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A rapid determination of the effective antioxidant agents using their Fe(III) complexes
⁎Corresponding author at: Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131, Thailand. anana@buu.ac.th (Anan Athipornchai)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Abstract
A simple, cheap and rapid colorimetric method to determine the total amount of ortho-hydroxyphenolic compounds (TOHPC) as the effective antioxidant agents in food and natural products samples using ferric chloride (FeCl3) reagent was developed. The developed assay shows good linearity, reproducibility, LOD and LOQ. In addition, the performance of the method was validated against the conventional assay by analyzing the total amount of phenolic content (TPC) of twenty tea samples. Similarly, a high correlation was observed between TOHPC and TPC (R2 = 0.9651). In addition, the correlations between TOHPC and TPC and antioxidant activities including DPPH• and ABTS•+ assays were studied. It was found that when the total phenolic content in both assays increased the antioxidant activities also increased. The results demonstrated that the developed assay could be used as an alternative method to determine the total amount of effective antioxidant agents in several food, beverages and natural products samples.
Keywords
Phenolics
Ortho-hydroxyphenolics
Ferric chloride
Colorimetric method
Antioxidant activity
1 Introduction
Biological reactive oxygen species (ROS) and reactive nitrogen species (RNS) are normal products of human metabolism and are also generated from exogenous sources such as smoking, environmental pollution and UV-light exposure. Once this balance is disturbed, free radicals can damage cells and tissue which causes several human diseases including cancer, cardiovascular diseases, diabetes, Alzheimer’s disease, Parkinson’s disease and age-related macular degeneration (Valko et al., 2004; Floyd et al., 2011; Popa-Wagner et al., 2013; Kawagishi and Finkel, 2014; Zhang et al., 2014; Zhou et al., 2015). Currently, dietary research findings suggest that consuming greater amounts of antioxidant-rich foods and beverages have the potential to mitigate the presence of excess free radicals in the body (Frankel and Meyer, 2000; Choleva et al., 2015). These antioxidants mainly come from dietary resources in the form of phenolic compounds including phenolic acid, flavonoids, stilbenes, vitamin C, vitamin E and carotenoids (Lindsay and Astley, 2002; Pisoschi and Negulescu, 2011). Phenolic compounds, as secondary metabolites, are widely distributed in plants. They have excellent antioxidant properties due to their ability to donate an electron or hydrogen from phenolic hydroxyl groups. In general the antioxidant activity of phenolic compounds depends on the numbers and substitution pattern of hydroxyl groups in the molecular structure. Lin and coworker reported the structure-activity relationships of the antioxidant activity of phenolic flavonoids isolated from Pyrethrum tatsienense (Lin et al., 2014). The positions of C-3′, 4′ in the B ring of flavonoids were all replaced by two hydroxyls, which lead to a significant increase of antioxidant activity. The results indicated that the catechol hydroxyls are the most important active sites because it can be related to the double oxidation mechanisms. After the first hydrogen abstraction, the intramolecular hydrogen bond is formed by semi-quinonoid free radicals with a 4′ hydroxyl and further quinone is formed after the second hydrogen abstraction occurs at the 4′ hydroxyl (Amić et al., 2003; Rösch et al., 2003; Lin et al., 2014). Moreover, several scientific reports have found that quercetin, which is the major constituent in onions and several vegetables and catechins or epigallocatechins found in green tea and cocoa, have been found to be potent antioxidant and anti-inflammatory agents in human cells (Sutherland et al., 2006; Kumar and Pandey, 2013). Therefore, the structural requirement of phenolic compounds considered to be essential for effective antioxidant properties is the presence of the ortho-dihydroxy, ortho-trihydroxy and ortho-hydroxybenzoyl moiety in the phenolic molecules.
Quantitative colorimetric Folin-Ciocalteu method which using a mixture of phosphomolybdate and phosphotungstate reagents and aluminium chloride (AlCl3) method were developed to determine the total phenolic content and total flavonoid content in samples, respectively (Stratil et al., 2006; Majhenic et al., 2007; Athipornchai and Jullapo, 2018). These methods require many solutions to be prepared and the reaction time is quite long. In addition, the Folin-Ciocalteu assay cannot specifically determine ortho-hydroxyphenolic compounds. Moreover, AlCl3 assay cannot specifically determine the total amount of phenolics. Therefore, the aim of this study was specific to determine the total amount of ortho-hydroxyphenolic agents which the structural requirements of these agents are the presence of the ortho-dihydroxy, ortho-trihydroxy and ortho-hydroxybenzoyl moiety in the phenolic molecules, in food and natural products samples using colorimetry with ferric chloride (FeCl3) reagent.
