Color and chemical stability of tea polyphenol (−)-epigallocatechin-3-gallate in solution and solid states

Food Research International xxx (2012) xxx–xxx

FRIN-04353; No of Pages 13

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Color and chemical stability of tea polyphenol (−)-epigallocatechin-3-gallate insolution and solid states

Na Li a, Lynne S. Taylor b, Mario G. Ferruzzi a, Lisa J. Mauer a,⁎a Department of Food Science, Purdue University, 745 Agriculture Mall Drive, West Lafayette, Indiana 47907, United Statesb Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, United States

Abbreviations: C, (−)-catechin; CG, (−)-catechinECG, (−)-epicatechin gallate; EGC, (−)-epigallocatechigallate; GC, (−)-gallocatechin; GCG, (−)-gallocatechinRT, retention time; TOF, time-of-flight.⁎ Corresponding author. Tel.: +1 765 494 9111; fax:

E-mail address: [email protected] (L.J. Mauer).

0963-9969/$ – see front matter © 2012 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.foodres.2012.11.019

Please cite this article as: Li, N., et al., Color aFood Research International (2012), http://dx

a b s t r a c t
a r t i c l e i n f o
Article history:Received 31 August 2012Accepted 17 November 2012Available online xxxx

Keywords:StabilityEGCGTheasinensinGCGEpimerizationOxidationSolid stateColor

(−)-Epigallocatechin-3-gallate (EGCG), the main component with the highest biological activity in green tea,has been extensively studied for its activities and degradation behaviors in infusions and solutions. However,neither the characterization of its degradation products in solutions, nor its color degradation and stability inthe solid state are well documented. Therefore, in this present study, the color and chemical stability of EGCGin solutions with various concentrations ranging from 0.05 mg/mL to 30 mg/mL and in the solid state followingstorage at 0% and 85% relative humidity (RH), 80 °C were investigated. Color parameters were monitored overtime, and the chemical stability was determined using high performance liquid chromatography (HPLC)–diode array detection (DAD) analysis and HPLC–mass spectrometry (MS). Mathematical models for color degra-dation of EGCG in both solution and solid stateswere established. The chemical stability of EGCGwas found to beaffected by its physical state, concentration of reactants, oxygen levels, pH, and RH conditions. Epimerization andoxidation were found to be the two major reactions causing the degradation of EGCG in the solution and solidstate, respectively. Major degradation products, catechin monomers and oxidation dimers, were identified.Cleavage products, gallic acid and catechin monomers, were also found. These results further elaborated thedegradation mechanisms of EGCG in solutions of varying concentrations and revealed the chemical changesresulting in color degradation of EGCG in the solid state.

© 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Due to its reported health benefits, green tea has garnered increas-ing worldwide interest. As the most abundant component in greentea, (−)-epigallocatechin gallate (EGCG) has one of the highest antiox-idant activities among catechin monomers. This component is believedto play an important role in both the prevention and treatment of awide variety of diseases, including cardiovascular diseases (McKay &Blumberg, 2002) and carcinogenesis in the lung (Suganuma et al.,1999), colon (August et al., 1999), and skin (Katiyar, Elmets, & Katiyar,2007). As a result, interest in application of EGCG in food fortificationand as a dietary supplementation or therapeutic agent has increased.

EGCG has been reported to undergo epimerization and oxidationunder thermal treatments or exposure to near neutral pH in solutions(Chen, Zhu, Tsang, & Huang, 2001; Neilson et al., 2007; Wang &Helliwell, 2000). GCG (Wang & Helliwell, 2000) and the dimers ofEGCG, such as theasinensins and the P-2 dimer (Neilson et al., 2007),

gallate; EC, (−)-epicatechin;n; EGCG, (−)-epigallocatechingallate; RH, relative humidity;

+1 765 494 7953.

rights reserved.

nd chemical stability of tea p.doi.org/10.1016/j.foodres.20

were identified major degradation products discovered to result fromepimerization and oxidation, respectively. Under alkaline conditions,oxidation is the predominant reaction; following the inhibition ofoxidation with the addition of superoxide dismutase, epimerization be-comes more appreciable (Sang, Lee, Hou, Ho, & Yang, 2005).

A full characterization of the degradation products of EGCG in bothsolution and solid states is of great importance in order to better un-derstand the degradation mechanisms of EGCG. This information,however, is lacking, even though previous studies have reported onthe degradation profiles of EGCG and its epimerization product GCGin aqueous solutions (Chen et al., 2001; Sang et al., 2005; Wang &Helliwell, 2000; Wang, Zhou, & Jiang, 2008). Also, although numerousstudies have reported the degradation of green tea catechins in vari-ous tea drinks, infusions, cell culture media, green tea powders, aswell as in the bread baking process (Chen et al., 2001; Li, Taylor, &Mauer, 2011; Neilson et al., 2007; Proniuk, Liederer, & Blanchard,2002; Sang et al., 2005; Su, Leung, Huang, & Chen, 2003; Wang &Helliwell, 2000; Wang, Zhou, & Jiang, 2008), the chemical stabilityof isolated EGCG in the solid state has not been well documented.

In the formulation design of solid state dietary supplements anddrugs, amorphous solids have been extensively used to enhance the dis-solution rate of the compound, and to increase drug concentrations insolution, which then leads to an elevated absorptive transport and en-hanced bioavailability (Brouwers, Brewster, & Augustijns, 2009). In

olyphenol (−)-epigallocatechin-3-gallate in solution and solid states,12.11.019

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2 N. Li et al. / Food Research International xxx (2012) xxx–xxx

certain processing operations involving crystalline drugs, such as mill-ing, lyophilization, spray drying, and wet granulation, partially or fullyamorphous material can also be produced (Hancock & Zografi, 1997).Due to the thermodynamically and chemically unstable nature of theamorphous form, chemical stability assessments of this form are essen-tial in the pre-formulation design stage (Tong & Zografi, 2004). Also,catechins exist in the amorphous state in spray dried green tea pow-ders. Therefore, the stability study of amorphous EGCG is important.

