Eur Food Res Technol (2002) 215:437–442 DOI 10.1007/s00217-002-0582-2
O R I G I N A L PA P E R
Kerstin Höner · Rinaldo Cervellati
Measurements of the antioxidant capacity of fruits and vegetables using the BR reaction method
Received: 3 May 2002 / Revised: 18 June 2002 / Published online: 6 September 2002 © Springer-Verlag 2002
Abstract The antioxidant capacity of aqueous extracts of 20 vegetables and 16 fruits have been determined using the Briggs-Rauscher (BR) reaction method. Like other methods, the BR reaction method is also based on the generation of free radicals in the reaction mixture. Antioxidant scavengers of free radicals added to an active oscillatory BR regime cause an immediate cessation of the oscillatory regime, an inhibition time that linearly depends on the amount of the antioxidant added, and subsequent regeneration of oscillations. The BR reaction method works at pH≈2, which is similar to that of the fluids in the human stomach. It is known that a vegetarian diet can reduce the risk of stomach cancer and it is therefore interesting to determine the activity of antioxidants at low pH values. Different plants were tested with the BR reaction method, recording potentiometrically the inhibition times produced by their extracts on an active BR mixture. The results concerning the order of the antioxidant activity of the examined plants are illustrated and discussed. A comparison with the ranking order obtained with other methods is also given. Keywords Antioxidants · Briggs-Rauscher measurements · Fruits · Vegetables Abbreviations BR: Briggs-Rauscher · DPPH: 2,2-Diphenyl-l-picrylhydrazyl · DMPD: N,N-Dimethyl-p-phenylendiamine · FRAP: Ferric reducing ability of plasma · LDL: Lower density K. Höner (✉) Institut für Fachdidaktik der Naturwissenschaften, Abt. Chemie und Chemiedidaktik, Technische Universität Braunschweig, Pockelsstrasse 11, 38106 Braunschweig, Germany e-mail:
[email protected] Tel.: +49 5313912876, Fax: +49 5313912845 R. Cervellati Dipartimento di Chimica ‘G. Ciamician’, Università di Bologna, Via Selmi, 2, 40126 Bologna, Italy
lipoproteins · ORAC: Oxygen radical absorbance capacity · PCL: Photochemluminescence · TEAC: Trolox equivalent antioxidant capacity · TRAP: Total radical-trapping antioxidant parameter
Introduction There is considerable evidence that the antioxidants contained in fruits, vegetables and beverages play an important role in the maintenance of health and in prevention of disease [1, 2, 3, 4]. A connection has been made between the intake of high carotenoid-containing fruits and vegetables and protection from certain cancers [5, 6], and recent work is also beginning to highlight the role of the phenolic constituents of the diet in contributing to these protective effects. The phenolic OH group(s) contained in their molecules have radical scavenging properties and allow them to act as reducing agents, hydrogenor electron-donating agents or oxygen scavengers [7, 8]. As far as the antioxidant properties of dietary agents are concerned, there are situations in which knowledge of the total antioxidant potential might be more useful than the individual polyphenolic contents [9]. Recently Schlesier et al. [10] published an assessment of the antioxidant activity by using six different in vitro methods. The six common tests for measuring antioxidant activity (TEAC I-III, TRAP, DPPH, DMPD, PCL and FRAP) were evaluated by comparing the results of four antioxidants and applying the tests to some beverages. The assays differed in the pH (3.3–10.5) of the testing system and in the nature and type of production of radicals. Some assays are suitable only for hydrophilic antioxidants, others for hydrophilic and lipophilic substances when the solvent of the system is changed. The results showed that these six methods were not comparable because the ranking order of the antioxidant activity of the examined antioxidants differed from assay to assay. Also, different solvent systems for the same assay led to different antioxidant activities in some cases. The authors concluded that this effect is probably caused by
