Antioxidant activity of inclusion complexes of tea catechins with β-cyclodextrins by ORAC assays

Food Research International 43 (2010) 2039–2044

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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Antioxidant activity of inclusion complexes of tea catechins with β-cyclodextrins by ORAC assays C. Folch-Cano a, C. Jullian b, H. Speisky c, C. Olea-Azar a,⁎ a b c

Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1, Chile Departamento de Química Orgánica y Fisicoquímica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1, Chile Laboratorio de Antioxidantes, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Casilla 138-11, Santiago, Chile

a r t i c l e

i n f o

Article history: Received 11 March 2010 Accepted 8 June 2010 Keywords: Antioxidants Tea catechins ORAC-FL ORAC-PGR β-cyclodextrins

a b s t r a c t Inclusion complexes of native and two derivatizated β-cyclodextrins with some tea catechins (TCs) like catechin (C), epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG). The stoichiometry of TC complexes was determined and in all cases was 1:1. Stability constants were determined and their antioxidant capacity against reactive oxygen species (ROS) studied by means of the ORAC-fluorescein (ORAC-FL) and the ORAC-pyrogallol red (ORAC-PGR) assay. The antioxidant capacity of these TC in aqueous solution in the absence and presence of βCDs was studied using Multi-Detection Microplate Reader. The antioxidant reactivity mainly depends of the inclusion modes of the TC in the βCDs cavity. The difference between ORAC-PGR values for the same TC show that the inclusion structures should be different, maybe leading the same number of hydrogen atoms exposed (ORAC-FL values) outside or in the borders of βCDs cavity. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Cyclodextrins (CDs) are cyclic oligosaccharides composed of glucopyranose units and can be represented as a truncated cone structure with a hydrophobic cavity (Szejtli, 1998). The cavity is relatively hydrophobic, while the external faces are hydrophilic (Li & Purdy, 1992). The most extraordinary characteristic of a cyclodextrin is its ability to form inclusion complexes with a variety of compounds, i.e., by trapping foreign molecules (guest) in its cavity (host). Generally, hydrophobic molecules or those with hydrophobic residues have the highest affinity with the CD cavity in aqueous solution, and it is well established that the ability of β-cyclodextrin (βCD) to enhance drug stability and solubility depends on formation of inclusion complexes (Szejtli, 1994). Unmodified or unsubstituted βCD, i.e., those with no substituent on the glucopyranose unit, have poor water solubility and are parenterally unsafe due to nephrotoxicity. Therefore, several synthetically modified and relatively safe βCD have been made and used in parenteral formulations, such as hydroxypropyl-β-cyclodextrin (HPβCD) (Shuang, Pan, Guo, Cai, & Liu, 1997) and Heptakis-2,6-O-di methyl-β-cyclodextrin (DMβCD). The molecular encapsulation of drugs and food ingredients with CDs improves the stability of flavours, vitamins, colourants and unsatured fats, etc., both in a physical and chemical sense, leading to extended product shelf-life. (Brewster & Loftsson, 2007; Szente &

⁎ Corresponding author. Tel.: +56 2 9782844; fax: +56 2 7370567. E-mail address: [email protected] (C. Olea-Azar). 0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.06.006

Szejtli, 2004; Szente & Szejtli, 2005). HPβCD has been shown to be well tolerated in humans, with the main adverse event being diarrhea and there have been no reported adverse events on kidney function. In fact, is an alternative to βCD, with improved water solubility and may be more toxicologically benign (Gould & Scott, 2005). Tea is a beverage made from the leaves of Camellia sinensis species of the Theaceae family. This beverage is one of the most ancient and, next to water, the most widely consumed liquid in the world. Tea leaves are primarily manufactured as green, black, or oolong, with black tea representing approximately 80% of tea products consumed. Green tea is the non-oxidized/non-fermented product and contains several polyphenolic components such as catechin (C), epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG). EGCG is the major green tea catechin (TC) (N40% dry weight) and is probably, the principal responsible of chemoprevention and cardiovascular diseases prevention due to its free radical scavenging and antioxidant activities (Chemoprevention Branch and Agent Development Committee, 1996). Regular intake of tea is associated with an improved antioxidant status in vivo that may contribute to lowering the risk of certain types of cancer, coronary heart disease, atherosclerosis, stroke (Hong, Smith, Ho, August, & Yang, 2001; Sano et al., 2004; Uesato et al., 2001; Vinson, Teufel, & Wu, 2004), reduced mutagenicity (Gupta, Sahaa, & Giri, 2002) and inflammation (Katiyar, Matsui, Elmets, & Muktar, 1999), protection against neurodegenerative diseases (Choi et al., 2000; Choi et al., 2001; Datla et al., 2004) and increasing insulin sensitivity (Wu, Juan, Ho, Hsu, & Hwang, 2004). Research on the