Beyond actual chemical stability, color is another important attri-bute in shelf-life and product quality determinations. Because coloranalysis is non-destructive, and is easy and quick to perform, colorparameters are often monitored alone or in tandem with chemicalconcentrations of a target compound. A pinkish-brown color hasbeen observed in EGCG powders after long-term storage withoutthe appearance of chemical degradation beyond a trace amount ofGCG (Sang et al., 2005). Therefore, both catechin content and productcolor are important when monitoring the stability of EGCG formula-tions, and understanding the chemical changes resulting in color deg-radation is an essential first step in controlling quality.

To fully understand the stability of EGCG, a more detailed study ofits stability in solution and solid states is needed. In the present study,the color and chemical stability of EGCG in solutions with variousconcentrations and in the solid state under differing RH conditionswere investigated.

2. Materials and methods

2.1. Materials

The crystalline EGCG powder (Teavigo®) prepared from green teaextract was obtained from DSM Nutritional Products (Heerlen, NL).The composition of this crystalline EGCG powder and the amorphouspowder created from it are listed in Table 1, in agreement with lite-rature values (Wang, Zhou, & Wen, 2006). (−)-catechin, (−)-catechingallate, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin,(−)-epigallocatechin gallate, (−)-gallocatechin gallate, theaflavin, andgallic acid were all purchased from the Sigma-Aldrich Chemical Co.(St. Louis, MO). The structures of these catechins and gallic acid areshown in Fig. 1. Methanol (HPLC grade), acetonitrile (MeCN) (HPLCgrade), and glacial acetic acid were purchased from MallinckrodtBaker (Phillipsburg, NJ). All other chemicals were purchased fromSigma-Aldrich Chemical Co. (St. Louis, MO).

2.2. Preparation of amorphous EGCG samples

The amorphous form of EGCG was prepared by solvent evaporationof an ethanol/dichloromethane (50:50, v/v) solution using a rotaryevaporator. About 7.5 g of crystalline EGCG powders was dissolvedinto 100 mL of the solvent system, and solvent was removed undervacuum at 60 °C for 10 to 15 min. The samples were then furtherdried at room temperature using a vacuum oven (Precision Scientific,Chicago, IL) for 48 h. Samples were then cryomilled with a SpexCryomill (SPEX CertiPrep LLC., Metuchen, NJ) for 10 min to achieve areasonably uniformly distributed particle size. The amorphous naturewas verified by the use of powder X-ray diffraction, as is shown inFig. 2. Amorphous EGCG powders were then stored in desiccators withDrie-Rite® (WA Hammond Drierite Co. LTD., Xenia, OH) at −20 °Cuntil further use.

Table 1Compositions of crystalline and amorphous EGCG powders.

EGCG (%) ECG (%)

Crystalline 89.25±3.04a 2.40±0.05b

Amorphous 87.08±4.52a 2.36±0.11b

a, b: same letter indicating no significant differences between the means.

Please cite this article as: Li, N., et al., Color and chemical stability of tea pFood Research International (2012), http://dx.doi.org/10.1016/j.foodres.20

2.3. Powder X-ray diffraction (PXRD)

Powder X-ray diffractograms were obtained using a ShimadzuLabX XRD-6000 (Kratos Analytical, Chestnut, NY) diffractometer thatwas operating in the Bragg–Brentano configuration and equippedwith a fine-focus Cu Kα source rated at 1500 W when operating at40 kV/30 mA. A silicon standardwas employed for calibration purposesprior to each analysis. The aluminum sample holders used had an innerdiameter (i.d.) of 1 cm and a depth of 0.5 mm. The EGCG powders werepacked into the well and a smooth surface was produced using a glassslide. Samples before and after storage treatments were analyzed: thesamples that recrystallized during storage were ground into evenly dis-tributed powders prior to X-ray analysis. Samples were scanned from 5to 35° with a rate of 8° per min and an incremental increase of 0.04°.

2.4. Solubility determination

Triplicate sampleswere preparedwith the addition of approximately100 mg of crystalline EGCG powders or 200 mg amorphous EGCG pow-ders into a 1.0 mL centrifuge tube (Eppendorf, Hamburg, Germany)containing 1 mL distilled water in the absence and presence of 2% aceticacid. After 24 h of equilibration at room temperature on a shakingplatform (VWR International LLC., West Chester, PA), the tubes werethen centrifuged for 30 min at 10,000 rpm a micro centrifuge (VWRInternational LLC., Bristol, CT). Subsequently, 100 μL of the supernatantwas separated and filtered through 0.45 μMmembrane filters, followedby dilution with 2% acetic acid to achieve the concentration range cov-ered by the standard curve (0.0075–0.18 mg/mL) prior to HPLC analysis.

2.5. Storage treatments

Storage experiments were conducted using a temperature con-trolled incubator (Precision, Winchester, VA) for solid samples and adigital heat block (VWR International LLC., Bristol, CT) for solutions,both at 80 °C. The temperatures were verified by a thermometer. RHfor solid state samples was controlled using Drierite® for 0%RH and po-tassium chloride saturated solutions for 85%RH, and thewater activitieswere verified with an AquaLab 4TE water activity meter (DecagonDevices Inc., Pullman, WA). EGCG solutions with differing concentra-tions (0.05, 0.15, 0.3, 1.5, 5, 15, and 30 mg/mL) were used to study thestability of EGCG, in order to cover the range of concentrations typicallyfound in most animal studies and tea drinks up to near saturatedconcentrations (Sang et al., 2005). Crystalline EGCG powders weredissolved in distilled water to achieve differing concentrations, andthe pH of each was measured. Aliquots from the stock solution weredistributed in 20 mL scintillation vials and then flushed with N2.These vials were then sealed and heated at 80 °C for up to 720 min.All solution state samples were analyzed within 24 h: the more dilutesolutions (0.05, 0.15, 0.3, and 1.5 mg/mL) were diluted and analyzedwithin 4 h after heat treatments.

