Article pubs.acs.org/JAFC
Assessment of the Degree of Interference of Polyphenolic Compounds on Glucose Oxidation/Peroxidase Assay Adeline Ik Chian Wong† and Dejian Huang*,†,‡ †
Food Science and Technology Program, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore ‡ National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street, Suzhou Industrial Park, Jiangsu 215123, China ABSTRACT: The glucose oxidase/peroxidase assay (GOP) is a coupled enzymatic assay commonly used in measuring glucose concentrations in biological sciences and food chemistry, particularly for quantification of α-glucosidase activity. However, we found that the GOP assay is prone to interference, especially from reducing substances such as polyphenolic compounds, which are commonly found in botanical materials. To establish the scope and limitation of the assay in measuring α-glucosidase inhibition activity, we systematically investigated the structural features of the polyphenolic compounds that can lead to false positives. Utilizing sodium dodecyl sulfate (SDS) as surrogate for the meriquinone intermediate formed during the reaction, we measured the reactivity of this redox active intermediate toward common flavonoids. Our results show that flavonoids with o-dihydroxy groups in the B-ring cause strong interference and that compounds with little DPPH scavenging activity do not have interference. Our results highlight the need for checking the suitability of the GOP assay first before it is applied in measuring α-glucosidase inhibition activity. In addition, when the literature data on α-glucosidase inhibition activity of botanical extracts or polyphenolic compounds using GOP assay are interpreted, potential false positives due to interference on the assay will need to be taken into consideration. KEYWORDS: α-glucosidase, glucose oxidase/peroxidase assay, o-dianisidine, flavonoids
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INTRODUCTION Due to the increasing global population of type II diabetes patients, research on finding ways to mitigate this dreadful disease have intensified in recent years.1 One way to control hyperglycemia is through starch hydrolase inhibitors from edible plants, which contain structurally diverse α-glucosidase inhibitors including sugar mimics2 and polyphenolic compounds.3,4 To uncover novel α-glucosidase inhibitors from plants, particularly edible plants, the p-nitrophenyl glucopyranose (pNPG) assay and coupled glucose oxidase/peroxidase (GOP) bienzyme assay are widely applied as convenient guides to fractionation and purification of inhibitors. Whereas the pNPG assay uses a synthetic probe that may not be truly reflective of natural substrates of the enzymes, the GOP assay was developed initially to quantitate the amount of D-glucose in human fluids.5 Principally, this assay uses glucose oxidase to oxidize β-D-glucose by molecular oxygen to D-glucono-δ-lactone and hydrogen peroxide. In the presence of peroxidase (e.g., horseradish peroxidase), hydrogen peroxide reacts with colorless chromogenic molecules to form colorful dyes that can be quantified conveniently by colorimeters. Many chromogenic compounds have been used as substrates for horseradish peroxidase.6 Among them, o-dianisidine has been more widely applied than others. In acidic conditions, the oxidized o-dianisidine becomes rose-pink with absorbance maximum at 540 nm (Figure 1).7 Due to its convenience, the GOP assay has been applied in many other systems including measurement of α-glucosidase activity and screening for its inhibitors. Although the GOP assay is highly substrate specific and relatively convenient to use, it is highly susceptible to various interferences.8,9 The GOP assay utilizes two oxidases and © 2014 American Chemical Society
involves complicated redox reaction mechanisms; as such, any compounds that can inhibit the two enzymes or react with the reactive intermediates of reaction will cause interferences.10 Indeed, there are scattered reports on the interference issue of the GOP assay. Using 4-aminoantipyrine and 3-hydroxybenzoic acid as chromogenic substrates in the GOP assay, Shaukat and co-workers recently found that green tea polyphenolic compounds cause interferences and lead to false-negative results in blood serum glucose concentrations, and the interference was suggested to be due to the radical scavenging activity of the polyphenolic compounds.11 In another study, polyphenols were also found to interfere because of their reducing activity.12 Despite the number of studies addressing the issue of interferences, the GOP assay continues to be applied in the quantification of α-glucosidase inhibitory activity of natural products due to its convenience and selectivity of glucose.13 Because natural products often contain polyphenolic compounds, it is important to systematically address the scope and limitations of the GOP assay in quantifying botanical samples on α-glucosidase inhibitory activity. Therefore, we investigated the interference of common polyphenolic compounds found in the plant kingdom on the degree of interference on GOP assays by adding sodium dodecyl sulfate (SDS) to stabilize meriquinone intermediates formed during the oxidation of o-dianisidine by peroxidase. Our results reported herein will help researchers in using the GOP assay in plant extracts to assess the suitability of Received: Revised: Accepted: Published: 4571
January 28, 2014 March 29, 2014 April 27, 2014 April 28, 2014 dx.doi.org/10.1021/jf500431z | J. Agric. Food Chem. 2014, 62, 4571−4576
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RESULTS AND DISCUSSION
During the quantification of α-glucosidase activity and measurment of the activity of inhibitors, maltose or sucrose was hydrolyzed into glucose (and fructose in the case of sucrose). The α-glucosidase activity was measured by the generated glucose by the GOP assay. The commonly used chromophore in GOP assay is o-dianisidine, which is oxidized by peroxidase to give brown azo dye via a blue charge transfer complex, meriquinone (Figure 1). The polyphenolic compounds may interfere with the assay by reacting with meriquinone, which will lead to false negatives of the glucose or false positives in α-glucosidase inhibitory activity. To assess the severity of interference by different polyphenolic compounds by reducing meriquinone intermediate, we applied SDS at pH 4 according to literature studies to stabilize the positively charged meriquinone intermediates through electrostatic interaction with the negatively charged surfactant.14,15 Ascorbic Acid. When ascorbic acid was added to the meriquinone solution at concentrations of 25 and 50 μM (1:4 and 1:2 ratios with reference to H2O2), there was an initial marked decrease in absorbance but also a regain of the blue color from 10 to 250 s (Figure 2A). Apparently, the ascorbic acid is consumed
Figure 1. Reaction scheme of o-dianisidine oxidation. Hydrogen peroxide reacts with colorless reduced o-dianisidine in the presence of peroxidase to form the dianisidine quinonediimine intermediate. Application of SDS suspends the meriquinoid complex intermediate (λmax= 395; 650 nm). Irreversible coupling reaction with two quinonediimines forms the brown bisazobiphenyl product. Bisazobiphenyl product reacts with H2SO4 to form a more stable pink product (λmax= 540 nm).
the assays and provide a guide for avoiding unnecessary false results.
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MATERIALS AND METHODS
Reagents and Instruments. Chemical reagents were obtained from commercial sources. These compounds include caffeic acid, ferulic acid, rutin, apigenin, chrysin, coumaric acid, gallic acid, hesperedin, hesperetin, kaempferol, luteolin, morin, naringin, quercetin, hydrogen peroxide solution, o-dianisidine dihydrochloride, glucose oxidase, and peroxidase from horseradish, sodium dodecyl sulfate, (-)-Epigallocatechin gallate, (-)-Epigallocatechin, (-)-Epicatechin, Quinic acid (Sigma-Aldrich, Singapore); 3,5-dihydroxyflavone, 3,7-dihydroxyflavone, 3′,3,4-trihydroxyflavone, 5,3′,4′-trihydroxyflavone, 5,7,3′,4′,5′-pentahydroxyflavone, 7,3′,4′-trihydroxyflavone, 7,8-dihydroxyflavone, fisetin, galangin, gossypetin, and myricetin (Indofine Chem. Co., Hillsborough, NJ, USA); tannic acid (Merck, Germany); and ascorbic acid, chlorogenic acid (Spectrum Chemical Co., Singapore). A microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA) was used to measure the spectrophotometric readings. Generation of the Meriquinone Intermediate and Its Reactivity with Polyphenolic Compounds. o-Dianisidine (0.1 mM), SDS (1.0 mM), H2O2 (0.1 mM), and peroxidase (0.25 units) in citrate buffer (pH 4) were reacted for 1 min to generate the meriquinone intermediate, which was quantified by UV absorbance at 395 nm. Polyphenolic compounds with various concentrations were reacted with meriquinone. The decrease of the UV absorbance over time was monitored for selected compounds including vitamin C, coumaric acid, caffeic acid, gallic acid, and EGCG. For the rest of the samples, end point readings were carried out at 650 nm. The final concentrations of the polyphenolic compounds were kept at 100 μM. The absorbance readings were taken after 5 min of reaction. The degree of interference was quantified through the equation
Figure 2. (A) Reduction kinetics of the meriquinone structure monitored at 395 nm for 15 min at various stoichiometries of H2O2 concentration to ascorbic acid (ratios of 4:1, 2:1, and 1:1). (B) Reduction kinetics (monitored at 395 nm for 15 min) of the meriquinone structure of coumaric acid, caffeic acid, gallic acid, ascorbic acid, and EGCG at a stoichiometric ratio of 1:1 H2O2/sample concentration.
quickly by oxidation with meriquinone, leading to the fading of blue color. The increase in absorbance is due to the regeneration of meriquinone intermediates formed by enzymatically catalyzed oxidation of o-dianisidine as there was a surplus of H2O2. When ascorbic acid was added at a ratio of 1:1 with H2O2 (100 μM), there was a dramatic reduction in the meriquinone solution to a colorless solution without any regain of color. This finding
% interference = 100(A blk − A s)/A blk where As is the absorbance of the reaction mixture of the sample with meriquinone at 650 nm and Ablk is the absorbance of blank (water). All measurements were performed in triplicates, and the results were expressed as mean ± standard deviation. 4572
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decrease was not significant (p > 0.05). Ortho substitution with an electron donor of a methoxy group stabilizes the aryloxyl radical and thus its antioxidative action. However, the impact of a methoxy group is far from being equivalent to the addition of a hydroxy group. The removal of either the methoxy or hydroxy group from the phenolic ring resulted in a sharp decrease in the percent interference as seen by the coumaric acid structure. Monophenols are known to be less efficient in antioxidant activity.19 Flavan-3-ols. Similar to previous results, we found that all flavan-3-ols (catechins) exhibit strong interferences (Figure 3D). At the tested concentration, there was no difference among the catechins. The catecholic units in the B ring and/or the o-trihydroxy groups in the B ring and galloyl units are certainly potent in reducing meriquinones.20 Flavonols. With 11 compounds, this group is the largest category, and all of them exhibit a certain degree of interference (Figure 3E). The degree of interference is roughly correlated to the number of OH groups except rutin, which has four OH groups, but the interference activity is much weaker than that of its aglycone quercetin. Therefore, the 3-OH group contributes significantly to the activity. In this series, the o-OH group at the B ring increases the activity as is apparent by comparing the values of kaempferol and quercetin. 3,5-Dihydroxyflavone, 3,7-dihydroxyflavone, and galangin showed intermediate interference. These three compounds have a similar feature in that they lack any hydroxy group in the B ring of its structure but differ in the OH group present in the A and C rings. The hydroxy position of 3,7-dihydroxyflavone showed a significantly stronger interference compared to 3,5-dihydroxyflavone. Galangin, which has OH groups on the 3-, 5-, and 7-positions, did not significantly increase interference compared to 3,7-dihydroxyflavone. The addition of a hydroxyl group (monohydroxyl substitution) in the B ring as seen in kaempferol increased the percent interference significantly (p < 0.05). It has been proposed that the single 4′-hydroxy group in the B ring has the potential to conjugate with the 3-hydroxy group through conjugation of the C ring. The phenoxyl radical formed could abstract an electron from the radical cation to generate a dication and the phenolate.18 The addition of another hydroxyl group in the 5′-position of the B ring as seen in myricetin did not significantly (p > 0.05) alter the interference as compared to quercetin. This is on par with other studies, which have shown that the presence of a third hydroxy group in the B ring at the C-5 position did not enhance the effectiveness of myricetin compared to quercetin.19 The stabilizing effect of the extra radical is provided by only one of the two hydroxyls ortho to the hydrogen-donating hydroxyl group.20 Studies have also suggested that a hydrogen atom is most likely donated from the 3OH, which leads to a more stable radical tautomer.21,22 In a broad stroke, there seems to be an overall structure− activity relationship between the compound and its ability to reduce the meriquinone radical. Within the “high interference” category, the compounds generally possess a larger number of hydroxy groups (OH). The radical scavenging property of flavonoids has been attributed to the number of OH groups that are present in the ring structure.23,24 In addition, the configuration of the H-donating hydroxy groups is also one of the main structural features, which influences the antioxidant capacity of phenolics.25 Furthermore, compounds in the “high interference” group possess many OH groups that are in the ortho position. The ortho arrangement of OH groups causes the development of an intramolecular hydrogen bond (IHB), which is able to stabilize the radical, leading to a lower O−H bond dissociation enthalpy (BDE). The BDE governs the ability of a particular flavonoid to
suggests that vitamin C reacts only with meriquinone but does not inhibit peroxidase. We further monitored the reduction kinetics of the meriquinone intermediate reaction with coumaric acid, caffeic acid, gallic acid, and epigallocatechin gallate with concentration equal to that of H2O2 (Figure 2B). Coumaric acid with only one hydroxy group (OH) did not show much reactivity, whereas caffeic acid can lead to 50% interference at steady state throughout the remaining time. A further addition of an OH group in gallic acid was shown to reduce 75% of the absorbance values, from 0.8 to 0.2. The dependence on the number of OH groups in the phenolic acid is consistent with the increasing reducing activity from mono- to tri-OH moieties. These preliminary results prompted us to systematically evaluate the interference of common polyphenolic compounds. Reduction of Meriquinone Intermediate by Polyphenolic Compounds. To delineate the structure−activity relationship, we measured 31 commonly found polyphenolic compounds, and the results are shown in Figures 3 and 4 arranged by their structural groups. We will discuss each class of compounds, respectively. Flavones. As shown in Figure 3A, chrysin and apigenin have no interference (p < 0.05) indicating that the A ring OH groups at the 5- and 7-positions do not enable reduction of meriquinone. In contrast, apigenin isomer, 7(or 5),3′,4′-trihydroxyflavone, exhibits significant activity. This plus the fact that luteolin has a similar activity suggests that the catecholic unit is sufficient for the reduction of meriquinone. In agreement with this, 7,8-dihydroxyflavone also has a comparable activity, although it has only two OH groups. In this series, pentahydroxyflavone possesses the strongest activity because it has three o-OH groups at the B ring. Flavanones. The common flavanones naringin, hesperetin, and its glycoside counterpart hesperedin all showed intermediate interference activity as shown in Figure 3B. The reason was that the B rings of the tested flavones do not contain catecholic units. Apparently, methoxylation of the catechol unit will lead to a reduced activity. In addition, there was no difference between hesperidin and hesperetin, suggesting that the OH group in the C-7 position of the A ring does not contribute to the activity of reducing meriquinones. These flavanones do possess some antioxidant activity, although the activity is much weaker compared with those of many other flavonoids.16,17 Similar to the case of rutin, the glycosylation of hesperedin did not influence the interference percentage (p > 0.05). Phenolic Acids. Coumaric acid shows a partial interference even though it has only one OH group; this is very different from that of isoflavones, which have multiple OH groups but have only very weak interferences (Figure 3C). In addition, ferulic acid and caffeic acid show stronger interferences that are comparable to that of chlorogenic acid. Gallic acid also exhibits strong interference. Therefore, it is reasonable to conclude that hydrolyzable tannins, which are the major group of polyphenolic compounds in the plant kingdom, will also have strong interference because gallates are the main moiety in hydrolyzable tannins. Indeed, tannic acid shows a strong interference. Similarly, caffeic acid has two OH groups arranged in the ortho position and was shown to have a percent interference of 88.91 ± 2.35, which was comparatively high. The percent interference of caffeic acid was not significantly different (p > 0.05) from those of gallic acid (due to the additional OH group) and ferulic acid (due to the CH3O group). In the case of ferulic acid, one methoxy substitution at the ortho position relative to the hydroxyl group slightly decreased the interference (84.89 ± 2.8), but the 4573
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Figure 3. Interference of various compounds (represented in numerics) and subdivided according to their respective classes: (A) flavones; (B) flavanones; (C) phenolic acids; (D) flavan-3-ols; (E) flavanols. Compound identities for Figure 3E are: 21, 3,5-dihydroxyflavone; 22, rutin; 23, 3,7-dihydroxyflavone; 24, galangin; 25, kaemferol; 26, 3'3,4-trihydroxyflavone; 27, morin; 28, gossypetin; 29, fisetin; 30, myricetin; 31, quercetin.
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Funding
donate hydrogen atoms, and the lower the BDE value, the higher the ability of a flavonoid to donate hydrogen.26 Correlation of Degree of Interference and Antioxidant Activity Measured by DPPH Radical Scavenging Activity. Because meriquinone is an oxidant, its reaction with polyphenolic compounds has some similarity to the reaction used in measuring the antioxidant activity with the DPPH assay. If there is a good correlation of interference and the DPPH assay, one might be able to apply the DPPH assay to conveniently distinguish the likelihood of interference of any given plant extract. Therefore, we attempted to correlate the degree of the interference and the IC50 of the polyphenolic compounds for DPPH assay, and the results are shown in Figure 4. In general,
The authors would like to acknowledge the financial support from the National University of Singapore (Suzhou) Research Institute under the grant number NUSRI2011-007 and The Industrialization-Academic-Research Platform grant of Jiangsu Province, China. Notes
The authors declare no competing financial interest.
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Figure 4. Correlation between percent interference of compounds and the IC50 of DPPH (mM).
those compounds with a high interference also had a high antioxidant activity (i.e., low IC50 of DPPH assay), but there was no linear correlation. Therefore, DPPH may be qualitatively applied to judge the potential interference of the GOP assay. Measuring the turbidity decrease during enzymatic starch hydrolysis is an accurate way to test enzyme activity, and it is not subject to interference due to reducing activity of the phenolic compounds. We applied this method to evaluate the inhibitory activity of α-glucosidase and found that these compounds have no measurable activity. These results highlighted the limitation of the GOP assay in natural products. In conclusion, we have illustrated that the majority of polyphenolic compounds found in the plant kingdom have a significant interference in the GOP assay. Because the reaction with the free radical intermediate is likely to be a reduction reaction, it is reasonable to assume that radical scavengers will have a falsepositive interaction. This effectively makes the GOP assay not useful in measuring the α-glucosidase inhibition activity of botanical extracts. It is essential to test the interference first before the GOP assay is applied for quantification of α-glucosidase activity. Recently, our group developed a high-throughput method for screening α-glucosidase and α-amylase inhibition activity of natural products using turbidity changes.27 In this assay, natural starch was applied as the substrate and acarbose was used as a reference standard. Because turbidity is a physical parameter of the starch solution, one would expect that it would be more resistant to the interference of chemical reactions.
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