Optimized enzymatic colorimetric assay for determination of hydrogen peroxide (H2O2) scavenging activity of plant extracts

Graphical abstract

Half maximal effective concentration required to scavenge 50% of H 2 O 2 in the system (EC 50 value) calculated for several plant extracts and standard antioxidants resulted in coefficient of variance (CV%) of the EC 50 values less than 3.0% and correlation coefficient values (R 2 ) > 0.95 for all dose response curves obtained.

Background information
Hydrogen peroxide (H 2 O 2 ) scavenging activity of natural antioxidants present in plant extracts has been determined widely [1][2][3][4][5] by measuring decrement of H 2 O 2 in an incubation system containing H 2 O 2 and the scavenger using the classical UV-method at 230 nm [6]. The main disadvantage of this method is the possible interference from secondary metabolites present in plants which absorb in UV region [7]. Therefore, a simple and rapid colorimetric assay was developed to determine H 2 O 2 scavenging activity of plant extracts and standard antioxidants based on the reaction system where H 2 O 2 rapidly reacts with phenol and 4-aminoantipyrine in the presence of horseradish peroxidase (HRP) to produce a pink coloured quinoneimine dye ( Fig. 1) [8]. H 2 O 2 scavengers will eventually result in decreased production of this particular chromophore. This method was applied to standard antioxidants ascorbic acid, gallic acid and tannic acid in addition to selected plant extracts to determine their hydrogen peroxide scavenging abilities.

Preparation of the plant extracts
All the plant materials A. ceylanica (leaves), E. quinquangulare (whole plant), M. cerviana (whole plant), S. parvifolia (leaves) were prepared separately as decoctions according to the proportions followed by Ayurvedic practitioners. The plant materials described above were washed separately with tap water followed by distilled water and de-ionized water, dried to achieve a constant weight. Each plant material was cut into small pieces and ground to a fine powder using a clean kitchen blender. Powdered samples (30 g) except S. parvifolia were boiled with 800 ml of deionized water until the total volume reduced to 100 ml (1/8th of the original volume) using a beaker. Powdered leaves of S. parvifolia (30 g) was refluxed with 800 ml of deionized water to prepare the aqueous extract of the plant material. The decoctions were sonicated and filtered through a cotton wool plug and then using filter paper (Whatman No. 1). The filtrates were centrifuged at 2000 rpm for 10 min. The supernatants were freeze dried. The freeze dried samples were weighed, and stored at À20 C in sterile tubes until further use. A weight of 2.0 g of C. sinensis (black tea powder) was added into 200 ml of boiling water and allowed to stand in a closed beaker to prepare black tea infusion. The solution was allowed to cool to room temperature, filtered through filter paper (Whatman No. 1) and the filtrate was used for the experiments.

Determination of incubation period, enzyme concentration and H 2 O 2 concentration
A mixture containing phenol (12 mM, 350 ml), 4-aminoantipyrene (0.5 mM, 100 ml), H 2 O 2 (0.7 mM, 160 ml) and phosphate buffer at pH 7 (84 mM, 350 ml) was prepared in separate tubes for each incubation period (5-60 min) to optimize the incubation time needed for the completion of the reaction. HRP (0.1 U/ml, 40 ml) was added to each tube and incubated at 37 C. At the end of the each incubation period the absorbance was measured at 504 nm against reagent blank consisting of phosphate buffer instead of phenol.
Varying concentrations of HRP (0.01-1 U/ml) were incubated with substrates at pH 7 for 30 min. at 37 C as described above to optimize the enzyme concentration needed for the assay.
Similarly different concentrations of H 2 O 2 (0.175-0.70 mM), phenol, 4-aminoantipyrene, phosphate buffer (pH 7) were incubated with HRP (1 U/ml) as described above and the absorbance was read to study the best H 2 O 2 concentration to be used for the assay.
In the present colorimetric method to determine H 2 O 2 scavenging activity, maximum wavelength of absorbance (l max ) of the quinoneimine dye at pH 7 was observed at 504 nm ( Fig. 2A). At 37 C, pH 7, H 2 O 2 concentration of 0.7 mM and horse radish peroxidase enzyme concentration of 0.1 U/ml, maximum intensity of the quinoneimine dye was resulted at 30 min and the colour was stable until 60 min (Fig. 2B). When solutions having enzyme concentrations of 0.01-1 U/ml (maintained at 37 C, pH 7 with H 2 O 2 concentration of 0.7 mM) were incubated for 30 min, the colour increased drastically from enzyme concentration of 0.01-0.08 U/ml but very slight increment of the colour was observed from 0.08 to 1 U/ml (Fig. 2C). Although enzyme concentration of 0.1 U/ml turned out to be suitable for the experiment, further studies were carried out keeping enzyme concentration 10 folds higher (1 U/ml) to compensate for possible inhibition of enzyme that can be caused by phytochemicals present in plant extracts. When varying the H 2 O 2 concentration from 0.175 to 0.70 mM of the solutions having enzyme concentration of 1 U/ml (maintained at pH 7, 37 C for an incubation period of 30 min), it was observed that the enzyme is being saturated at H 2 O 2 concentration of 0.70 mM (Fig. 2D) and therefore this concentration was used for the assay.
The absorbances of the resulting solutions were measured at 504 nm against the reagent blank consisting of phosphate buffer at pH 7 instead of phenol.
Substrates were incubated with HRP (1 U/ml) at different temperatures (18-54 C) at pH 7 for 30 min and absorbance was measured at 504 nm as described above.
When pH was varied from 1 to 11 of the solutions having H 2 O 2 and peroxidase concentrations of 0.7 mM and 1 U/ml, respectively, maintained at 37 C and incubated for 30 min, maximum intensity of colour of the resultant dye was yielded at pH 7 (Fig. 2E). When temperature was varied from 18 to 54 C of the solutions maintained at similar conditions as above and at pH 7, maximum intensity of the colour of the dye was observed at 37 C (Fig. 2F). Determination of limit of detection (LOD) and limit of quantitation (LOQ) The LOD and LOQ values were determined according to the ICH guidelines provided [9]. Microsoft Excel was used to perform regression analysis of the calibration curve constructed using diluted samples of H 2 O 2 . The standard deviation of the y-intercepts (d) of the regression line and the slope of the calibration curve (S) were estimated. LOD and LOQ were calculated as 3.3 Â d/S and 10 Â d/S, respectively [9]. The calculated values were 136 mM and 411 mM for LOD and LOQ, respectively.

