 ± 0.0011
0.025 mg/ml k’2 0.2545 ± 0.0516 0.0036 ± 0.00074 0.0451 ± 0.0045
k’2max 0.2475 ± 0.0231 0.0213 ± 0.0013 0.0555 ± 0.0081
IC100 (mg/ml)
Stoichiometry (n, (mmol/l). ml/mg)
0.0318 ± 0.0024
6.289 ± 0.8724
0.1531 ± 0.0431
1.300 ± 0.424
0.0365 ± 0.0024
5.479 ± 0.524
(A') 2.0
(B)
(A) 0.15
5.10-4 mol/l -5 25.10 mol/ l 62.5.10 -6 mol/ l 31.25.10 -6 mol/ l -6 11.5625.10 mol/ l
0.4 0.3 0.2
5.10-4 mol/l 25.10-5 mol/l 62.5.10-6 mol/l 31.25.10-6 mol/l 11.5625.10-6 mol/l
0.1 0.0
1.5
0
1
2
3
4
5
Time (s)
1.0
k 1 (s-1 )
ln([DP PH . ] t/[DPP H .] 0 )
ln([DPPH .] t /[DPPH .] 0 )
2.5
0.10
0.05
0.5 0.0 0
5
10
15
20
0.00 0.0
25
Time (s)
0.1
0.2
0.3
c0 ascorbic acid (.10 -3 mol/l)
Figure 1: (A), variation of the logarithm of the DPPH• concentration on the time in reaction with different concentrations of ascorbic acid at 25 °C. (A’) show the magnification from t=0 to t=4 (s), and (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of ascorbic acid.
(A)
(B) 0.020
-4
5.10 mol/l 25.10-5 mol/l
0.015
5
62.50.10-6 mol/l 4
100
200
Time (s)
0.010
31.25.10-6 mol/l 11.56.10-6 mol/l
0.005
300
0.000 0.0
3 0
k 1 ( s -1 )
ln ([D P PH .] t /[D PPH .] 0 )
6
0.2
0.4
0.6
c0 gallic acid (.10 -3 mol/l)
Figure 2: (A), variation of the logarithm of the DPPH• concentrations on the time in the reaction with gallic acid at 25 °C at the indicated concentrations, and (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of gallic acid.
4 -5
25.10 mol/l 62.50.10-6 mol/l 31.25.10-6 mol/l 11.56.10-6 mol/l
0 0
200
400
600
800
0.006 0.004 0.002 0.000 0.0
1000
Time (s)
(C) 12.5 10-5 mol/l
0.002
0.001 -5
62.510 mol/l 31.25 10-6 mol/l
0.000 0
500
1000
Time (s)
0.2
0.4
0.6
c0 (+)-catechin (.10 -3 mol/l)
0.003
k'2 (.10-3 mol/l -1.s -1)
slope (10 -3 (mol/l) -1.s -1)
6
1500
k'2max (10-3 (mol/l)-1.s-1)
1 /[ D P P H .] t -1 /[ D P P H .] 0 ( 1 0 - 3 (m ol/l) - 1 )
5.10-4 mol/l
2
(B)
0.008
(A)
8
(D)
0.015
0.010
0.005
0.000 0.0
0.2
0.4
0.6
-3
c0 (+)-catechin (.10 mol/l)
Figure 3: (A), dependence of the reciprocals of the DPPH• concentrations on the reaction time with (+)catechin at the indicated concentrations at 25 °C, (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of (+)-catechin. (C), variations of k’2 as function of time. (D), dependence of k’2max on the initial concentration of (+)-catechin.