2 Materials and methods
2.1 Materials and instruments
All chemicals and the analytical grade organic solvents used in this study were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Twenty tea samples were purchased from a local market in Chonburi province, Thailand during the month of March 2018. Ferric chloride (FeCl3), Folin-Ciocalteu’s reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Colorimetric measurements were recorded using a UV–Visible Specord-210 Plus spectrophotometer. Microplate assays involved a 96-microplate reader (EPOCH-2, BioTek, USA) used with flat-bottomed 96-well microplates (NUNC, Sigma-Aldrich, USA).
2.2 Sample preparation
Twenty tea samples including black and green teas (2 g) were successfully extracted with 50 mL boiling water for 5 min. After the solution cooled down to room temperature it was filtered and the extract was made up to a volume of 50 mL in a volumetric flask. All extracts were refrigerated until further use.
2.3 Solution preparation
1% Ferric chloride (FeCl3) solution was prepared in six solvents; methanol, ethanol, deionized water, and acetate buffers pH 3.0, 3.6 and 4.0. All phenolic compounds were prepared to 10 mM in methanol.
2.4 Method validation
The assay was validated according to conventional validation procedure, which included parameters such as stability of FeCl3 solution, ratio of FeCl3 methanolic solution and ortho-hydroxyphenolic compounds, incubation time, linearity, reproducibility, limit of detection (LOD) and limit of quantitation (LOQ), to evaluate the performance of the developed method. Total ortho-hydroxyphenolic content (TOHPC) was developed. Briefly, 100 µL of the methanolic solution of FeCl3 was mixed with 200 µL of gallic acid standard solutions or the samples and placed into the 96–well plate and the solutions were incubated at room temperature for 30 min in the dark. The absorbance of the reaction mixture was measured at 695 nm using EPOCH-2 microplate reader spectrophotometer.
2.5 Total phenolic content (TPC)
TPC was determined spectrophotometrically using the Folin-Ciocalteu’s reagent. Gallic acid standard solution or sample (20 µL) was reacted with 80 µL of 10% Folin-Ciocalteu’s phenol reagent and placed into the well plate. The solutions were shaken and pre-incubated at room temperature for 5 min. 2.5% of Na2CO3 solution (100 µL) was added and kept for 30 min in the dark. The absorbance was measured at 760 nm (Athipornchai and Jullapo, 2018).
2.6 Antioxidant activity
Reactive oxygen species (ROS) or free radicals play important roles in a number of biological processes and regulate cell physiology and functions. In vitro experiment, DPPH and ABTS radical scavenging assays are widely used methods to evaluate the free radical scavenging ability of natural phenolic compounds. These assays are based on the measurement of the scavenging ability of antioxidant substances towards the stable radicals and decreases in the absorbance were monitored using a spectrophotometer.
2.6.1 DPPH free radical scavenging activity
The electron or hydrogen atom donation ability of the extracts was measured from the bleaching of a purple colored methanol solution of DPPH. Briefly, 180 µL of the methanolic solution of DPPH was mixed with 20 µL of gallic acid standard solutions or samples and added to a 96–well plate. The mixture solution was incubated at room temperature for 30 min in the dark. The absorbance was measured at 517 nm (Braca et al., 2002).
2.6.2 ABTS radical scavenging activity
The procedure followed the method of Re et al. with some modifications. The working solution was prepared by mixing 7 mM ABTS solution and 2.45 mM potassium persulfate (K2S2O8) solution in equal quantities and allowing them to react for 16 h at room temperature in the dark. The solution was then diluted by phosphate buffer saline pH 7.4 to obtain an absorbance of 0.700 ± 0.02 units at 734 nm using a spectrophotometer. Fresh ABTS•+ solution was prepared for each assay. Briefly, 180 µL of ABTS•+ solution was reacted with 20 µL of gallic acid standard solution or samples into the well plate and the reaction mixture was incubated at room temperature for 5 min in the dark. The absorbance was measured at 734 nm (Re et al., 1999).
2.7 Statistical analysis
All analyses in this study were conducted in triplicate and averaged. Microsoft Excel was used for statistical analyses. Quantification of all activities was based on a standard curve generated with gallic acid and the results were expressed as gallic acid equivalents (GAE).