For solid state samples, triplicate samples of 15.0 mg EGCG amor-phous powders were prepared in 8 mL scintillation vials and stored atdifferent RHs (0% and 85%) at 80 °C. Crystalline EGCG powders werealso stored at 85% RH as a control. An additional heating studyconducted at 120 °C was used to compare the stability of crystallineversus amorphous EGCG powders. After the storage treatments, allsamples were capped and immediately frozen at−20 °C until analysis.

2.6. Color analysis

Several color parameters were analyzed throughout the storagetreatments. The L (brightness), a (redness), and b (yellowness) colorspace parameters were monitored using Color Companion software(DMI digital interactive LLC) analysis of digital photographs of thesamples. Both the powder samples and 5 mg/mL EGCG solutions wereanalyzed. To examine the color stability of EGCG aqueous solutions, a



O

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EGCG: R1=OH, R2=galloylECG: R1=H, R2=galloylEGC: R1=OH, R2=OHEC: R1=H, R2=OH

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Galloyl

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Theasinensin A

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Theasinensin D

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Fig. 1. Structure of EGCG and its degradation products.

3N. Li et al. / Food Research International xxx (2012) xxx–xxx

5 mg/mL EGCG solution was heated at 80 °C over a period of up to720 min. This concentration was chosen based both on the relativelyhigh color intensity of the solution and on the short reaction time deter-mined to be necessary from the preliminary experiments. The color ofsolid state EGCG samples was monitored daily for a period extendingup to 33 days. Samples were prepared in triplicates, three photographsof each samplewere taken under the same light and distance conditions,and then five points were taken from each picture for color analysis. Awhite background was used as the blank.


The total color changes (TCD), La/b, and hue angle (h) are alsocommonly used color variables to describe color degradation, andthese were calculated as follows (Ávila & Silva, 1999; Topuz, 2008):

TCD ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiΔL2 þ Δa2 þ Δb2

pð1Þ

h ¼ tan−1 b=að Þ: ð2Þ



5 10 15 20 25 30 350

100

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Rel

ativ

e pe

ak in

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Fig. 2. X-ray powder diffraction pattern of EGCG powders. Upper: crystalline EGCG.Middle:crystallized EGCG from amorphous powder stored at 85% RH after 12 h. Lower: amorphousEGCG.


The TCD and La/b suggest a total color change of the food system,while the hue angle indicates the actual color, i.e., how similar the coloris to red, green, blue and yellow. A hue angle of 180 °C is the recognizedgreen color, while 0 °C represents red (Arias, Lee, Logendra, & Janes,2000).

All of these color parameters, i.e. L, a, b, TCD, La/b, and h, were used inthe development of mathematical models for color degradation inEGCG samples. Zero order reaction kinetics is the most commonly ob-served kinetics seen for color degradation in food systems (Ibarz,Pagán, & Garza, 1999; Lavelli & Vantaggi, 2009):

C ¼ C0−kt ð3Þwhere C is the measured value of color variables at time t, C0 is the colorvariable at time0, t is the reaction time, and k is the reaction rate constant.

The fraction first order kinetic model has also been used to describechemical reactions (Ávila & Silva, 1999; Ibarz et al., 1999; Steet & Tong,1996; Topuz, 2008). For an irreversible first order reaction, the fraction fis defined as:

f ¼ C0−CC0−C∞

ð4Þ

where C∞ is the variable at infinite time.

Table 2Kinetic parameters for the color degradation of EGCG in solution and solid states.

Samples Model C0

80 °C, 5 mg/mLEGCG solution

L Zero-order 103.2a Logistic –

b – –

La/b Logistic –

TCD Fractional conversion 3.66h Logistic –

80 °C, 0%RH, EGCGAmorphous powder

L Fractional conversion 80.83a Fractional conversion 9.91b Fractional conversion 15.20La/b Fractional conversion 53.02TCD Fractional conversion 2.65h Fractional conversion 56.78

80 °C, 85%RH, EGCGinitially amorphous powder

L Fractional conversion 74.97a Zero-order 34.89b Zero-order 41.29La/b Zero-order 53.10TCD Zero-order 16.00h Fractional conversion 56.59


For chemical reactions,C is the concentration of a reactant. ThereforeC∞ would be zero at infinite time. Thus the first order rate equation canbe described as:

lnCC0

¼ ln 1−fð Þ ¼ −kt: ð5Þ

In the case of color degradation, the C∞ values are not zero. Thereforein physical property modeling, the model becomes:

C−C∞C0−C∞

¼ exp −ktð Þ: ð6Þ

Logistic (sigmoid) functions have also been commonly used tomodel color changes in food systems (Calligaris, Falcone, & Anese,2002; Tijskens & Evelo, 1994):

C ¼ a1a2 þ exp −ktð Þ þ a3 ð7Þ

where a1, a2, and a3 are the constants.To evaluate the color of the EGCG samples, different models were

chosen for different color parameters and different samples, dependingon the degradation pattern. The regression coefficients and parametersare listed in Table 2. Color swatcheswere also produced using PhotoshopCS3 10.01 (Adobe Systems Incorporated, San Jose, CA) according theaverage L, a, b values obtained for each sample.

The correlations between color and chemical stability wereestablished using an exponential regression model:

y ¼ A1e− x

t1 þ y0

where y is the corresponding color variable, x is the peak area of the col-ored compound with HPLC–DAD at 280 nm.A1, t1, and y0 are constants.