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putative health effects of tea has demonstrated the contribution of phytochemicals, particularly phenolic acids and flavanoids (mainly catechins) to the above-mentioned benefits. The actions ascribed to polyphenols are almost certainly mediated partly by their free radical scavenging, antioxidant and metal complexing actions (Bahorun, Luximon-Ramma, Crozier, & Aruoma, 2004). However, TC powders are bitter, brown, and easily oxidized and hence difficult to use as a medicine or a natural food additive. In fact, when incubated in various solutions and soft drinks these catechins had a poor stability and decayed by at least 50% during the first month of storage at room temperature (Su, Leung, Huang, & Chen, 2003). For this reason we have proposed the encapsulation of some TC with βCDs. Our investigation group has studied the antioxidant activity of inclusion complexes of flavonoids with native and derivatives of βCDs, and observed an increase or maintenance in their antioxidant reactivity, respect to their respective free flavonoids (Jullian, Moyano, Yañez, & Olea-Azar, 2007; Jullian, et al., 2008). This complexation phenomenon increased the antioxidant activity of some flavonoids, which reached a maximum level when each flavonoids had been complexed in the hydrophobic cavity of CDs. The antioxidant activity increased because the flavonoids were protected against rapid oxidation by free radicals (Mercader-Ros, Lucas-Abellán, Fortea, Gabaldón, & Núñez-Delicado, 2010). Here, we report the preparation of inclusion complexes of βCD, HPβCD and DMβCD with some TC like C, EGCG and GCG (Fig. 1). Stability constants were determined and their antioxidant capacity against reactive oxygen species (ROS) studied by means of the ORACfluorescein (ORAC-FL) (Ou, Hampsch-Woodill, & Prior, 2001) and the ORAC-pyrogallol red (ORAC-PGR) (Alarcón, Campos, Edwards, Lissi, & López-Alarcón, 2008) assay. The antioxidant capacity of these TC in aqueous solution in the absence and presence of βCDs was studied using Multi-Detection Microplate Reader. 2. Materials and methods 2.1. Chemicals (−)-catechin, (−)-epigallocatechin gallate, (−)-gallocatechin gallate, β-cyclodextrin, Heptakis(2,6-di-O-methyl)-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin [M.S. (average molar degree of substitution) = 1.0], AAPH (2,2_-azobis(2-methylpropionamidine) dihydrochloride), FL (Fluorescein disodium salt), PGR (pyrogallol red) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), were from Sigma-Aldrich, Inc., St. Louis, MO. 2.2. Preparation of TC complexes For all assays inclusion complexes were obtained by mixing appropriate amounts of C, EGCG and GCG with βCDs in nanopure

water. The resulting mixture was equilibrated in a Julabo thermostatic shaking water bath for 24 h at 30 °C after which the equilibrium was reached. The continuous variation method (Job, 1928) were determined from Jenway 6405 UV/vis spectrometer in aqueous solution. The total molar concentration (i.e., the combined concentration of TC and βCDs) was kept constant (0.1 mM), but the mole fraction of TC (i.e., [C] / ([C] + [CD])) was varied from 0.1 to 0.9. 2.3. Determination of stability constants Steady-state fluorescence measurements were performed using a Perkin Elmer LS 55 spectrofluorimeter. Emission fluorescence spectra were acquired in the 320–480 nm range (1 nm step), at a fixed excitation wavelength of 276 nm. The reaction medium contained 50 μM TC and increasing concentrations of βCDs, prepared in water to reach a final volume of 3 mL at 30 °C. Assuming that the increase in the TC fluorescence intensity observed in the presence of increasing concentrations of βCDs is due to the formation of the inclusion complexes between TC and βCDs, the following equilibrium can be described: TC + βCDs

↔ TC–βCDs KC

The fluorescence intensity at any wavelength (F) can be related to the βCDs concentration by the Eq. (1) (Connors, 1987): F = F0 +