40
25.10-5 mol/l
20
62.50.10-6 mol/l 31.25.10-6 mol/l 11.56.10-6 mol/l
0 0
50
100
150
200
slope (.10 -3 (mol/l) -1.s -1)
5.10-4 M
0.20 0.15 0.10 0.05 0.00 0.00
250
Time (s)
(C) 5.10 mol/l 25.10-5 mol/l 62.50.10-6 mol/l 31.25.10-6 mol/l 11.56.10-6 mol/l
0.6 0.4 0.2 0.0 100
200
0.10
0.15
0.20
0.25
300
Time (s)
(D)
0.8 -4
0
0.05
c0 tannic acid (.10 -3 mol/l)
400
500
k'2max (.10-3 (mol/l)-1.s -1)
1/[DPPH.] t -1/[ DPPH.] 0 (.10-3 (mol/l) -1)
60
0.8
k'2 (.10-3 (mol/l)-1.s-1)
(B)
0.25
(A)
First peak Second peak
0.6 0.4 0.2 0.0 0.0
0.2
0.4
0.6
c0 tannic acid (.10 -3 mol/l)
Figure 4: (A), dependence of the reciprocals of the DPPH• concentrations on the time in the reaction with tannic acid at a temperature of 25 °C at the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l, (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of tannic acid, (C), variations of k’2 versus time, (D), dependence of k’2max on the initial concentration of tannic acid.
40
62.5.10-6 mol/l 20 -6
31.25.10 mol/l
slope (.10 -3 (mol/l) -1.s -1)
25.10-5 mol/l 12.5.10-5 mol/l
60
0
200
400
600
800
1.5 1.0 0.5 0.0 0.0
0 1000
0.2
0.4
0.6
c0 (-)-epigallocatechin (.10 -3 mol/l)
Time (s)
(D)
(C) 2.0
k'2max (.10-3 (mol/l)-1.s-1)
1/[DPPH.] t -1/[ DPPH .] 0 (.10-3 (mol/l) -1)
2.0
5.10-4 mol/l
2.0
k'2 (.10-3 (mol/l)-1.s-1)
(B)
(A)
80
5.10-4 mol/l 25.10-5 mol/l 12.5.10-5 mol/l 62.5.10-6 mol/l 31.25.10-6 mol/l
1.5 1.0 0.5 0.0 0
100
200
300
Time (s)
400
500
1.5 1.0 0.5 0.0 0.0
0.2
0.4
0.6
c0 (-)-epigallocatechin (.10 -3 mol/l)
Figure 5: (A), Dependence of the reciprocals of the DPPH• concentrations on the time in the reaction with (-)epigallocatechin at a temperature of 25 °C at the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l, (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of of (-)epigallocatechin, (C), variations of k’2 versus time, (D), dependence of k’2max on the initial concentration of (-)epigallocatechin.
20
0
50
100
150
2.0 1.5 1.0 0.5 0.0 0.0
0 200
Time (s)
0.4
0.2
0.0 100
200
Time (s)
0.4
0.6
300
(D)
4
10-4 mol/l 8.10-5 mol/l 6.10-5 mol/l 4.10-5 mol/l 2.10-5 mol/l
0
0.2
c0 caffeic acid (.10 -3 mol/l)
(C)
0.6
k'2 (.10-3 (mol/l) -1.s-1)
slope (.10 -3 (mol/l) -1.s -1)
5.10-4 mol/l 25.10-5 mol/l 12.5.10-5 mol/l 62.5.10-6 mol/l 31.25.10-6 mol/l
40
(B)
2.5
400
k'2m ax (.10-3 (mol/l) -1.s -1)
1/[DPPH.]t-1/[ DPPH.]0 (10-3 (mol/l)-1)
(A)
3 2 1 0 0.0
0.2
0.4
0.6
-3
c0 caffeic acid (.10 mol/l)
Figure 6: (A), Dependence of the reciprocals of the DPPH• concentrations on the time in the reaction caffeic acid at a temperature of 25 °Cat the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l, (B), ), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of caffeic acid, (C), variations of k’2 versus time during the reaction of 0.2 mmol/l, (D), dependence of k’2max on the initial concentration of caffeic acid.