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different pH values. The ranking of the examined beverages differed from assay to assay which led to the conclusion that it is only possible when comparing antioxidant activities to use a ranking order for each assay. The results can give an idea of the protective efficacy of secondary plant products [10]. A lot of studies have been made to evaluate the antioxidative activities of several types of fruits and vegetables with usual methods [9, 11, 12, 13, 14, 15, 16, 17, 18, 19]. The aim of our study is to complete the information about the antioxidant potential of several fruits and vegetables using the BR reaction method recently reported, which is based on the inhibitory effects by antioxidants on the oscillations of the Briggs-Rauscher (BR) reaction [20, 21]. The BR system consists of hydrogen peroxide, acidic iodate, malonic acid, Mn(II) as catalyst and works at pH≈2. Therefore, the BR reaction method works for water-soluble antioxidants at a pH value that is similar to that of the fluids in the human stomach. Since fruits and vegetables are like all other foods usually consumed per os, it is conceivable that they effectuate their first antioxidant capacity against free radicals produced in the stomach, and in this way, prevent medical problems such as cancer to this organ [22]. The BR reaction method can give useful in vitro information on the antioxidant activity at acidic pH values, which had been difficult prior to the advent of this method. Recently, a comparison of the results obtained from 16 German white wines, using the new BR reaction method and the commonly used method, TEAC (trolox equivalent antioxidant capacity), has been reported taking into account the different pH values [21]. It showed that some antioxidants are more active under acidic conditions. This can also be interesting for the in vivo effects of digested antioxidants. Serafini et al. [23] evaluated the in vitro activity of green and black teas and their in vivo effect on plasma antioxidant potential in man. After digestion of the tea there was a prompt in vivo response, tested by the human plasma antioxidant capacity assay. This fact suggests that the absorption of the bioactive components of tea takes place in the upper part of the gastrointestinal system, probably starting from the stomach [23]. Also notable was the fact that the black tea, which contains condensed polyphenols because of the fermentation, showed a lower in vitro action than green tea but an equal in vivo antioxidant action. This led to the conclusion that the condensed polyphenols appear to develop an appreciable in vivo action because of structural modifications in the stomach caused by acid gastric secretion, similar to the effect caused by lemon juice when added to a cup of black tea [23]. Therefore, it might also be useful to utilise an assay that works at acid pH for the determination of the in vitro antioxidant activity of food and other nutrition products. Some black and green teas have been tested with a modified BR reaction method and gave similar results regarding their in vitro antioxidant activities [24]. It was also shown that there exists a relationship between the
total phenolic content and the BR inhibition times as found with other methods [21]. The BR reaction method is based on the inhibitory effects by antioxidant scavengers of free radicals on the oscillations of the BR reaction. The generated hydroperoxyl radicals (HOO·) are among the main intermediates of the BR system. The radicals produced in most of the other assays do not occur in the human body [10]. The mechanism of the action of antioxidants against HOO· radicals in the BR system has recently been described [20, 21]. When antioxidant scavengers of free radicals are added to an active oscillating BR mixture there is an immediate quenching of the oscillations, an inhibition time that linearly depends on the concentration of the antioxidant added in a wide range of concentration, and a subsequent regeneration of the oscillations. Relative antioxidant activities with respect to a substance chosen as a standard are determined on the basis of the inhibition times. The inhibition time is defined as the time elapsed between the end of the addition of the antioxidant and the first regenerated oscillation. We have investigated the antioxidant capacities of aqueous extracts of several fruits and vegetables.