3 Results and discussion
3.1 Method validation
First of all, we were studied to establish the effects of the different substitutions of the hydroxyl group on the aromatic ring of phenolic compounds. By mixing a 10 mM methanolic solution of the phenolic compound with 1% FeCl3 solution in a test tube, shaking was continued for 5 min. The colors of the phenolic-Fe3+ complexes (Supporting Information, Fig. S1) were observed and compared with that of the reference solution. From the results, the phenolic compound which have ortho-dihydroxyl and ortho-trihydroxyl substitution on the aromatic ring (catechol) and ortho-hydroxyl substitution on benzoyl moiety developed significant color changes to green or dark green. On the other hand, phenolic compounds with only one or two hydroxyl groups (meta- and para-substitution) on the benzene ring (phenol, resorcinol and hydroxyquinone) did not develop significant color changes when compared with the corresponding FeCl3 reference solution (yellow solution). In addition to the mononuclear hydroxo-species, binuclear cations, in which the iron atoms are bridged by OH groups, may have formed (Paiva-Martins and Gordon, 2005; Wang, 2013; Wang et al., 2015). The results demonstrated that the developed assay could be used as an alternative method to determine the total amount of ortho-hydroxyphenolic compounds using colorimetry with ferric chloride (FeCl3) reagent.
After that the stability of FeCl3 solution in six solvents including methanol, ethanol, deionized water and acetate buffers pH 3.0, 3.6 and 4.0 were studied as descripted above. All solutions were measured using a UV–Vis spectrophotometer with a full scan mode from 200 to 900 nm. First of all, the suitable concentrations of FeCl3 solution in each solvent were studied and found that the concentration of FeCl3 solution were prepared for 8 ppb in ethanol and methanol and for 4 ppb in deionized water and acetate buffers. The absorbance was measured with the series of times 0, 10, 20, 30, 60, 120, 180 min. The results (Supporting Information, Fig. S2) showed that the absorbance peak of FeCl3 in deionized water and acetate buffers pH 3.6 and 4.0 increased with time while the spectrum of FeCl3 in methanol, ethanol, and acetate buffer pH 3.0 remained the same over the full scan from 200 to 900 nm thus showing good stability for at least 180 min. In addition, a single sharp peak for FeCl3 in methanol was observed while the spectrum of FeCl3 in ethanol showed more absorbance peaks and the acetate buffer at pH 3.0 precipitated slightly with time. As a result methanol was selected as the solvent for FeCl3 in this study.
The ratio of FeCl3 methanolic solution and ortho-hydroxyphenolic compounds catechol, protocatechualdehyde, protocatechuic acid, caffeic acid and gallic acid were prepared at 1:1, 1:2, and 1:3 (v/v). The mixtures absorbances were measured from 400 to 900 nm. The results shown in the Supporting Information (Fig. S3) found that the spectra with complex ratios of 1:1 and 1:3 were less stable while the 1:2 ratio was the most stable. In addition, all phenolic-Fe3+ complexes showed the same highest absorbance peak at 695 nm. Thus the complex ratio of 1:2 and the absorbance peak at 695 nm were selected as the optimal conditions for further experiments.
The stability of complexes in a series of time 0, 5, 10, 15, 20, 30, 40, 50, 60 min was studied and the absorbance measured at 695 nm. From the results (Supporting Information, Fig. S4), the absorbance of all phenolic-Fe3+ complexes decreased rapidly within 5 min and then decreased slightly until after 30 min all of the complexes were stable. Thus our incubation time of 30 min at room temperature was chosen. Furthermore, both dark and light conditions for incubation of the complexes were studied using gallic acid (Supporting Information, Fig. S5). The solutions held in the dark (R2 = 0.9894) showed marginally more linearity than those in the light (R2 = 0.9843). Dark conditions were selected for incubating the ortho-hydroxyphenolic-Fe3+ complexes.
From the above studies the optimal conditions selected were methanol as the solvent for FeCl3 while the ratio of ortho-hydroxyphenolic: Fe3+ was 2:1 and the absorbance peak at 695 nm was measured. The incubation time was 30 min at room temperature in the dark. Five standard ortho-hydroxyphenolic compounds; catechol, protocatechualdehyde, protocatechuic acid, caffeic acid, and gallic acid were used to study the performance of the developed method. For all standard analyses the results given in Fig. 1 showed that as the concentration increased the absorbance of ortho-hydroxyphenolic-Fe3+ complexes increased initially and became steady at higher concentrations. The linear range of the calibration curves for each standard is also indicated on separate graphs.
Calibration curves in the linear range for all ortho-hydroxyphenolic standards: catechol (A), protocatechualdehyde (B), protocatechuic acid (C), caffeic acid (D), and gallic acid (E).