ðF∞  F0 ÞKC ½βCDS 0 1 + KC ½βCD0

ð1Þ

where F∞ is the fluorescence intensity when total TC has been complexed in βCDs and F0 is the fluorescence of TC in the absence of βCDs. Experimental data of F as a function of [βCDs] can be fitted to Eq. (1), using as initial parameters (Kc and F∞) those obtained from the analysis of the experimental data using the Benesi–Hildebrand Eq. (2) for 1:1 complexes (double reciprocal plot) (Benesi & Hildebrand, 1949): 1 1 1 = + ðF−F0 Þ KC ðF∞ −F0 Þ½βCDS 0 ðF∞ −F0 Þ

ð2Þ

2.4. ORAC-FL assay The ORAC analyses were carried out on a Synergy HT multidetection microplate reader, from Bio-Tek Instruments, Inc. (Winooski, USA), using 96-well polystyrene white microplates, purchased from Nunc (Denmark). Fluorescence was read from the top, with an excitation wavelength of 485/20 nm and an emission

Fig. 1. Structure of TC used in this study.

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Table 1 Mole fraction in the absorbance maximum (χmax), stoichiometry values, stability constant values (KC) and adj. R-square of Benesi–Hildebrand plots for TC complexes.

Fig. 2. Job’s plot of GCG complexes formed with native βCD, DMβCD and HPβCD, represented by maximum absorbance as a function of mole fraction.

filter of 528/20 nm. The plate reader was controlled by Gen 5 software. The oxygen radical absorbance capacity was determined as described by Ou et al. (2001), with slight modifications. The reaction was carried out in 75 mM sodium phosphate buffer (pH 7.4), and the final reaction mixture was 200 μL. FL (150 μL; 40 nM, final concentration) and C, EGCG or GCG, in the absence or presence of βCDs (25 μL) solutions, were placed in the wells of the microplate. The mixture was preincubated for 15 min at 37 °C, before rapidly adding the AAPH solution (25 μL; 18 mM, final concentration) using a multichannel pipette. The microplate was immediately placed in the reader and automatically shaken prior to each reading. The fluorescence was recorded every 1 min for 90 min. A blank with FL and AAPH using sodium phosphate buffer instead of the antioxidant solution and five calibration solutions using Trolox (0.5, 1.0, 1.5., 2.0 and 2.5 μM) as antioxidant were also used in each assay. The inhibition capacity was expressed as Trolox equivalents (M), and is quantified by integration of the area under the curve (AUC). All reaction mixtures were prepared in triplicate and at least three independent assays were performed for each sample.

Complex

χmax

Stoichiometry

Kc(M−1) × 103

R2

EGCG + βCD EGCG + DMβCD EGCG + HPβCD GCG + βCD GCG + DMβCD GCG + HPβCD C + βCD C + DMβCD C + HPβCD

0.51 0.49 0.52 0.52 0.53 0.49 0.50 0.52 0.51

0.96 1.04 0.92 0.92 0.89 1.04 1.00 0.92 0.96

7.47 23.80 14.60 15.50 17.40 26.90 3.00 8.67 3.41

0.98 0.93 0.99 0.93 0.96 0.98 0.98 0.99 0.99

The area under the fluorescence decay curve (AUC) was calculated integrating the decay of the fluorescence where F0 is the initial fluorescence read at 0 min and F is the fluorescence read at time. The net AUC corresponding to the sample was calculated by subtracting the AUC corresponding to the blank. Data processing was performed using Origin Pro 8 SR2 (Origin Lab Corporation, USA).

2.5. ORAC-PGR assay The ORAC analyses were carried out on a Synergy HT multidetection microplate reader, from Bio-Tek Instruments, Inc. (Winooski, USA), using 96-well polystyrene clear microplates, purchased from Nunc (Denmark). Absorbance was read from the bottom, at 540 nm wavelength. The plate reader was controlled by Gen 5 software. The oxygen radical absorbance capacity was determined as described by Alarcón et al. (2008), with slight modifications. The reaction was carried out in 75 mM sodium phosphate buffer (pH 7.4), and the final reaction mixture was 200 μL. PGR (150 μL; 70 μM, final concentration) and C, EGCG or GCG, in the absence or presence of βCDs (25 μL) solutions, were placed in the wells of the microplate. The mixture was preincubated for 15 min at 37 °C, before rapidly adding the AAPH solution (25 μL; 0.1 M, final concentration) using a multichannel pipette. The microplate was immediately placed in the reader and automatically shaken prior to each reading. The absorbance was recorded every 0.25 min for 90 min. A blank with PGR and AAPH using sodium

Fig. 3. Increase of the fluorescence intensity for EGCG, when [HPβCD]/[EGCG] concentration fraction increase. On the right the representation of 1/(F – F0) vs. 1/[CDs] (double reciprocal plot) for the same complex.