Experimental Materials, methods and procedure Malonic acid (Merck, reagent grade, >99%), manganese(II) sulfate monohydrate (Merck, reagent grade, >99%), NaIO3 (Merck, reagent grade, >99.5%) were used without further purification. HClO4, H2O2, and other chemicals were of analytical grade. All stock solutions were prepared from doubly distilled, deionised H2O. Perchloric acid was analysed by titration vs. a standard 0.1 M NaOH solution (from Merck). H2O2 was standardised daily by manganometric analysis. Oscillations in the BR mixtures were followed potentiometrically by recording the potential of a combined redox electrode (Mettler Toledo InLab 501). The electrode was connected to a pH multimeter (WTW, model pH 540 GLP) controlled by an IBMcompatible PC. The accuracy of the multimeter is±1 mV. The data-acquisition program Multi Achat II (WTW) was used. The multimeter is equipped with a temperature sensor with an accuracy of±0.1 °C. All solutions and reaction mixtures were maintained at constant temperature (25.0 °C) using a thermostating system (accuracy±0.1 °C). BR mixtures were prepared by mixing the appropriate amounts of stock solutions of reagents using pipettes or burettes in a 100 mL beaker to a total volume of 30 mL. The order of addition was: malonic acid, MnSO4, HClO4, NaIO3 and H2O2. Oscillations started after the addition of H2O2. Diluted fruit or vegetable extract (1.0 mL) was added to 30 mL of an active, well-stirred BR mixture (initial composition: [H2O2]=1.5 M, [HClO4]=0.0266 M, [IO3-]=0.0667 M, [malonic acid]=0.05 M, [Mn2+]=0.0063 M), after the third oscillation. Typical potentiometric recordings of the oscillating potential of the solution for a non-inhibited and an inhibited BR mixture are shown in Figs. 1a and b, respectively. The inhibition times were then measured. The pH value of the mixture was 1.58. All samples were analysed in triplicate and results were expressed as mean values inhibition times±standard deviation.
439 tained (filtered extract of 200 mg vegetable in 1 mL water) was diluted in a suitable way with doubly distilled water. Antioxidant potential The antioxidant potential (AP) of fruit and vegetable extracts were estimated on the basis of the BR inhibition times measured potentiometrically for different dilutions of the stock solutions as described above. This potentiometric technique furnishes indirect measures of the relative capacity of the antioxidants present in the extracts to scavenge the HOO· radicals generated in the oscillating BR mixture.
Results and discussion
Fig. 1 a Non-inhibited reaction. Recording of the potential of a combined platinum electrode vs. time of an oscillating BriggsRauscher (BR) mixture. b Inhibited reaction. Recording of the potential of a combined platinum electrode vs. time when 1 mL of a solution of caffeic acid (0.03 mg/mL) was added to 30 mL of an oscillating Briggs-Rauscher (BR) mixture after the third oscillation. Initial conditions: [H2O2]=1.5 mol/L, [HClO4]= 0.0334 mol/L, [IO3-]=0.0667 mol/L, [malonic acid]=0.050 mol/L, [Mn2+]=0.0063 mol/L
Preparation of the aqueous extracts Aqueous extracts of fruits The fruit extracts were prepared at room temperature because fruits normally are consumed raw. An amount of each fruit was ground in a mortar and exactly 20 g of this material was put into a measuring flask of 100 mL, which was then filled up with doubly distilled water. The slurry (20 g/100 mL) was stirred for 10 min in the closed flask thermostated at 24 °C. Longer extraction times did not lead to a higher antioxidant activity. Therefore, it seems that most of the water-soluble substances were passed in the extract. This extract was then filtered before taking aliquots for the antioxidant activity measurements. The stock solutions obtained (filtered extract of 200 mg fruit in 1 mL water) were diluted in a suitable way with doubly distilled water. Aqueous extracts of vegetables The vegetable extracts were prepared under refluxed conditions because vegetables are consumed cooked. An amount of each vegetable was ground in a mortar and exactly 20 g of this material was put into a measuring flask of 100 mL, which was then filled up with doubly distilled water. The slurry (20 g/100 mL) was stirred and warmed at 100 °C under reflux for 10 min. Shorter refluxing times gave less antioxidant activity; longer refluxing times did not result in higher antioxidant activity. The extract was then cooled down on an ice bath and filtered before taking aliquots for the antioxidant activity measurements. The stock solution ob-
The linear behaviour of the inhibition time vs. concentration of extracts added are shown in Fig. 2 and Fig. 3 for some fruits and vegetables studied. The parameters of the straight lines together with the R-squared values are reported in Tables 1 a and b. It has been discussed recently that below a certain concentration of the extract added (different for each extract) the behaviour deviates from linearity. In fact, at low concentration of extract added, the inhibition times become too low to be measured, as shown in reference [25]. There is a threshold under which inhibition times cannot be detected. We believe that under these lower limits, the straight lines curve towards zero. This is the reason why, in a few cases, the order of activity can be reversed (see Tables 2 a and b) as happens for Brussels sprout and red cabbage. The antioxidant potentials expressed by BR inhibition times for 80 mg/mL and 100 mg/mL dilutions of extracts of fruits and vegetables are presented in Tables 2 a and b. The BR method measures the total antioxidant activity of the aqueous extracts. The ranking order considered is for the 100 mg/mL extracts and can be seen directly by the order in the third column of Table 2 a and b. In a few cases, the order differs for the 80 mg/mL extracts because of the different slopes of the straight lines. The carrot extract does not show any appreciable activity because the main antioxidants, carotenes, are not water-soluble. The turnip extract also did not show antioxidant activity. There is a great difference between the two different types of apples, the Braeburn apple extract being much more active than the Elstar. Differences are also found for different types of other fruits or vegetables (see for example oranges, grapes and peppers). The assessment of the antioxidant activities of a serving of 100 g freshly weighed fruit or vegetable shows, for example, that the antioxidant activity of 100 g mandarins = 135 g oranges = 254 g grapefruit = 1360 g pears and so on. All equivalences are reported in Table 3 a and b. The equivalences were calculated with the parameters of the straight line of each sample to give the same inhibition time given by 100 g of mandarins or 100 g of Brussels sprouts. In the literature, there are a lot of studies in which the antioxidant activities of fruits and vegetables have been
440 Table 1 Parameters of the straight lines and R-squared values for the a fruits and b vegetables
Fig. 2 Inhibition time vs. concentration of extract added for some fruits
Fig. 3 Inhibition time vs. concentration of extract added for some vegetables
examined [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19]. Fruits and vegetables rich in anthocyanins (e.g. strawberry, raspberry and red plum) demonstrated the highest antioxidant activities, followed by those rich in flavanones (e.g. orange, grapefruit) and flavones (e.g. onion, leek, spinach, green cabbage), while the hydroxycinnamaterich fruits (e.g. apple, tomato, pear, peach) consistently elicited the lower antioxidant activities [13]. Proteggente et al. [13] found that the TEAC, FRAP and ORAC values for each extract were relatively similar and well correlated with the total phenolic and vitamin C contents. The order of the TEAC values of the fresh weight extracts was: strawberry = raspberry = red plum = red cabbage >>grapefruit = orange >spinach >broccoli >green grape = onion >green cabbage >pea >apple >cauliflower, pear >tomato, peach = leek >banana, lettuce. In another study the ORAC assay was used to determine the antioxidant activities of several fruit with the following ranking: strawberry >plum >orange >red grape >kiwi fruit >pink grapefruit >green grape >banana >apple >tomato >pear >melon. The order changes in some cases for the dry weight measurements. The vitamin C content was also examined with the result that the
Slope, m s mL mg–1
Intercept, q s
R2
a Fruit Mandarin Orange Red orange Braeburn apple Grapefruit Pear Strawberry Elstar apple Lychee Red grape Wild strawberry Mango Banana Green grape Kiwi fruit Nectarine
44.72 33.25 22.93 17.77 16.88 14.92 7.06 5.31 5.20 4.12 3.01 2.50 2.10 1.83 1.24 1.24
–394 –313 –67 –25 –125 –243 +69 –129 –141 –142 –74 –26 –67 –93 –40 –82
0.994 0.998 0.999 0.997 0.999 0.997 0.990 0.996 0.994 0.996 0.994 0.998 0.979 0.999 0.988 0.972
