Effective use of reducing agents and nanoparticle encapsulation in stabilizing catechins in alkaline solution
Introduction
Catechins are a class of polyphenolic flavonoids predominantly found in foods and beverages, such as apples, chocolate, red wine and green tea (Hackman et al., 2008, Rice-Evans et al., 1995). The main catechin species include epicatechin (EC), catechin (C), epicatechin gallate (ECG), epigallocatechin (EGC) and epigallocatechin gallate (EGCG) (Fig. 1). Currently, there is much research interest in catechins, and this is attributed to their potent antioxidant activities and diverse biological properties, which include anti-inflammatory, vasodilatory, neuroprotective and chemopreventive effects (Kuzuhara et al., 2008, Zaveri, 2006).
However, one of the major pitfalls of catechins is that they are chemically unstable (Mochizuki et al., 2002, Neilson et al., 2007, Zhu et al., 1997). In solution, they readily undergo oxidation, involving the loss of hydrogen atoms, generation of a semiquinone radical intermediate and formation of quinone oxidised products (Janeiro and Oliveira Brett, 2004, Mochizuki et al., 2002). A number of factors, including oxygen concentration and pH, influence the stability of catechins. pH is the most crucial factor and it has been shown that the rate of oxidation increases as the pH increases (Mochizuki et al., 2002). For example, Su, Leung, Huang, & Chen (2003) found a mixture of catechins to have a half life of more than 24 h at pH 5.0, whereas, at pH 7.4, the half life was reduced to approximately 1 h. Unfortunately, catechins are often exposed to alkaline environments. Upon consumption, catechins are exposed to alkaline fluids present in some regions of the gastrointestinal tract, under which they have a propensity to degrade. This instability has been cited as one of the reasons for the poor bioavailability of these compounds (Zhang, Zheng, Chow, & Zuo, 2004). Furthermore, in in vitro experimental studies, it is often necessary to study and/or quantify catechins under conditions which mimic the environment present in the gastrointestinal tract or other fluids such as plasma. One example is in the evaluation of the performance of dosage formulations incorporating catechins, where dissolution testing for prolonged periods in simulated intestinal fluids (pH 6.8 and 7.4) is required. Therefore, in order to accurately quantify catechins in such solutions, stabilization strategies are necessary.
Currently, there are a number of strategies that have been developed to protect catechins from degradation in vitro. The most common approach is the addition of ascorbic acid (AA), which is preferentially oxidised in place of the catechin (Chen, Zhu, Wong, Zhang, & Chung, 1998). Another approach is the use of the reducing agent dithiothreitol (DTT), which acts by providing hydrogen atoms to the semiquinone intermediate, thereby driving the reaction back to the catechin-reduced state (Jovanovic et al., 2007, Webb and Ebeler, 2004). However, despite the use of these chemical additives, high levels of protection for prolonged periods have not been achieved. In a study by Chen et al. (1998) demonstrating the protective effect of AA against catechin degradation, it was observed that in the presence of AA, not more than 50% of the initial catechin remained after 24 h of incubation at pH 7.4. This observation may be related to the fact that reducing agents, such as AA, also undergo oxidative degradation in such solutions (Yuan & Chen, 1998).
Tris(2-carboxyethyl)phosphine (TCEP) is a relatively new reducing agent used in various chemical applications. It has been found to be more effective in the reduction of protein disulphide bonds compared to agents such as DTT (Han & Han, 1994). Therefore, TCEP may be able to provide higher levels of protection to catechins for more prolonged periods.
Apart from the use of chemical additives to stabilize catechins in vitro, an alternative approach, suitable particularly for the stabilization of catechins in vivo, is encapsulation in nanoparticles. Recently, nanoparticles prepared from non-toxic and biodegradable polymers have been used to stabilize pharmaceutical actives against pH-mediated or enzymatic degradation in the gastrointestinal tract. An example is chitosan–tripolyphosphate nanoparticles (CS–TPP-NPs), which have been shown to protect insulin from degradation in the gastrointestinal tract, subsequently resulting in the increased bioavailability of this protein (Sarmento, Ribeiro, Veiga, Ferreira, & Neufeld, 2007).