Determination of H 2 O 2 scavenging activity
According to the above experiments the optimum conditions selected for the reaction catalyzed by horse radish peroxidase is stated in Table 1. These conditions were maintained when plant extracts were introduced into these systems to assess their scavenging ability of H 2 O 2 molecules. Phenol (12 mM) and 4-aminoantipyrene (0.5 mM) were chosen and used for all the above tests as these concentrations led to maximum intensity of the resultant chromophore. The percentage inhibition (% I) of H 2 O 2 caused by plant extracts and standard antioxidants was calculated as follows. Reaction mixture comprising of test sample (plant extract/standard antioxidant; 350 ml), phenol solution (12 mM, 350 ml), 4-aminoantipyrene (0.5 mM, 100 ml), H 2 O 2 (0.7 mM, 160 ml) and HRP (1 U/ml) prepared in phosphate buffer (84 mM, pH 7) was incubated at 37 C for 30 min. The absorbances of the resulting solutions were measured at 504 nm against reagent blank consisting of phosphate buffer instead of plant extract/standard antioxidant and phenol. The control was made out of same reagents except plant extract replaced by phosphate buffer. Interference for the assay from the plant extracts was minimized as follows. For each concentration of plant extract, samples for background subtraction were made using the plant extract with other reagents replacing phenol by phosphate buffer. Each resulting absorbance value was subtracted from the relevant original absorbance reading. Five types of plant extracts known for their antioxidant properties were tested for their H 2 O 2 scavenging activities. L-Ascorbic acid, gallic acid and tannic acid were used as reference standard antioxidants. The percentage inhibition of hydrogen peroxide was calculated by the equation as described as for many antioxidant assays [3][4][5]: %Inhibition ¼ Abs: of control À Abs: of sample Abs: of control Â 100% The effective concentration required to scavenge 50% of H 2 O 2 in the system (EC 50 value) was calculated from either linear or logarithmic dose response curves plotted between % Inhibition of hydrogen peroxide versus concentration of test samples/standards. EC 50 values were presented as mean AE standard deviation (Mean AE SD) of six independent experiments. Student's t-test was used to compare mean EC 50 values of standard antioxidants/plant extracts and p value <0.05 was considered as significant. Coefficient of variance (CV %) was computed for the EC 50 values obtained for plant extracts/standard antioxidants. All regression and statistical analyses were performed using Microsoft Excel software.
In this study, various plant extracts and standard antioxidants were investigated for their hydrogen peroxide scavenging ability utilizing the developed method by comparing the EC 50 (half maximal effective concentration) values obtained from the corresponding dose response curves via linear or  C. sinensis black tea infusion had the highest ability to scavenge H 2 O 2 molecules followed by S. parvifolia. There was no significant difference in H 2 O 2 scavenging ability between A. ceylanica and E. quinquangulare (p > 0.05) but both of these extracts had lesser H 2 O 2 scavenging ability than S. parvifolia. M. cerviana had the least ability to scavenge hydrogen peroxide molecules. However, the scavenging ability of hydrogen peroxide was superior in the standard antioxidants (i.e., L-ascorbic acid, gallic acid and tannic acid) than all the plant extracts studied. H 2 O 2 scavenging ability of L-ascorbic acid was significantly lower (p < 0.001) than gallic acid and tannic acid. There was no significant difference in H 2 O 2 scavenging ability observed between gallic acid and tannic acid (p > 0.05).
In addition, we have conducted most widely used antioxidant assay i.e.; DPPH test [10] for some of the above mentioned plant extracts and antioxidants. With respect to this assay, the antioxidant potential for these substances varied according to L-ascorbic acid > C. sinensis > S. parvifolia > A. ceylanica > M. cerviana [10][11][12][13]. Similar pattern of variation in the antioxidant potential of the same substances was observed in the method developed for the determination of H 2 O 2 scavenging activity.
For the current method for analysis of H 2 O 2 scavenging activity, coefficient of variance (CV%) for the EC 50 values obtained were less than 3.0% for all the plant extracts and standard antioxidants studied. The correlation coefficient values (R 2 ) of the dose response curves were greater than 0.95 (Table 2).
When considering other methods used for the determination of H 2 O 2 scavenging activity, the classical UV-method is widely used where the decrement of H 2 O 2 in an incubation system containing H 2 O 2 and the scavenger is measured at 230 nm. According to this method described by Ruch et al. [6], we experienced fluctuations in absorbance. This encountered with less reproducible and reliable results. The interference from secondary metabolites present in plants which absorb in UV region affects the results [7]. The present method involves background correction for endogenous interfering substances which improves the reproducibility. Czochra and Widénska [14] have developed fluorescence spectroscopic method using homovanillic acid (4-hydroxy-3-methoxyphenylacetic acid) and peroxidase for the determination of H 2 O 2 scavenging activity of plant extracts and standard antioxidants. Although it is a highly selective and sensitive method, fluorescence generated can be lost by quenching, resonance energy transfer and inner filter effect in the presence of various endogenous phytochemicals apart from the fact that fluorescence spectrometry being a costly method [15]. UV-vis spectrophotometers are widely used in most of the laboratories in low-income countries, and the current method can be used without additional burden. Zhang [16], has estimated hydrogen peroxide scavenging activity of plant extracts by replacement titration method. This method described based on iodide oxidation by hydrogen peroxide is a time consuming macro method where a titration is involved and may not be feasible with higher number of analytes [17]. Many different systems too have been described and are commercially available for quantification of H 2 O 2 in experimental systems, for instance, chemiluminescent, fluorogenic substrates like Amplex Red [18], chromogenic substrates like tetramethylbenzidine (TMB) [19] and 2,2 0 -azino-bis(3-ethylbenzothiazoline-6sulphonicacid) (ABTS) [20]. The reaction of peroxidase catalyzed conversion of H 2 O 2 , phenol and 4-aminoantipyrine to a chromogenic substance is utilized in many commercially available assay kits designed for bio-analytical tests which include determination of blood glucose [21] and cholesterol [22]. However this reaction is not involved so far for the determination of H 2 O 2 scavenging activity of various antioxidants. Therefore use of the same reagents for determination of H 2 O 2 scavenging activity gives a dual purpose for the reagents which in turn reduces costs for the purchase of special chemicals like Amplex red, TMB and ABTS.
In conclusion, the colorimetric method developed and optimized in the current study for the quantification of H 2 O 2 scavenging activity of standard antioxidants as well as natural antioxidants present in plant extracts is less expensive, precise, rapid and yields reproducible results. Therefore this method is ideal for routine laboratory analyses.

Additional information
During aerobic metabolism as well as in the process of drug biotransformation, reactive oxygen species (ROS) are produced as by-products. These include radicals such as superoxide anion (O 2 À ), hydroxyl radical (HO ), alkoxyl radical (RO ), peroxyl radical (ROO ) and non radicals such as hydrogen peroxide (H 2 O 2 ) and singlet oxygen ( 1 O 2 ) [23]. ROS can cause lipid oxidation, protein oxidation, DNA strand breaks, and modulation of gene expression. Experimental evidences show that these ROS are involved in liver diseases and also lead to atherosclerosis, cancer, stroke, asthma, arthritis and other age related diseases [24]. In order to combat ROS, living organisms have developed defense mechanisms consisting of variety of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase [25] as well as non-enzymatic antioxidants such as ascorbic acid, a-tocopherol, b-carotene, flavonoids and many phenolic compounds [26]. Antioxidants are compounds that when present in low concentration in relation to the oxidant prevent or delay the oxidation of a particular oxidizable-substrate [27]. Since natural antioxidants are capable of scavenging various ROS, many methods have been developed for the estimation of these properties. Hydrogen peroxide can be formed in vivo by various oxidizing enzymes such as superoxide dismutase. It can permeate through biological membranes slowly oxidizing number of compounds. Hydrogen peroxide is used in the respiratory burst of activated phagocytes [28]. Although hydrogen peroxide itself is not very reactive [29], it can generate the highly reactive hydroxyl radical (HO ) through the Fenton reaction [30] and is found to be main reason for toxicity associated with hydrogen peroxide. Hydrogen peroxide can deactivate enzymes involved in cellular energy production such as glyceraldehyde-3-phosphate dehydrogenase found in glycolytic pathway [31] as well as aconitase and a-ketoglutarate dehydrogenase found in Krebs cycle [32] by oxidation of essential thiol (À ÀSH) groups. Therefore, scavenging of hydrogen peroxide is considered as an important feature of antioxidants [33]. Accepting electrons in the presence of electron donors, hydrogen peroxide is decomposed into water [34]. Hydrogen peroxide scavenging activity especially of phenolic compounds is assigned to their electron-donating ability [35].