b Vegetables Brussels sprout Red cabbage Garlic Black olive Bean (kidney) Button mushroom Broccoli Red pepper Spinach Green bean Corn Pea Cauliflower Onion Celery Leek Zucchini Yellow pepper Turnip Carrot
36.55 32.77 12.72 11.18 8.50 7.84 7.52 6.68 5.48 5.10 4.35 3.14 2.98 2.37 2.09 2.08 1.32 1.07 0.52 0.06
–58 +138 –629 –145 +27 –102 –156 –148 –31 –152 –22 –107 –52 –81 –71 –38 –38 –34 –21 +11
0.999 0.981 0.990 0.999 0.988 0.992 0.997 0.999 0.960 0.950 0.994 0.995 0.997 0.986 0.995 0.992 0.992 0.973 0.908 0.837
major source of antioxidant activity of most fruits may not be from vitamin C [14]. Vinson et al. [15] studied the phenol antioxidant quantity and quality in vegetables and used a phenol antioxidant index, which is a combined measure of the total phenolic content and the IC50 values obtained by the LDL method. They found for the wet weight measurements the following order: Kidney bean >garlic >onion >beet >potato >broccoli >tomato >corn >pepper >carrot >cauliflower >mushroom >celery >cabbage >lettuce >cucumber. In another study, 22 fresh weight vegetables were examined using the ORAC assay but with three different reactive species (ROO·, HO·, Cu2+). The findings showed that the ranking of the antioxidant activities depend on the type of the reactive species in the reaction mixture [16]. Velioglu et al. [17] found a relationship between the total phenolic content and the antioxidant activity of
441 Table 2 Inhibition times for 80 mg/mL and 100 mg/mL dilutions of extracts of a fruits and b vegetables Inhibition time for 80 mg/mL s
Inhibition time for 100 mg/mL s
a Fruit Mandarin Orange Red orange Braeburn apple Grapefruit Pear Strawberry Elstar apple Lychee Wild strawberry Red grape Mango Banana Kiwi fruit Green grape Nectarine
3079±46 2288±34 1777±27 1228±19 1226±19 978±15 609±10 252±4 274±4 195±3 180±3 169±3 105±2 58±2 54±2 18±1
4163±63 3067±46 2236±34 1570±24 1561±23 1265±19 797±12 391±6 388±6 245±4 273±4 222±4 126±3 84±3 92±3 49±2
b Vegetables Red cabbage Brussels sprout Black olive Bean (kidney) Button mushroom Spinach Broccoli Garlic Red pepper Corn Green bean Cauliflower Pea Onion Celery Leek Zucchini Yellow pepper Carrot Turnip
2899±44 2861±43 742±11 661±10 545±9 469±7 434±7 420±6 377±6 310±5 254±4 178±3 138±2 94±2 88±1 74±1 61±1 48±1 15±1 15±1
3563±53 3600±54 968±15 890±14 703±11 511±8 581±9 588±9 526±8 419±7 302±5 244±4 199±3 150±3 138±2 116±2 95±2 74±2 17±1 16±1
plant materials using the β-carotene bleaching method. This was also found in other studies [21, 26, 27]. However, despite these differences, the BR reaction method also gives an idea of the protective efficacy like all the other in vitro assays. The differences found in the ranking order of the antioxidative activity in comparison to other assays surely depends on the different pH and the different radicals formed in the reaction mixture. From the experimental results and the data treatment presented here, the oscillating BR reaction is suitable as an analytical method to measure the relative in vitro antioxidative potential of fruits and vegetables at a low pH value, similar to the pH in the stomach. It has recently been shown for some pure antioxidants that the inhibition time linearly depends on the antioxidant concentration between 0.001 and 0.16 mM in mixture [20]. Therefore, the sensitivity of the BR assay is comparable with those of other methods.
Table 3 Equivalents of a fruits in relation to 100 g of mandarin and b of vegetables in relation to 100 g of Brussels sprout
Portion (g) a Fruit Mandarin Orange Red orange Braeburn apple Grapefruit Pear Strawberry Elstar apple Lychee Wild strawberry Mango Banana Green grape Kiwi fruit Nectarine
100 135 184 247 254 295 580 811 825 1408 1676 2014 2326 3390 3423
b Vegetable Brussels sprout Red cabbage Black olive Garlic Bean (kidney) Button mushroom Broccoli Red pepper Spinach Green bean Corn Pea Cauliflower Onion Leek Celery Zucchini Yellow pepper Turnip
100 105 335 335 420 472 500 561 663 736 833 1181 1226 1554 1749 1757 2756 3396 6963
For the study of antioxidative activity, some factors of the method have to be considered i.e. the practicability, instrumental requirements and the time, expertise and cost necessary for the analysis. Concerning these aspects, the BR method reaction described and used here has many advantages. The analysis is inexpensive and rapid and the reagents and apparatus are commonly used in all chemical laboratories.
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