DPPH.scavenging percentage (%)
100 80 60 40 20 0 0.00
0.02
0.04
0.06
0.08
0.10
c0 tannic acid (.10 -3 mol/l)
Figure 7: graphical determination, of IC100 value of tannic acid during its reaction with DPPH•. The scavenging • • percentage (%) was calculated as at a given concentration of an antioxidant. •
0.66 mg/ml 0.25 mg/ml 0.21 mg/ml 0.18 mg/ml 0.15 mg/ml 0.11 mg/ml
60 40 20 0 0
5
10
15
(B)
4
slope ((mg/ml)-1.s -1)
1/[DPPH.] t-1/[ DPPH.] 0 (mg/ml) -1
(A) 80
3 2 1 0 0.00
20
0.05
Time (s)
(C)
3 2 1 0 0
100
200
0.15
0.20
0.25
300
Time (s)
400
(D)
4
0.2 mg/ml 0.75 mg/ml 0.65 mg/ml 0.55 mg/ml 0.45 mg/ml 0.35 mg/ml
k'2max ((mg/ml)-1.s-1)
-1 -1
k'2 ((mg/ml) .s )
4
0.10
c0 green tea (mg/ml)
500
3 2 1 0 0.00
0.05
0.10
0.15
0.20
0.25
c0 green tea (mg/ml)
Figure 8: (A), dependence of the reciprocals of the DPPH• concentrations on the time in the reaction with green tea extract at a temperature of 25 °C at the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l, (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of green tea extract, (C), variations of k’2 versus time, (D), dependence of k’2max on the initial concentration of green tea.
40 30 20 10
50
100
0.04 0.03 0.02 0.01 0.00 0.00
0 0
(B)
0.05
0.75 mg/ml 0.45 mg/ml 0.35 mg/ml 0.25 mg/ml 0.1 mg/ml
slope ((mg/ml)-1.s -1)
1/[DPPH.] t -1/[ DPPH.] 0 (mg/ml) -1
(A)
150
0.05
0.10
0.15
(C)
0.25
(D) 0.20
0.75 0.65 0.55 0.45 0.35
0.4
mg/ml mg/ml mg/ml mg/ml mg/ml
0.2
0.0 0
100
200
300
Time (s)
400
500
k'2max ((mg/ml)-1.s -1)
0.6
k'2 ((mg/ml)-1.s-1)
0.20
c0 black tea (mg/ml)
Time (s)
0.15 0.10 0.05 0.00 0.00
0.05
0.10
0.15
0.20
0.25
c0 black tea (mg/ml)
Figure 9: (A), dependence of the reciprocals of the DPPH• concentrations on the time in the reaction with black tea extract at a temperature of 25 °C at the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l. (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of black tea, (C), variations of k’2 versus time, (D), dependence of k’2max on the initial concentration of black tea extract.
0.3
0.5 mg/ml 0.4 mg/ml 0.38 mg/ml 0.333 mg/ml 0.25 mg/ml
40
20
slope ((mg/ml)-1.s -1)
1/[DPPH.] t -1/[ DPPH .] 0 (mg/ml)-1
(B)
(A)
60
0 0
100
200
300
400
0.2
0.1
0.0 0.00
500
0.05
0.0 200
Time (s)
300
400
k'2max ((mg/ml)-1.s -1)
k'2max ((mg/ml)-1.s-1)
0.5 mg/ml 0.4 mg/ml 0.35 mg/ml 0.3 mg/ml 0.25 mg/ml
0.1
100
0.20
0.25
0.3
0.2
0
0.15
(D)
(C)
0.3
0.10
c0 coffee (mg/ml)
Time (s)
0.2
0.1
0.0 0.00
0.05
0.10
0.15
0.20
0.25
c0 coffee (mg/ml)
Figure 10: (A), dependence of the reciprocals of the DPPH• concentrations on the time in the reaction with coffee extract at a temperature of 25 °C at the indicated concentrations, with fixed DPPH• concentration at 0.2 mmol/l. (B), kinetic dependence of the slope of the linear curve from (A) on the initial concentration of coffee extract, (C), variations of k’2 versus time, (D), dependence of k’2max on the initial concentration of coffee extract.
Research highlights:
Kinetic and endpoint analysis were combined for reliable comparison of antioxidants activities
Parameter k’2 offers a better understanding of the antioxidant’s kinetic behavior
Parameters k ’2max, IC100 and n can be used to compare antioxidants potential
The kinetic analysis was applied to compare antioxidant activity of 3 natural beverages