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phosphate buffer instead of the antioxidant solution and five calibration solutions using Trolox (50, 100, 150, 200 and 250 μM) as antioxidant were also used in each assay. The ORAC-PGR indexes were calculated like previously we indicated in Section 2.4. 3. Results and discussion The stoichiometry of TC complexes was determined from the representation of the absorbance maximum of all the TC and βCDs versus the mole fraction (Fig. 2), as explained in Section 2.2. In all cases we obtained an inclusion stoichiometry 1:1, as indicated by the results showed in Table 1. These results are in agreement with the values obtained in previous works (Ishizu, Kajitani, Tsutsumi, Yamamoto, & Harano, 2008; Jullian, Miranda, Zapata-Torres, Mendizábal, & Olea-Azar, 2007). In the case of GCG with βCD, Ishizu et al. (2008) proposed an inclusion stoichiometry 1:2, but their stoichiometrics results were obtained by NMR determinations, and they suggested interactions between the hydrogen atoms of B ring with βCD cavity. Our fluorimetric results indicated that the inclusion stoichiometry was 1:1 and we believe that the interactions indicated by Ishizu et al. (2008) were overall interactions with the borders of the βCD cavity rather than an effective inclusion of the B ring. Moreover, based on the results obtained in the ORAC assays, we postulate that for EGCG and GCG the B ring and galloyl groups were either exposed or in the borders of βCDs cavities. We used a fluorimetric method (Section 2.3) to calculate the Kc values for the complexation of TC with βCDs. In this method, the increase in fluorescence intensity as a function of TC complexation by βCDs was used to calculate the Kc. When increasing concentrations of βCDs were added to the reaction medium, the fluorescence intensity increased, until a maximum was reached (Fig. 3). The representation of 1 / (F − F0) vs. 1 / [CDs] (double reciprocal plot), known as a Benesi– Hildebrand plot (Fig. 3), leads to a straight line for the three type of βCDs used, and corroborate the 1:1 stoichiometry obtained by the continuous variation method for the complexes. This linear relation agrees with to that described by the Benesi–Hildebrand Eq. (1). The linear plots of Fig. 3 can be used to determine Kc values by simply dividing the intercepts by the slopes (Table 1). But since Benesi– Hildebrand plots tend to place more emphasis on low CD concentrations than on higher values, the slope of the line is more sensitive to the ordinate values of the points for the lowest concentrations. To corroborate de values of Kc obtained, a non-linear regression analysis of the plots in Fig. 4 was carried out by applying the Eq. (2) and using as initial parameters those estimated by the Benesi–Hildebrand plot. These representations showed a good correlation with the experimental comportment observed when the βCDs concentration was increased. As can be seen in Table 1, the values of stability constant for derivatized βCDs complexes are higher than those for the native βCDs complexes. This may indicate that the terminal groups of the

Table 2 ORAC indexes and free TC concentration percentages in the inclusion equilibrium (% [TC]F) for free and complexed TC. (ND = not determinated indexes). Antioxidant

%[TC]F

ORAC-FL

ORAC-PGR

TX EGCG EGCG + βCD EGCG + DMβCD EGCG + HPβCD GCG GCG + βCD GCG + DMβCD GCG + HPβCD C C + βCD C + DMβCD C + HPβCD

– 100.00 12.80 4.28 7.02 100.00 6.66 5.97 3.94 100.00 26.45 11.23 24.08

1 1.89 3.42 3.54 3.48 1.87 3.45 3.77 3.31 3.99 2.82 2.79 2.68

1 9.48 7.77 6.07 9.46 13.32 11.24 6.41 11.86 0.45 ND ND ND

derivatized βCDs permit more interactions with TC than the native βCD. The most effective inclusions were EGCG with DMβCD (EGCG + DMβCD) and GCG with HPβCD (GCG + HPβCD) with the higher stability constant values. The ORAC-FL indexes were calculated by fluorescence measurements (Table 2) as indicated in Section 2.4. In general, the antioxidant activity of flavonoids depends on the structure and substitution pattern of their hydroxyl groups. Recently, Mercader-Ros et al. (2010) showed that the presence of a 3-OH group is relevant for the antioxidant activity of flavonoids. Additionally, hydroxyl groups at positions C5 and C7 of the A ring appear to be less important. The flavanols tested in this work have a different number of OH in ring B and the presence of galloyl group in C3. We observed (Fig. 5) that the ORAC-FL indexes show the tendency: C N EGCG ≈ GCG, which is inversely proportional to the number of hydroxyl groups in the B ring. This relevant result indicates that the most labile hydrogen atoms for C could be in the A ring. Such contention is corroborated by the decrease of the hydrogen atom donating capacity of the C + βCDs inclusion complexes respect to free C. Jullian, Miranda et al. (2007); Jullian, Moyano et al. (2007) determined the inclusion complexes structure of C with DM, HP and the native βCDs, obtained through molecular modeling and NMR spectroscopy, showing that the 3-OH group was totally included in the βCDs cavity. Such results are in agreement with the results obtained here. On other hand for EGCG and GCG complexes, we observed an almost two-fold increase of their capacity respect to the free TC; probably due to a complexation of the A ring, leading the hydroxyl groups of B and galloyl groups exposed outside or in the borders of the βCDs cavity. This is in agreement with the inclusion structures determined by Ishizu et al. (2008) through molecular modeling and NMR spectroscopy, for EGCG and GCG with native βCD. Therefore our

Fig. 4. Comparison between experimental increase of fluorescence intensities when increase the βCDs concentrations, with Connors equation representation (solid line) for C complexes.

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Fig. 5. Columns bar graph of ORAC-FL indexes and stability constants (KC) for free and complexed TC.

results corroborate that the inclusion stoichiometry for GCG + βCD must be 1:1, because the ORAC-FL indexes were similar to EGCG inclusion complexes indicating the same number of available hydrogen atoms. The ORAC-PGR indexes were calculated by UV/vis measurements (Table 2) as indicated in Section 2.5. This methodology permits estimating the antioxidant reactivity of TC. Alarcón et al. (2008) explained the differences between ORAC values obtained employing PGR and FL as target molecules in terms of differences in their reactivities towards peroxyl free radicals. This would lead to ORAC-FL values conditioned by stoichiometric factors, while ORAC-PGR values would be more dependent upon the antioxidants reactivity. In fact, we didn't observe a general correlation between ORAC-FL and ORAC-PGR indexes. We observed (Fig. 6) the ranking of antioxidant activities to be as follows: GCG N EGCG N C. Moreover we observed no perceptible activity from C inclusion complexes, because their antioxidant activities were lower than free C. On other hand, the ORAC-PGR indexes showed a lower decrease or maintenance of antioxidant activity of EGCG and GCG complexes with βCDs, respect to free EGCG and GCG. The most reactive complexes were with HPβCD and the complexes with lowest reactivity were with DMβCD. This assay permits estimating the different antioxidant reactivities between the inclusion complexes with the same TC, indicating that the inclusion structure could be different for the βCDs used in this study, in example EGCG + HPβCD complex showed practically the same reactivity of free EGCG, however EGCG + DM and native βCDs showed a decrease in their antioxidant reactivities. Therefore the results showed that the spatial distribution of B and galloyl groups in EGCG and GCG had an important influence in the antioxidant

reactivity of these TC with peroxyl free radicals. In fact for inclusion complexes formed with GCG we observed that only GCG + DMβCD showed a great decreasing on its reactivity compared with EGCG + DM and native βCD, indicating that the B and galloyl groups were more available in the complexes formed for GCG. In order to prove the influence of inclusion complexes formed in this study, we decided to compare the antioxidant activities with the free TC fractions present in the inclusion equilibrium TC-βCDs, calculated from the stability constants values with Eq. (3) (Mercader-Ros et al., 2010):

½TCF =

ð−ð½CD0 KC −½TC0 KC + 1Þ +

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð½CD0 KC −½TC0 KC + 1Þ2 + 4KC ½TC0 Þ 2KC

ð3Þ

where [TC]0 is initial tea catechins concentration and [CD]0 is initial CDs concentration. For GCG + βCD, GCG + HPβCD and EGCG + HPβCD complexes we showed an antioxidant activity maintenance respect to their free TC, with low free TC percentages, indicating that the antioxidant activity is due to the contribution of the inclusion complexes formed. No relationship between the KC values and the antioxidant activity of the inclusion complexes formed was found in this study, indicating that the antioxidant reactivity mainly depends of the inclusion modes of the TC in the βCDs cavity. The difference between ORAC-PGR values for the same TC show that the inclusion structures should be different, maybe leading the same number of hydrogen atoms exposed (ORAC-

Fig. 6. Columns bar graph of ORAC-PGR indexes and free TC concentration percentage of EGCG and GCG inclusion complexes.

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