2.7. High performance liquid chromatography (HPLC)

2.7.1. HPLC–DAD analysisPrior to HPLC analysis, all of the samples were diluted with 2% acetic

acid to achieve a final concentration of 0.15 mg/mL (EGCG/H2O) andwere then filtered with 0.45 μM membrane filters. HPLC analysis wasthen performed using the method described by Li, Taylor, Ferruzzi,and Mauer (in press) with minor modifications (Li et al., 2011). Inbrief, a Waters 2690 separation module equipped with a Waters 996photodiode array detector along with Masslynx V 4.1 software (WatersCorp., Milford, MA) was applied. Separation was achieved using aWaters XTerra RP-18 column (100 mm×3.9 mm, i.d. 3.5 μm). A binary

C∞ k (min−1) a1 a2 a3 R2

– −0.05938 – – – 0.8995– 0.01411 0.6946 0.01375 0 0.9555– – – – – –

– 0.01274 1.375 0.01901 0 0.946276.22 0.00777 – – – 0.9786– 0.00845 −4.381 0.07414 97.2 0.967354.34 0.2251 – – – 0.899836.12 0.4061 – – – 0.905031.06 0.6921 – – – 0.841870.29 0.1668 – – – 0.311342.97 0.3730 – – – 0.874137.44 0.1340 – – – 0.662850.93 0.1652 – – – 0.6482– −0.6373 – – – 0.6314– −0.9493 – – – 0.7143– −0.0704 – – – 0.0013– 0.9153 – – – 0.653644.91 0.2142 – – – 0.3048



Fig. 3. Color changes of EGCG in solution and solid states.


solvent systemwas utilized: Phase A—water, acetonitrile, and TFA (919/80/1, v/v) and Phase B — water, acetonitrile, methanol, and TFA (699/270/30/1, v/v). An initial gradient of 95:5 (A:B) was used, followed by a40:60 gradient from 2.85 to 6.42 min (convex), and then 1:99 at15 min (concave), with a return to 95:5 at 17.7 min (immediate) for atotal run timeof 20 min. Theflow ratewas 0.7 mL/min, the injection vol-ume was 10 μL, and the wavelength scan was from 200 to 800 nm. Cat-echins and gallic acid were verified by the elution time of standards andwere quantitated by the corresponding standard curves (R2, 0.9997–1.0000). Integrations of each compound were conducted at 280 nm.

2.7.2. HPLC–MS analysisBoth the solution and solid state samples were analyzed with an

HP1100 HPLC separation module (Agilent, Waldbronn, Germany)equipped with Waters XTerra RP-10 columns (guard column:20 mm×2.1 mm, i.d. 3.5 μm; analytical column: 100 mm×2.1 mm,i.d. 3.5 μm) and MS detection. The elution method for the detection oftheasinensin used was described by Neilson et al. (Neilson, Song,Sapper, Bomser, & Ferruzzi, 2010) with minor modifications. Briefly, abinary solvent system was used: Phase A — formic acid, and water (4/996, v/v) and Phase B — isopropyl alcohol, formic acid, and methanol(40/4/956, v/v). An initial gradient of 85:15 (A:B) was used, followedby linear gradient: 30:70 at 8 min, 20:80 at 9 min, then 0:100 at9.5 min, with a return back to 85:15 at 9.5 min for a total run time of18 min. The flow rate was 0.3 mL/min, and the columns weremaintained at 40 °C. The column and elution method used for the de-tection of the colored compound are the same as the method forHPLC-DAD detection described previously. After separations, the liquideluent was introduced into the (−)-electrospray ionization interfaceinto a Waters LCT premier time-of-flight (TOF) mass spectrometer(Waters Corp., Milford, MA). The ESI capillary and cone voltages usedwere −3.5 kV and 35 V, and the desolvation and source temperatureswere maintained at 350 °C and 150 °C, respectively. N2 was used asboth the desolvation and cone gas, at the flow rate of 400 L/h and60 L/h, respectively. Degradation products were monitored betweenm/z 100–1500 and extracted ion responses (EIR) were followed at m/zvalues corresponding to their deprotonated pseudomolecular ionsfor monomeric catechins and known dimerized oxidation products(m/z=289, 305, 441, 457, and 913 for EC/C, EGC/GC, ECG/CG, EGCG/GCG, and theasinensin A/D, respectively).

2.8. Statistical analysis

All of the data were presented in the form of means±standard de-viations. A pb0.05 level was chosen for the significance tests. All sta-tistical analyses and regressions were conducted using OriginPro8(Origin Lab Corp., Northampton, MA). Model fittings and parametercalculations were performed using Matlab R2009a (The MathWorksInc., Natick, MA).

3. Results and discussion

3.1. Color degradation of EGCG solutions

The effects of heat treatment on the color of EGCG solutions are sum-marized in Figs. 3 and 4A, respectively.With increased heating time, theEGCG solutions turned from clear to yellow/orange. This shift corre-sponds to a decrease in the L value and increases in a and b values(Fig. 4A1). The changes in the L value fit into a zero-order kineticmodel: it decreased in a linear manner, with a corresponding increasein absorbance at 420 nm (data not shown). The perceived yellow/orange color is considered to be related to the larger molecular weightoxidation products of catechins, such as theaflavins, thearubigins, andtheabrownins (Bailey, Nursten, & McDowell, 1992; Bark et al., 2011;Gong, Zhang, Peng, Fan, & Dong, 2012; Sang et al., 2005). The a value in-creased over time following a sigmoid pattern. The b value reached a


maximum value at 240 min, and then decreased linearly over time.The La/b and h parameters changes followed the sigmoid patterns,and the TCD values increased over time, gradually reaching a measuredplateau level (Fig. 4A2). Therefore, for establishing the kinetic modelsfor the color data, a zero-order kinetic model was utilized for the L pa-rameter with the application of Eq. (3), sigmoid functions were usedfor the a, La/b, and h parameters with Eq. (6), and a fractional conver-sion kinetic model was used for the TCD value with Eq. (7). The param-eters and rate constants for the kinetic models are listed in Table 2, withR2 values ranging from 0.8995 to 0.9786.

3.2. Color Degradation of EGCG powders

Crystalline EGCG in the solid state was reported to be very stable,demonstrating color changes but no detectable new compounds andonly a trace amount of GCG formation over prolonged storage, as deter-mined by both HPLC and NMR (Sang et al., 2005).We heated both crys-talline and amorphous EGCG powders at an elevated temperature(120 °C) in order to compare the differences in color and chemical sta-bility between its crystalline and amorphous form. The compositions ofthe initial crystalline EGCG sample and the amorphous EGCG powderprepared from it were not significantly different (Table 1); however,these powders responded differently to heat treatment. CrystallineEGCGpowderwhichwas heated at 120 °C had 98.89±3.86% remainingafter 16 h,while only 69.40±3.48% of amorphous EGCG remained afterthe same treatment. The crystalline powder turned to a light pinkishcolor, while the amorphous powder exhibited a darker crimson color.Therefore, in order to further investigate the relationship between thecolor and chemical stability of EGCG in the solid state, amorphousEGCG powder was used throughout this study. Also of note, the solubil-ity of crystalline EGCG in water was 47.74±2.94 mg/mL at 25 °C, com-pared to the amorphous powder solubility of 108.18±7.53 mg/mL.

The effects of RH treatment on the color and color parameters of solidstate EGCG powders are summarized in Figs. 3 and 4B and C. AmorphousEGCG powders stored at 0% RH changed from a pale pink color to a darkred color over time (Fig. 3). These samples remained X-ray amorphousthroughout the 0% RH storage treatment but turned dark, which is indi-cated in decreases in the L values (Fig. 4B1). Increases in the a and bvalues suggested that there was a formation of reddish and yellowishpigments, which are likely due to the formation of oxidation products,as will be discussed later (Guyot, Vercauteren, & Cheynier, 1996). The



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Fig. 4. Color stability of EGCG in solution and solid states. A) 5 mg/mL solution. B) Solid state at 0% RH. C) Solid state at 85% RH. D) Correlations of L and h in solution state. E) Correlation ofa and h in solid state. Data are shown by: ■ L ● a ▲ b □ La/b ○ TCD △ h.


Please cite this article as: Li, N., et al., Color and chemical stability of tea polyphenol (−)-epigallocatechin-3-gallate in solution and solid states,Food Research International (2012), http://dx.doi.org/10.1016/j.foodres.2012.11.019



La/b, TCD, and h parameters are shown in Fig. 4B2. Fractional conversionmodels (Eq. (7)) were applied to L, a, and b values, as well as La/b, TCD,and h parameters for the EGCG powders at 0% RH. The parameters andrate constants are listed in Table 2, with R2 values ranging from 0.8418to 0.9050, with the exception of 0.6628 for h and 0.3113 for La/b. Suchfraction conversion kinetics was commonly observed in previous studiesinvolving pear puree and peas (Ávila & Silva, 1999; Ibarz et al., 1999;Steet & Tong, 1996). The color of the amorphous powders stored at 0%RH had a maximum absorbance at around 497 nm, while the solutionsamples had a shifting and increasing absorbance between 300 and500 nm over time (data not shown). The differences in the color andcolor degradation profiles in the EGCG solution and solid samples possi-bly suggest different chemical degradation products and pathways.

Amorphous EGCG powders stored at 85%RH crystallized rapidly(verified by PXRD analysis, Fig. 2) and collapsed into a solid cake within12 h. This collapsed structure created an interior that was moreprotected from the environment than the surface, andwhile the interiorof these samples remained ivory the surface underwent significantcolor changes over time. The surface of the crystallized samples shiftedfrom the pale pink of the starting amorphous powder to a light red colorafter 1 day of storage (Fig. 3). Therefore, the color parameters at day0 from the amorphous powders were not used in the development ofkinetic models for the EGCG powders that crystallized during storage

A

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Fig. 5. HPLC chromatogram and UV spectrum of EGCG degradation products. A) Concentratidegradation.


at 85% RH: only the color changes on the surface of the crystallizedEGCG samples were used in the models. It is worth noting that whenthe amorphous EGCG powder (0% RH storage) and the recrystallizedEGCG powder (85% RH storage) were reconstituted, the solutionswere different colors: red-orange and yellow-gold, respectively (Datanot shown). The color changes of amorphous EGCG powders stored at0% and 85% RH showed different kinetics. This could be due to changesin reaction mechanisms resulting from different RHs and the alteredphysical state of the samples. Also, for the crystallized samples, colorchanges occurred on the surface, whereas for amorphous powders auniform color was developed.

Crystalline EGCG powders stored at 85% RH developed different andlighter colors than the amorphous powders (Fig. 3). Different patternsin the X-ray diffractograms (Fig. 2) indicate the possibility that a differ-ent polymorph of crystalline EGCG was formed when the amorphousEGCG recrystallized during storage at 85% RH. This structural differencecould contribute to the differences in color development of these twocrystalline solids during storage.

The surface color of recrystallized EGCG shifted from a light red to adark brown color over time (Fig. 3), which is indicated in decreases in L,a, and b values (Fig. 4C1). The La/b, TCD, and h parameters are shown inFig. 4C2. Zero-order and fractional conversion kinetic models were ap-plied to the color parameters as shown in Table 2, and although these

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Fig. 7. Thermal degradation of EGCG in 0.3 mg/mL aqueous solution at 80 °C. A) EGCG,EGC, GCG, and gallic acid. Data are shown by: –■– EGC –●– EGCG –▲– GCG –▼– gallicacid. B) ECG, CG, and C. Data are shown by: –□– CG C –△– ECG.


models fit the data the best, the R2 values weremuch lower than for theamorphous EGCG powders stored at 0% RH.

The changes of hue angle were found to be linearly correlated withcertain color parameters (Tolvaj &Mitsui, 2010). In EGCG solutions, a lin-ear correlationwas obtained between redness a and hue angle h (Fig. 4D,R2=0.9740), whereas in amorphous EGCG powders stored at 0% RH, asimilar correlation was obtained between brightness L and hue angle h(Fig. 4E, R2=0.9167). This suggests that the changes in the hue angleof solution samples can possibly be attributed to the changes in redness.For EGCG powders at 0% RH, the linear correlations established the pos-sibility of merely monitoring the brightness as a means to follow thecolor degradation. A weak linear correlation between L and h wasobtained for amorphous powders stored at 85% RH, resulting in crystal-lization and uneven color change, with an R2 of 0.6770 (data not shown).

3.3. Chemical stability of EGCG in aqueous solutions

To study the degradation products of EGCG in dilute aqueous solu-tions, a 0.3 mg/mL EGCG solution was heated at 80 °C for up to35 min. The chromatograph profiles of EGCG before and after 35 minof heat treatment are shown in Fig. 5A. The starting material was com-posed of 89.25±3.04% of EGCG (RT: 10.95 min) and 2.40±0.05% ofECG (RT: 13.40 min). The degradation products, i.e. catechin monomersand gallic acid, were identified and confirmed with the elution time ofthe corresponding standard, as well as with the LC/MS technique. It isclear that after 35 min of thermal treatment, the EGCG content de-creased while its epimer GCG (RT: 12.03 min) was produced. Similarly,CG (RT: 14.53 min), the epimerization product of ECG, was producedfollowing the heat treatment. Varied degradation products, i.e. gallicacid (RT: 2.89 min), C (RT: 9.24 min), and EGC (RT: 8.73 min) werealso observed. In the LC/MS extracted ion chromatogram, trace amountsof both EC and GC were also noted (data not shown). The production ofGCG and CG result from the epimerization of EGCG and ECG within thestartingmaterial. It is probable that gallic acid, ECG, and EC are the resul-tant cleavage products from the EGCG and ECG (Nam, Smith, & Dou,2001), whereas the production of CG and C are possibly the result ofthe epimerization fromECG and EC. A schematic of the possible degrada-tion pathways of EGCG and ECG in solutions is shown in Fig. 6. Morecomplex reactions might also be involved, with no detectable productswithin the experimental conditions. No colored compounds were elutedunder the HPLC conditions used in this study.

A concentration dependency of EGCG degradation was also found(Fig. 5A). After heating for 35 min, EGCG was more stable in a 5 mg/mLsolution than in 0.15 mg/mL or 0.05 mg/mL solutions. Furthermore, theproduction of gallic acid increased with a reduction in concentrationfrom 5 mg/mL to 0.15 mg/mL, but was not found to be stable in0.05 mg/mL solutions. GCG demonstrated an increase in peak heightwhen the starting EGCG concentration in solution increased from0.05 mg/mL to 1.5 mg/mL but decreased in the 5 mg/mL solutions.

The changes of catechin content in 0.3 mg/mL aqueous solutionsover 35 min at 80 °C are shown in Fig. 7. The contents of both EGCGand ECG decreased over time. However, EGC increased over time toreach a maximum level at approximately 20 min, with decreasingEGC concentrations found with further heating. A possible cause ofthis shift is that the EGC production from epimerization was dominantduring the initial period of 0 to 20 min, followed by a consumption ofEGC through degradation with longer heating times. ECG was more

Fig. 6. Possible degradation pathways of EGCG in solution.


stable than EGCG over time. After 30 min of heat treatment, roughly53.20±1.17% of EGCG and 64.68±4.50% of ECG remained, similar toresults reported in a previous study (Sang et al., 2005). The contentsof GCG, CG, and C all increased during the 35 min at 80 °C. Similar in-creased productions of GCG and CG had also been observed in previousstudies which reported that epimerization occurred in EGCG aqueoussolutions, green tea powder, and green tea infusions upon heat treat-ment (Chen et al., 2001; Li et al., 2011; Wang et al., 2008b).

EGCG solutions ranging from0.05 to 30 mg/mLwere heated at 80 °Cfor up to 420 min and the concentrations of EGCG, gallic acid, GCG, andEGC were monitored (Fig. 8). The stability of EGCG appeared to beconcentration dependent, with higher concentrations exhibiting morestability. EGCG in 0.05 mg/mL solutions degraded rapidly and was notdetected by the HPLC analysis after a 5 min. The concentration ofEGCG decreased to 6.39±1.63% in 0.15 mg/mL solutions after 5 minof heat treatment. As EGCG concentration increased, the t1/2 valuesincreased, varying from 25.36 min for 0.3 mg/mL solutions to2292.19 min for 30 mg/mL solutions.

The production of degradation products from EGCG was also con-centration dependent (Fig. 8). The degradation products (gallic acid,GCG, EGC and GC) could all be quantitated, except in the case of GCwhich was found to be below the limit of detection by our currentHPLC–DAD method. The production of gallic acid increased with de-creasing EGCG concentration, except in the case of 0.05 mg/mL solu-tions (Fig. 8B). In more dilute solutions (0.05 and 0.15 mg/mL), the



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Fig. 8. Concentration dependent stability of EGCG in aqueous solutions during heating at 80 °C for up to 420 min. A) EGCG. B) Gallic acid. C) GCG. D) EGC. Data are shown by:–■– 0.05 mg/mL –●– 0.15 mg/mL –▲– 0.3 mg/mL –▼– 1.5 mg/mL –□– 5 mg/mL 15 mg/mL –△– 30 mg/mL.


Please cite this article as: Li, N., et al., Color and chemical stability of tea polyphenol (−)-epigallocatechin-3-gallate in solution and solid states,Food Research International (2012), http://dx.doi.org/10.1016/j.foodres.2012.11.019



concentration of gallic acid increased to a maximum level at 15 to20 min, and then decreased with further heating. Such results suggestthat other than being produced in cleavage reactions, gallic acid isconsumed in other reactions. Similar trends are also to be expectedin more concentrated solutions (above 0.15 mg/mL) with prolongedheating times beyond those in our experiments.

GCG is the major degradation product in the more concentratedEGCG solutions; in solutions with lower concentrations of EGCG(0.05 mg/mL and 0.15 mg/mL), GCG showed a decreasing trend overtime (Fig. 8C). The relatively high starting concentration of GCG is likelydue to the rapid production of GCG over the initial minutes during dilu-tion, followed by rapid degradation of GCG over time. In solutions withEGCG concentrations of above 0.15 mg/mL, GCG increased with in-creases in heating time. This observation is consistent with a previousreport that epimerization of EGCG to GCG is more dominant when thestarting concentration of EGCG exceeds some threshold level. Sang etal. (2005) reported that, in pH 7.4 phosphate buffers, epimerizationwas dominant when the concentration of EGCG was 4.12 mM(1.89 mg/mL) or higher, while oxidation becomes significant andEGCG dimers were primarily formed in less concentrated solutionsranging from 20 to 100 μM (0.009–0.046 mg/mL).

A similar concentration-dependent stability of green tea catechinswas observed in our previous work (Li et al., in review). EGCG is aweak acid, with a pKa1 value of 7.55±0.03 (Inoue, Inoue, Fernando,Valcic, & Timmermann, 2002). The ionization state of EGCG, as affectedby the pH, influences its chemical stability. Increases in pH values havebeen reported to facilitate catechin degradation (Li et al., in press;Yoshioka et al., 1991; Zhu, Zhang, Tsang, Huang, & Chen, 1997). In thispresent study, the pH values of EGCG aqueous solutions increasedfrom 4.92±0.32 to 5.95±0.31 as the concentration of EGCG solutions

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Fig. 9. Identification of theasinensin A/D and the colored compound in amorphous EGCG powspectra of the colored compound at 10.27 min with noise subtraction.


decreased from 30 mg/mL to 0.05 mg/mL. Therefore, the shift in pH ofEGCG solutions is likely a factor that contributes to the concentrationdependency of EGCG stability in this study. The production of EGC in-creased with decreases in EGCG concentration from 15 mg/mL to0.3 mg/mL (Fig. 8D). In more dilute solutions (0.05 and 0.15 mg/mL),only a trace amount of EGC was found. This suggests that the cleavageof EGCG occurs primarily when the concentration of EGCG is 0.3 mg/mLor higher, where the pH levels of the solutions are lower than 5.90. How-ever, less cleavage occurred as concentration increased, which is likelydue to the enhanced stability of EGCG as pH decreased with increasingconcentration.

A previous study (Sang et al., 2005) reported on both the concentra-tion dependent stability of EGCG and the formation of theasinensin inEGCG aqueous solutions, in which phosphate buffer at pH 7.4 and nitro-gen flushing were used. Theasinensin was not found in solution statedegradations in our study. Therefore, another possible explanation ofsuch concentration dependence is that the overall degradation of EGCGdoes not follow first order reaction kinetics. EGCG is involved in manyreactions in solutions, as illustrated in Fig. 6, including reversibleepimerization between EGCG and GCG and cleavage reactions fromEGCG to EGC and gallic acid. Therefore, as a combination of many reac-tions, the overall reaction order for EGCGdegradation could be fractional.

3.4. Chemical stability of EGCG in the amorphous solid state

A comparison of HPLC chromatograms of EGCG heated at 80 °C for720 min in solution and amorphous solid state before and after stor-age at 0% RH for 40 days is shown in Fig. 5B. In EGCG amorphouspowders, storage treatment induced both the formation of gallicacid (RT: 2.82 min) and an unknown peak (RT: 10.02 min). The UV

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spectrum for the unknown peak eluted at 10.04 min indicated thatthe maximum absorption was around 497 nm. Therefore, the reddishcolor of amorphous EGCG powder upon degradation is likely due tothe formation of this unknown compound. The colored compoundwas not observed in solid state samples stored at 85% RH. GCG wasnot detected in amorphous solid state degradation at 0% RH, butwas found in samples stored at 85% RH.

Mass spectrometry was used to identify the possible oxidationproducts in the EGCG solid state samples. Theasinensin A/D, anEGCG dimer, was found in solid state samples (m/z 913) after a stor-age period of 40 days at 80 °C and 0% RH. The inline TOF spectraof theasinensins are shown in Fig. 9A. The peak area of theasinensinA/D was also found to increase over an increasing storage time (datanot shown). For the colored compound, a fragment with an m/z valueof 285 was observed with LC/MS as shown in Fig. 9B, suggesting a mo-lecular weight of 286 for the colored compound. Since catechin has amolecular weight of 290, this compound might be a diquinone of cate-chin, or some other catechin derivative. Further experiments with col-umn chromatography separation, MS/MS, and 2D NMR identificationmight be needed to identify the structure of this compound.

Theaflavins and theasinensins are the major oxidation productsfound in black tea, an oxidized product from green tea (Tanaka,Watarumi, Matsuo, Kamei, & Kouno, 2003). Theaflavin is a reddishorange compound, which has maximum absorbance at around 280,373, and 450 nm (Ho, Lin, & Shahidi, 2009). However, it was not ob-served in solid state degradation samples (data not shown).Theasinensin is a colorless dimer of EGCG linked through C2′\C2′bond in the B-ring of EGCG (Fig. 1). Other than these two compounds,a few red, reddish brown, and brown colored compounds, primarilydehydrotheasinensin, EGCG dimers, and their quinoxaline derivativeswere identified in the enzymatic catalyzed oxidation of EGCG (Li,

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Fig. 10. Thermal degradation of amorphous EGCG powder at 80 °C. A) 0% RH: EGCG and ECG–■– EGC –●– GCG. C) 0% RH: Gallic acid and colored compounds. Data are shown by: –■– gstability at different RHs. Data are shown by: –■– 0% amorphous –●– 85% amorphous –▲–


Tanaka, & Kouno, 2007; Tanaka, Matsuo, & Kouno, 2005; Tanaka,Mine & Kouno, 2002; Tanaka, Mine, Watarumi, et al., 2002; Tanakaet al., 2003). However, in order to better identify the colored com-pound which was found in this study, further oxidation treatments,isolation, and identification would be required along with using col-umn chromatography and NMR techniques.