All analytical figures of merit including linearity, reproducibility, limit of detection (LOD) and limit of quantitation (LOQ) were measured and summarized in Table 1. The reproducibility of the proposed assay was quantified as the relative standard deviation (%RSD) for three replicate analyses of ortho-hydroxyphenolics at five different concentrations in the linear range. Good reproducibility was obtained with the %RSD being in the range 1.2–11.3%. In addition, most standards also showed good linearity within the range 2–10 mM with the exception of catechol which was 0.4–1.2 mM with correlation coefficients (R2) of at least 0.996. The LOD and LOQ for each standard were calculated by taking 3.3 and 10 times the standard deviation of a blank, respectively, and dividing by the slope of the calibration line (i.e., molar absorption coefficient). All standard phenolics were obtained the lowest LOD and LOQ values. These results confirmed that the developed assay could be used as an alternative method to determine the total amount of ortho-hydroxyphenolic compounds.
Phenolic standard
Analytical characterization
Linear range (mM)
Calibration function
R2
%RSD
LOD
LOQ
Catechol
0.4–1.2
y = 1.6526x-0.3997
0.9968
3.4–11.3
0.21
0.64
Protocatechualdehyde
2.0–10
y = 0.0799x + 0.2448
0.9955
3.9–6.3
0.72
2.18
Protocatechuic acid
2.0–10
y = 0.0633x + 0.2150
0.9989
1.2–5.2
1.33
4.02
Caffeic acid
2.0–10
y = 0.0392x + 0.2232
0.9974
3.6–4.8
0.76
2.31
Gallic acid
1.0–10
y = 0.0831x + 0.0955
0.9978
1.8–2.7
0.42
1.26
3.2 Tea sample analysis
The performance of the developed TOHPC method was validated against the conventional TPC assay by analyzing the total phenolic content of twenty tea samples. The results for two methods are compared in Fig. 2. Summarizes the results of GAE in each tea samples showed in the Supporting Information (Fig. S6). From these results found that a correlation R2 was studied by comparing the two series of GAE data and the proposed TOHPC assay gave a preponderant of results than the conventional TPC methods. Similarly, a high correlation was observed between TOHPC and TPC (R2 = 0.9651) confirming that the developed assay could be used for determine the ortho-hydroxyphenolic content in food, beverages and natural products samples.
Correlation plot of GAE determined by TOHPC and TPC methods.
3.3 Correlations between TOHPC and TPC and antioxidant activities
Several reports have shown that the structural requirement of phenolic compounds essential for effective antioxidant properties is the presence of the ortho-dihydroxy, ortho-trihydroxy and ortho-hydroxybenzoyl moiety in the phenolic molecules (Amić et al., 2003; Rösch et al., 2003; Lin et al., 2014). Therefore, the correlations between TOHPC and TPC and antioxidant activities including DPPH• and ABTS•+ assays were studied. All results of antioxidant activities in each tea samples are shown in the Supporting Information (Fig. S7). It was found that when the total phenolic content in both assays increased the antioxidant activities also increased. The correlation was studied by comparing the two series of data (Figs. 3 and 4). High correlations (R2 = 0.9548 and 0.9744) were observed between TOHPC and TPC with the DPPH• assay, respectively, while the correlations with the ABTS•+ assay were 0.9443 and 0.9559, respectively. These results confirmed that the phenolic compounds are likely to contribute to the radical scavenging activities of these teas and it confirmed that the catechol moiety of phenolic compounds is essential for effective antioxidant properties.
Correlation between DPPH radical scavenging activity and TOHPC and TPC.
Correlation between ABTS radical scavenging activity and TOHPC and TPC.
4 Conclusion
This work reports a simple and rapid method to determine specifically the total amount of ortho-hydroxyphenolic compounds using ferric chloride (FeCl3) reagent and this phenolic compound is essential for effective antioxidant properties. This developed assay shows good linearity, reproducibility, LOD and LOQ. Therefore, the developed assay could be used as an alternative method for determine the total ortho-hydroxyphenolic content in several in food, beverages and natural products samples.
Acknowledgement
Authors to thank the Project for the Promotion of Science and Mathematics Talented Teachers (PSMT) Scholarship in the management of the Institute for the Promotion of Teaching Science and Technology (IPST), The Research Unit in Synthetic Compounds and Synthetic Analogues from Natural Product for Drug Discovery (RSND) and The Center of Excellence for Innovation in Chemistry (PERCH-CIC), Department of Chemistry, Faculty of Science, Burapha University for providing research facilities. Special thanks to Professor Dr. Ronald Beckett for comments and grammatical suggestions on the manuscript.
Declaration of Competing Interest
ve no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Appendix A
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2021.102986.
Appendix A
Supplementary material
The following are the Supplementary data to this article:
