Food Chemistry 170 (2015) 10–15

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Analytical Methods

Effect of esterification condensation on the Folin–Ciocalteu method for the quantitative measurement of total phenols Liang-Yu Chen a,1, Chien-Wei Cheng b,1, Ji-Yuan Liang a,⇑ a b

Department of Biotechnology, Ming-Chuan University, Gui-Shan 33343, Taiwan, ROC Department of Restaurant and Institutional Management, Shih-Chien University, Taipei 10462, Taiwan, ROC

a r t i c l e

i n f o

Article history: Received 9 January 2013 Received in revised form 1 July 2014 Accepted 10 August 2014 Available online 19 August 2014 Keywords: Polyphenols Molybdate Photometry Alkaline reduction Esterification

a b s t r a c t The Folin–Ciocalteu method is widely applied for the determination of the total phenolic contents in natural products. This method is significantly affected by the addition of sodium carbonate. The currently applied Folin–Ciocalteu methods may have been modified without any validation in the quantitative standards and the order of processes. In this study, serial experiments were performed to investigate the effect of phenolic calibrations based on the classic Folin–Ciocalteu method. Esterification condensations were observed in the assays with prior basification for gallic acid and catechin used as quantitative standards. The phenolic contents obtained in the samples differed depending on when basification occurred compared with the gallic acid calibration. The bias of the classic Folin–Ciocalteu method derived from cross-linkage of molecules was first defined in this study. The performance of the Folin–Ciocalteu method is optimised and validated again. Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

1. Introduction The antioxidant capacity of natural ingredients is an important issue for foods with special dietary purposes, such as functional foods, medical foods and dietary supplements. The antioxidant capacity is also an important research topic in different fields of human and animal biology, such as physiology and nutrition science (Perez-Jimenez, Neveu, Vos, & Scalbert, 2010). Polyphenols are normally polyhydroxy phenolic complexes and secondary metabolites in plants. These compounds possess an aromatic ring bearing one or more hydroxyl groups, and their structures may range from that of a simple phenolic molecule to that of a complex high-molecular mass polymer (Balasundram, Sundram, & Samman, 2006). Polyphenols are associated with a wide range of physiological properties, including anti-inflammatory (Yan, Wang, Chen, Zhuang, & Wang, 2013), anti-microbial (Michel, Destandau, Le Flochc, Lucchesi, & Elfakir, 2012) and antioxidant activities (Meng, Fang, Qin, Zhuang, & Zhang, 2012; Sekhon-Loodu, Warnakulasuriya, Rupasinghe, & Shahidi, 2013). Furthermore, phenolic compounds are considered as the most important antioxidants in plants and plant-based foods (Mariod, Matthäus, Idris, & Abdelwahab, 2010). The Folin–Ciocalteu (FC) method (Folin & Denis, 1915) is a convenient analytical technique with good reproducibility for the ⇑ Corresponding author. 1

The authors contributed equally to this work.

http://dx.doi.org/10.1016/j.foodchem.2014.08.038 0308-8146/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

determination of total polyphenols in biological materials. Many quantitative data have been reported based on the FC method (Everette et al., 2010; Huang, Ou, & Prior, 2005). The accuracy of the results obtained using this method is determined by the equivalent standards used, the order in which the reagents are added, and the Folin–Ciocalteu reagent (FCR), which contains molybdenum VI (Folin & Ciocalteu, 1927). Phenolic compounds are determined based on the reduction of Mo6+ to Mo5+, which is blue and can be measured optically at 730 nm (Huang et al., 2005). The mixture of FCR and phenolic compounds is stable in acid but unstable in an alkaline solution. Therefore, sodium carbonate, which is used to provide an alkaline environment, is critical to the reaction. The FC method is a convenient and simple method. The FC method requires no particular equipment and can be useful in characterising and standardising botanical samples (Prior, Wu, & Schaich, 2005). A major advantage of the FC method is that it has an equivalent response to different phenolic substances in wines, making it suitable for accurately measuring total phenolic compounds (Waterhouse, 2001). Accordingly, the total phenolic contents determination based on the FC method has been used extensively to characterise wines and spirits, fruit juices, plant tissues, sorghum grain and other similar products (Singleton, Orthofer, & Lamuela-Raventós, 1999). The FC method depends on the selective oxidation of similar easily-oxidised substances that contribute to the apparent total phenol content. However, there are still some disadvantages to using the FC method. For example, the FC method may lack

L.-Y. Chen et al. / Food Chemistry 170 (2015) 10–15

specificity. Although the FC method produces predictable results on a wide range of phenolics, these results can be affected by other non-phenolic reducing molecules (Rover & Brown, 2013). The FC method may also suffer from a number of interfering substances, such as sugars, aromatic amines, sulphur dioxide, ascorbic acid, organic acids, and iron (II), in addition to other enediols and reductones. Therefore, correcting for interfering substances is required to avoid the bias due to these substances (Prior et al., 2005). The amount of total polyphenols in a sample is usually indicated by its equivalent with the relative contents of catechin or gallic acid, which are used as the quantitative standards. Friedman and Jurgens (2000) reported that caffeic, chlorogenic, and gallic acids are not stable at higher pH levels. They also found that pH and time-dependent spectral transformations were irreversible (Friedman & Jurgens, 2000). This implies that the condensation products of phenolic acids would be produced by prior basification. In contrast, catechin, epigallocatechin, ferulic acid, rutin, and trans-cinnamic acid resisted pH-induced degradation. However, an irreversible compound was produced when 20% sodium carbonate was added prior to FCR. In the Lowry method of protein determination, an alkali is added and incubated with copper ions before the addition of FCR. This biuret-type reaction causes the conversion of non-phenolic dipeptides and enhances the reactive enolic compounds and cuprous ions. The FC method does not add copper and thus avoids this reaction. However, cuprous ions can interfere, albeit not significantly, at levels found in biological samples (Singleton et al., 1999). Uses of the FC method describe different orders of basification (addition of sodium carbonate). In the first type, FCR was added with sodium carbonate (Alonso Borbalan, Zorro, Guillen, & Barroso, 2003; Katsube et al., 2004; Maksimovic, Malencic, & Kovacevic, 2005); in the second, the addition of sodium carbonate was delayed for 1 min (Ao, Li, Elzaawely, Xuan, & Tawata, 2008; Dorman, Kosar, Baser, & Hiltunen, 2009; Du, Li, Ma, & Liang, 2009) or 5 min (Cízˇ et al., 2010) after the addition of FCR; and in the third method, the addition of sodium carbonate was 2 min (Chen, Lin, & Hsieh, 2007; Chen & Yen, 2007; Taga, Miller, & Pratt, 1984; Yen & Hung, 2000) or 5 min (Wang et al., 2011) before the FCR addition. Based on our previous study (Chen et al., 2012), gallic acid is unstable in alkaline solutions. Thus, the order that the reagents are added could affect the outcome and lead to incorrect comparisons in the studies with the gallic acid equivalents (GAE). Therefore, the widely accepted FC method must be validated to ensure high accuracy. The goals of this study were to investigate the effect of basification on the stability of phenols as assayed using the FC method and to validate the performance of the calibration curves in the determination of the quantitative standards of GAE and catechin equivalents (CaE). 2. Materials and methods 2.1. Chemicals The Folin–Ciocalteu reagent (FCR), (+)-catechin, oxalic acid, sodium carbonate and succinic acid were purchased from Sigma– Aldrich (St. Louis, MO, USA). Isophthalic acid was purchased from Bio Basic Inc. (Markham, ON, Canada). The ultra-pure water was obtained from a Milli-Q system (Millipore Corporation, Billerica, MA, USA) and was used as a solvent throughout this study. 2.2. The effect of incubation time on the FC method The method for the determination of the phenolic contents was modified from the FC method with GAE developed by Gahler, Otto,

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and Bohm (2003). The gallic acid solution (0.25 mL) was mixed with 1 mol/L FCR (0.25 mL) and incubated for 0, 2, 5 and 10 min. Next, the solution was mixed with 0.5 mL 20% sodium carbonate (Na2CO3) and 4 mL water. The final gallic acid concentrations were 0, 0.02, 0.04, 0.06, 0.08 and 0.1 mg-GAE/mL. The reaction solutions were incubated at room temperature for 25 min and then measured at 730 nm using a UV/Vis spectrophotometer (Lambda 35, PerkinElmer, USA). A homoscedastic two-sample t-test was used to assess whether the two sets of measurements differed. 2.3. RP-HPLC analysis of the gallic acid solution after basification Gallic acid solutions (1000 lg/mL) in 0.25 mL each were mixed with 0.5 mL of 20%, 2% and 0.2% sodium carbonate. The solutions were made up to 5 mL with water and then incubated for 25 min at room temperature. The final gallic acid concentration was 50 lg/mL. Moreover, the gallic acid solution with 0.2% Na2CO3 was incubated for 25 min and adjusted to pH 2.3 with 6 mol/L HCl. Chromatographic analysis was performed as follows: different reaction solutions were separated with a Mightysil RP-18 GP Aqua column (250/i.d. 4.6 mm, 5 lm, Kanto Chemical Co. Tokyo, Japan) at 27 °C on an HPLC system, equipped with a pump (HitachiL2130) and a photodiode array detector (Hitachi-L2450). The mobile phase was prepared with methanol and 0.25% H3PO4 in a ratio of 15:85. Samples of 20 lL were subsequently injected at the flow rate of 1.0 mL/min. 2.4. Effect of basification on the FC methods The effect of the order of addition of the reagents on the results using the FC method was investigated. The first method was abbreviated as FCR/Basf, where the Na2CO3 was added 5 min after the addition of FCR. The second method, which was abbreviated as Basf/FCR, was based on the method of Taga et al. (1984). FCR was added 2 min after the addition of Na2CO3. The gallic acid (or catechin) solution (0.25 mL) was added to 4.5 mL of 2% sodium carbonate. After 2 min, 1 N FCR (0.25 mL) was added to the mixture and incubated for 25 min at room temperature. The final concentrations of gallic acid or catechin were 0, 0.02, 0.04, 0.06, 0.08 and 0.1 mg/mL. The contents of the phenolic compounds in the samples were measured at 730 nm. 2.5. Analysis of total phenolic contents The total phenolic contents of soybean, black soybean and green tea extracts were determined using FCR/Basf and Basf/FCR of the FC method, as described in Section 2.4. The soybean and black soybean samples were purchased from a local market (Taoyuan, Taiwan). Green tea (Pi Lo Chun) was purchased from Ten-Ren Tea Co. (Taipei, Taiwan). The samples were lyophilised and ground through a 60-mesh sieve. One gram of each sample was placed in 5 mL 70% ethanol and sonicated for 20 min before being centrifuged at 4 °C, 3000 rpm for 10 min. The supernatants were collected. The process was repeated for the precipitates, and then the combined extracts were brought up to 10 mL with water and stored at 4 °C. The total phenolic contents of the extracts were determined as described in Section 2.4. The amount of total phenolic content was expressed as GAE in mg per gram of dried sample extract. 2.6. Catechin condensation with bicarboxylic acids Succinic acid in equal concentrations was added to the catechin solutions to polymerise the phenolic molecules. Mixtures of catechin and succinic acid were used to build the calibration curves in CaE for the two modified FC methods, which were marked as

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the Basf(Suc)/FCR and FCR(Suc)/Basf methods, respectively. The heating process (incubation at 75 °C for 10 min) was also utilised to enhance the efficiency of catechin condensation reactions with bicarboxylic acids, such as oxalic acid, succinic acid and isophthalic acid. All of the CaE calibrations were performed by multiple experiments, as described in Section 2.4. 2.7. Identification of the catechin condensation with oxalic acid The effect of oxalic acid on catechin condensation was determined via UV/Vis spectrophotometry. Four solutions were prepared: (a) 50 lg CaE/mL solution, (b) 50 lg CaE/mL solution containing an equal concentration of oxalic acid, (c) 0.25 mL of 1,000 lg CaE/mL solution containing an equal concentration of oxalic acid was mixed with 4.75 mL of 2% Na2CO3, and (d) 0.25 mL of 1000 lg CaE/mL containing equal concentrations of oxalic acid solution was mixed with 4.75 mL of 2% Na2CO3 and then incubated at 75 °C for 10 min. These solutions were measured at 250–800 nm via spectrophotometry. 2.8. RP-HPLC analysis of the catechin after basification The effect of oxalic acid on catechin condensation was examined via RP-HPLC analysis. Four solutions were prepared, as described in Section 2.7. Chromatographic analysis was performed with an RP-HPLC system. The analytical conditions were developed by Yang, Hwang, and Lin (2007). Gradient elution consisting of acetonitrile as solvent A and 0.9% acetic acid (prepared with ultra-pure water) as solvent B was used: a mixture of 5% A and 95% B was used during the first 5 min, and then the mixing was programmed to 13% A at the 6th min up to 25 min. Samples of 20 lL were injected subsequently at the flow rate of 1.0 mL/min. 3. Results and discussion 3.1. Effect of basification on using the FC method with gallic acid Gallic acid is extensively used as a comparative standard for the determination of the total phenolic content in various foods or natural products by the FC method. The results are generally reported as GAE. The GAE calibration curves in this study are shown in Fig. 1. In the late basification (addition of Na2CO3 after FCR with gallic acid), the t-test of GAE calibration curves reveals the consistency with the different reaction times (p > 0.75). In addition, the relative standard deviation of the signals measured is only 2.59% for the

Fig. 1. The GAE calibration curves of the FCR added after the addition of the sodium carbonate for 2 min (⁄) and of the sodium carbonate added after the FCR addition for 0 min, 2 min, 5 min, and 10 min, denoted by d, , j, and N, respectively.

different reaction times at 0.1 mg/mL of GAE. The GAE calibration curve for the prior basification (addition of Na2CO3 before FCR with gallic acid) examined was, however, found to be lower than that of the late basification. The slopes of the GAE calibration curves for late basifications were all greater than 4.7, while the slope for the prior basification was only 1.93, suggesting that earlier basification may decrease the reductive efficiency of phenols in the FC method. The slope of the calibration curve can be an index to the sensitivity of the analytical methods with respect to the same quantitative standard. The sensitivities of the GAE calibration curves for the late basification were higher than the prior basification despite the signal nuances in the reaction time. The spectrum of the gallic acid solution in acid (pH 3.27) has a maximum absorption at approximately 270 nm (Chen et al., 2012). The chromatograms of the gallic acid solutions at 270 nm, following different treatments, are shown in Fig. 2. The chromatogram of gallic acid without basification has a peak at 5.78 min (line d). However, only tiny signals were observed in chromatograms a, b and c for the samples of gallic acid treated with 20%, 2% and 0.2% Na2CO3, respectively. These indicate that the peak at 5.78 min would disappear when Na2CO3 was used to basify gallic acid. However, two peaks at 2.82 min and 5.80 min can be observed in line e, where the gallic acid solution was acidified to pH 2.3 by 6 mol/L HCl after basification with 0.2% Na2CO3. Some irreversible compounds might have been produced from gallic acid during the process of basification. 3.2. Effect of acid on catechin condensation The slopes of the CaE calibration curves for catechin standards in the prior and late basifications are displayed in Fig. 3A. As shown in Fig. 3A, the slope of CaE calibration for the prior basification is less than the rate for the late basification. Catechin is a flavan3-ol with five phenolic hydroxyl groups. In general, catechin is able to resist pH-induced degradation due to the absence of a carboxyl group in the molecule (Friedman & Jurgens, 2000). The slopes of all of the CaE calibration curves in Fig. 3A are similar, despite the existence of succinic acid. As a result, it seems that the order of basification does not affect the performance of the FC method with catechin equivalents. However, the cross-linkage of catechin and bicarboxylic acid can be observed in alkaline solutions when the reaction temperature is increased, which was observed in alkaline solutions when the reaction temperature was increased. Prior basifications with heating resulted in reduced slopes for the CaE calibrations

Fig. 2. The chromatograms of the 50 lg GAE/mL solutions treated with (a) 20%, (b) 2%, (c) 0.2%, (d) free Na2CO3, and (e) acidification to pH 2.3 by 6 mol/L HCl after the basification with 0.2% Na2CO3.

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Fig. 3. (A) The sensitivities of the CaE calibrations for the different processes in FCR, organic acids, and basification order. (B) The sensitivities of the CaE calibrations for the different processes and heating (Suc: succinic acid, Ox: oxalic acid, Isoph: isophthalic acid). Error bars represent the standard deviations.

containing bicarboxylic acids by approximately one-half as much as the slopes of the late basifications (Fig. 3B). The chromatograms for catechin solutions at 280 nm following different treatments are shown in Fig. 4A. The chromatogram of 50 lg CaE per mL and catechin with oxalic acid are shown in lines a and b, respectively. The catechin peaked at 11.6 min (line a), and the oxalic acid peaked at 3.1 min (line b). The chromatogram of a mixture of catechin, oxalic acid and 2% sodium carbonate is shown in line c. The catechin was still present at 11.6 min (line c). As to basification and heating, the chromatogram (line d) has no signal for catechin in the mixture of catechin, oxalic acid and 2% sodium carbonate incubated at 75 °C for 10 min. However, four peaks at 2.4 min, 3.1 min, 3.2 min and 6.2 min can be observed in line d. This result indicates that the peak at 11.6 min would have disappeared when catechin reacted with oxalic acid under the alkaline and high temperature scenario. The absorption spectra of a mixture with 50 lg CaE per mL and catechin with oxalic acid are shown in Fig. 4B (line a and line b), respectively. The absorbance of the mixture was greater than catechin alone at the peak at approximately 279 nm. Oxalic acid had no impact on the observed spectrum. However, the absorption spectrum of a mixture of catechin, oxalic acid and 2% sodium carbonate is shown in line c (Fig. 4B). The peaks indicate a red shift at 290 nm and 428 nm. Regarding basification and heating, the absorption spectrum of a catechin mixture incubated at 75 °C for 10 min is shown in line d. Three peaks were observed at 293 nm, 354 nm and 422 nm. Catechin derivatives were produced by the cross-linkage with oxalic acid under the alkaline and high temperature scenario. 3.3. Quantitative bias in the applications of the FC method Dissociation of a phenolic proton leads to a phenolate anion, which is capable of reducing FCR. Phenolic compounds react with FCR and change colour through an electron transfer mechanism only under an alkaline environment (Huang et al., 2005). For

Fig. 4. (A) The chromatogram spectra and (B) the absorption spectra of (a) catechin, (b) the mixture of catechin and oxalic acid, (c) the mixture of catechin and oxalic acid in 2% Na2CO3, (d) the mixture of (c) incubated at 75 °C for 10 min. All of the reaction solutions were measured in the spectral range of 250–800 nm.

comparison, the slopes of the GAE calibration curves in Fig. 1 are also examined and listed in Table 1. In Table 1, the sensitivity of the GAE calibration curve is 5.19 when the basification was performed after the addition of FCR. Gallic acid is a tri-hydroxyl-benzoic acid. The carboxyl group of gallic acid is reactive to the hydroxyl groups from the Basf/FCR treatment (prior basification). The sensitivity of the GAE calibration was reduced by esterification. The sensitivity was 2.7 times more than that for the basification before the FCR in the GAE calibrations. Therefore, the phenolic content of the same sample would appear to be different, despite using essentially the same method. As shown in Fig. 5, the total phenolic contents of soybean, black soybean and green tea (as GAE) were determined using the prior and the late basification. The phenolic contents determined were almost 2.2 times more with the prior basification compared to the late basification case. The carboxyl group of succinic acid is also reactive to the phenolic hydroxyl groups of catechin via the Basf(Suc)/Heating/FCR treatment. The sensitivity of the CaE calibration was reduced. When heat was applied, esterification of catechin occurs in an alkaline solution, while the phenolic hydroxyl groups are consumed. The quantitative efficiency depends on the relative contents of the carboxyl groups. The ratio for the sensitivity of the CaE

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Table 1 The sensitivities of the calibration curves for quantitative measurement in GAE or CaE by the different treatments using the FC method. Phenolic GAE standards

GAE

CaE

CaE

CaE

CaE

Treatments FCR/Basf Sensitivity 5.19 ± 0.06 [abs/conc.]

Basf/FCR 1.93 ± 0.23

FCR/Basf 4.73 ± 0.10

Basf/FCR 4.18 ± 0.02

Basf(Suc)/FCR 4.34 ± 0.05

Basf(Suc)/heating/FCR 2.22 ± 0.04

References

Fig. 5. A comparison of the total phenolic contents of soybean, black soybean and green tea tested by the FCR/Basf and Basf/FCR protocols of the FC method.

calibrations with the prior/late basifications was attenuated to approximately 0.5. Catechin condensation by the cross-linkage molecule is reported for the first time in this study. Heating is usually applied to reduce the time of analysis. The esterification is usually condensed with carboxyl and hydroxyl groups of organic molecules in an alkaline condition for the equivalent calculation. Thus, the determination of phenolic content in the raw samples by the FC method is affected significantly by the order of addition of sodium carbonate, i.e., after or before the addition of FCR. 4. Conclusion The FC method is a colourimetric assay to measure the antioxidant capacity of botanical materials in food science. The FCR in alkaline solution is used to assay phenolic antioxidants for the normalised quantisation, which is called GAE, compared to the standard. Our results demonstrated that the order of addition of sodium carbonate relative to the addition of the other reagents significantly affected the determination of the phenolic content by the FC method. Because the molecular condensation reaction reduces the reactivity of phenols to FCR, a quantitative bias would be derived from the modified FC method. To optimise the procedure, the sodium carbonate should be added after the FCR addition in the FC method. This study reveals the critical step in the popular method to determine total phenolic contents in natural products. Furthermore, the performance of the Folin–Ciocalteu method is optimised and validated again. Acknowledgements The authors thank the editor and anonymous reviewers for their assistance in the processing of this article. We appreciate Dr. Andrew Corbin from Washington University for his critical review and proof reading of this manuscript. We also appreciate the help of the students in the AA402 laboratory, namely HuiHsueh Chang, Ji-Shiuan Wang, Jui-Ling Li, Jun-Yun Wu, Shin You and Yun-Li Chang, who provided assistance in performing the experimental work. The financial support for this work is partially from the National Science Council, Taiwan, under contract no. NSC 100-2113-M-130-001-MY2 (Grant to L.-Y. Chen).

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Effect of esterification condensation on the Folin-Ciocalteu method for the quantitative measurement of total phenols.

The Folin-Ciocalteu method is widely applied for the determination of the total phenolic contents in natural products. This method is significantly af...
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