The stability of EGCG and the production of degradation productsin amorphous EGCG powder over time are shown in Fig. 10. Due to alack of authentic standard for the colored compound, the unknownpeak was not quantifiable. Instead, peak area was plotted as a func-tion of time to establish an indication of the production of the coloredcompound (Fig. 10C). When compared with the solution state EGCG,results indicated that EGCG degraded at a lesser rate in the powders,likely resulting from the limited molecular mobility, as well as an in-creased concentration in the solid state.

Previous studies have reported that a pinkish color was observed inpure EGCG crystalline powder upon long-term storage (Sang et al.,2005); however, no new compounds other than EGCG and a traceamount of GCG were observed. Similar pinkish color in crystalline EGCGpowders heated at 120 °C for 16 h was seen in our study. This pinkishcolor possibly results from the formation of the colored compound, asseen in the HPLC–DAD chromatogram (Fig. 5B). Since crystalline EGCGis more stable than the amorphous form, the discovered amount ofsuch compound would typically be much lower than what was foundin the amorphous powders, leading to the lighter color development.

For amorphous powders stored at 85% RH, crystallization occurredrapidly and therefore higher stability of EGCG compared to that at 0%RH was observed (Fig. 10D). Trace amounts of GCG and gallic acidwere found in these recrystallized samples. The colored compoundwhich elutes at 10.02 min was not seen. This colored compound wasnot observed in EGCG solutions either. For crystalline powders stored

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at 85% RH, no significant degradation was observed over 30 days ofstorage at 80 °C (Fig. 10D). The crystalline EGCG powders were morestable than the recrystallized EGCG solids, consistent with the differ-ences in their color development (Fig. 3).

Comparedwith the EGCG solutions, whichwere nitrogen flushed andsealed prior to heat treatments, the solid state samples were stored inopen vials in desiccatorswith controlled RHs, thereby exposing the solidstomore oxygen. Thewater vapor pressure at 85% RH is higher than at 0%RH, therefore the oxygen level is lower at 85% RH. Theasinensin forma-tion and color development increased as RH decreased (data notshown). Such results suggest that the reduced levels of oxygen resultingfromhigherwater vapor pressure likely suppress catechin oxidation, andenhance epimerization.

All of these results indicate that the two major reactions which con-tribute to the instability of EGCG are epimerization and oxidation. Therates of these two reactions are affected by various factors, such as mo-lecularmobility, oxygen levels, pH, reactant concentration, and the solidstate form of the reactant. Epimerization from EGCG to GCG becomesdominant under low oxygen levels, whereas oxidation is the dominantreaction in solid state under low RH conditions.

3.5. Correlation of color with chemical stability

There were no colored degradation products observed in the HPLCchromatograms of EGCG solution samples and solid samples stored at85% RH, thus the regressions between color and chemical stabilitywere only performed for the amorphous EGCG powders stored at 0%RH. The correlations between the HPLC peak area of the coloredcompound and total color changes are listed in Table 3. Exponentialregressionmodels produced good correlations (R2=0.9374–0.9897) be-tween the colored compound and color parameters. Exponential in-creases in all of the color parameters were found, followed bysaturation after a period of about 5 days. Although significant chemicaldegradation was not observed in EGCG in the first 5 days of storage(Fig. 10A), the sample color was almost fully developed within 5 days.This can possibly explain why color development was observed inEGCG powders without evidence of chemical degradation in previousstudies (Sang et al., 2005). Such non-linear correlations of colored com-pounds with color parameters were also observed in previous studies(Arias et al., 2000).

4. Conclusion

The physical state of EGCG, concentrations of reactant, oxygenlevels, pH, and the RH each had varied effects on the color and chem-ical stability of EGCG. Mathematical models for the color degradationkinetics of EGCG in both solution and solid states were establishedand can be used for color degradation predictions and shelf-life deter-minations. The epimerization from EGCG to GCG was found to bedominant in EGCG solutions and EGCG powders stored at 85% RH(which crystallized), whereas oxidation was dominant in EGCG pow-ders stored at 0% RH (which remained amorphous). In EGCG solu-tions, cleavage and epimerization products, gallic acid and other

Table 3Summary of the regressions of the color readings with the production of the coloredcompound of amorphous EGCG powders stored at 0% RH.

Factor Exponential regression R2 Parameters

A1 t1 y0

L 0.9897 27.17 286.51 53.69a 0.9374 −27.09 192.86 36.88b 0.9519 −15.78 7.66 31.23La/b 0.9660 −17.95 407.39 70.81TCD 0.9474 −40.44 232.71 42.93h 0.9645 20.36 393.57 36.55


catechin monomers, were found. The stability of EGCG was concen-tration dependent, with increasing stability evident as the EGCG con-centration increased. In amorphous EGCG powders at 0% RH,theasinensins were formed and red coloration developed, and thecolored compound detected had a molecular weight of 286. Thecolor development of EGCG was more noticeable as compared to itschemical stability. These results extended the understanding of thechemical mechanisms leading to EGCG degradation in both solutionand solid states.

Acknowledgments

This research was supported in part by USDA-NRICGP grant#07-35503-18405. Thanks are also given to the China ScholarshipCouncil.

The authors acknowledge Teavigo® for providing crystalline EGCGpowders. Thanks are also given to Tristan Lipkie and Brian Song forassistance with LC/MS analysis.

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Calligaris, S., Falcone, P., & Anese, M. (2002). Color changes of tomato purees duringstorage at freezing temperatures. Journal of Food Science, 67(6), 2432–2435.

Chen, Z. -Y., Zhu, Q. Y., Tsang, D., & Huang, Y. (2001). Degradation of green tea catechinsin tea drinks. Journal of Agricultural and Food Chemistry, 49(1), 477–482.

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Color and chemical stability of tea polyphenol (−)-epigallocatechin-3-gallate in solution and solid states