The aims of this study therefore were, firstly, to optimise, evaluate and compare the effectiveness of the reducing agents AA, DTT and TCEP in protecting (+)-catechin and (−)-epigallocatechin gallate from degradation in an alkaline medium. The findings of this investigation have implications for in vitro experimental studies, which are conducted in alkaline environments. The second aim was to evaluate the effect of encapsulation in CS–TPP-NPs on the stability of these catechins in an alkaline medium, which has implications in the stabilization of catechins once consumed and exposed to the alkaline regions of the gastrointestinal tract.
Section snippets
Materials
(+)-Catechin hydrate (C), (−)-epigallocatechin gallate (EGCG), lyophilised ascorbic acid (AA), dithiothreitol (DTT), tris[2-carboxyethyl]phosphine hydrochloride (TCEP), low molecular weight chitosan (CS) (85% degree of deacetylation, viscosity of 1% solution in 1% acetic acid = 20–200 centipoise) and sodium tripolyphosphate (TPP) were all obtained from Sigma–Aldrich Co (MO, USA). All other chemicals and solvents were of analytical grade and were used as received. The water used was obtained from
Stability of (+)-catechin and (−)-epigallocatechin gallate in alkaline solution
First, the stability of C and EGCG in 50 mM potassium hydrogen phosphate buffer, pH 7.4, at 37 °C, was determined. The stability profiles of C and EGCG are shown in Fig. 2, Fig. 3, respectively. These results confirmed previous reports demonstrating that catechins are unstable in alkaline solution and that EGCG is less stable than is C (Zhu et al., 1997). These results also suggested that the stability of catechins may be concentration-dependent. The lower concentrations appeared to degrade at a
Conclusion
First, this study evaluated and compared optimal concentrations of reducing agents for use in stabilizing catechins under in vitro experimental studies comprising an alkaline environment. Examples of such studies may include the analysis of catechins in alkaline biological fluids or, as in our case, the analysis of catechins following encapsulation or release from a dosage form in an environment which mimics the alkaline intestinal fluids. We found that TCEP provided greater protection than did
References (29)
- et al.
Mucoadhesive polymeric platforms for controlled drug delivery
European Journal of Pharmaceutics and Biopharmaceutics
(2009) - et al.
Transcytosis of nanoparticle and dendrimer delivery systems: Evolving vistas
Advanced Drug Delivery Reviews
(2001) - et al.
A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol
Analytical Biochemistry
(1994) - et al.
Catechin electrochemical oxidation mechanisms
Analytica Chimica Acta
(2004) - et al.
Green tea catechin as a chemical chaperone in cancer prevention
Cancer Letters
(2008) - et al.
Hydrophobically modified glycol chitosan nanoparticles-encapsulated camptothecin enhance the drug stability and tumor targeting in cancer therapy
Journal of Controlled Release
(2008) - et al.
Kinetic analysis and mechanistic aspects of autoxidation of catechins
Biochimica et Biophysica Acta (BBA) – General Subjects
(2002) - et al.
Epigallocatechin-3-gallate (EGCG): Chemical and biomedical perspectives
Phytochemistry
(2006) - et al.
Interactions between chitosan-modified particles and mucin-coated surfaces
Journal of Colloid and Interface Science
(2008) Green tea and its polyphenolic catechins: Medicinal uses in cancer and noncancer applications
Life Sciences
(2006)
Biopolymeric delivery system for controlled release of polyphenolic antioxidants
European Polymer Journal
Investigation of intestinal absorption and disposition of green tea catechins by caco-2 monolayer model
International Journal of Pharmaceutics
Chitosan and chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines
Pharmaceutical Research
Stabilizing effect of ascorbic acid on green tea catechins
Journal of Agricultural and Food Chemistry
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