Ameliorative effect of aspalathin from rooibos (Aspalathus linearis) on acute oxidative stress in Caenorhabditis elegans

Abstract

Rooibos leaves and fine stems (Aspalathus linearis; Fabaceae) are increasingly enjoyed as herbal tea, largely in fermented (oxidised) red-brown form, but also in unfermented (unoxidised) green form. Rooibos is rich in antioxidant polyphenols, with the dihydrochalcone, aspalathin, as a major active ingredient. We used Caenorhabditis elegans as model organism to investigate the effect of rooibos extracts against oxidative stress in vivo. In a high glucose environment, C. elegans treated with rooibos extract exhibited an extended lifespan. Furthermore, green rooibos was a more potent antioxidant than red rooibos, probably due to its substantially higher aspalathin content. In addition, rooibos decreased acute oxidative damage caused by the superoxide anion radical generator, juglone, with aspalathin playing a major role in improving the survival rate of C. elegans. Quantitative real-time PCR results demonstrated that aspalathin targets stress and ageing related genes, reducing the endogenous intracellular level of ROS. These findings suggest that rooibos increases stress resistance and promotes longevity under stress, probably mediated via a regulation of the DAF-16/FOXO insulin-like signalling pathway, supporting some of the health claims put forward for rooibos tea.

Introduction

Reactive oxygen species (ROS) are generated not only by endogenous metabolic functions but also by environmental stimuli such as ultraviolet light or other kinds of radiation. Excess free radicals disturb the dynamic balance between oxidants and antioxidative defence in organisms, leading to oxidative stress which can induce damage to proteins, biomembranes and DNA (Finkel and Holbrook, 2000). Oxidative stress is assumed to be involved in several health disorders, including diabetes, cardiovascular problems and neurodegenerative diseases such as Alzheimer's disease. Therefore, drugs with antioxidant properties are employed in medicine to reduce cellular stress and, in consequence, combat several severe diseases. Medicinal plants, rich in antioxidant polyphenols and terpenoids, could play an important role in this context (Van Wyk and Wink, 2004).

Rooibos (Aspalathus linearis), a traditional herbal tea from South Africa, is becoming increasingly popular as a health beverage. Since 2003, exports exceeded 5000 metric tonnes per annum and the product is currently sold in more than 37 countries (Joubert and De Beer, 2011). The cut leaves and young stems are subjected to “fermentation”, an oxidative process resulting in a colour change from green to red-brown, hence the final product is also referred to as fermented rooibos or red rooibos. To date studies on the phenolic oxidative changes have focussed on the conversion of aspalathin, a CC linked dehydrochalcone glucoside (Fig. 1) unique to rooibos, to the flavones, orientin and isoorientin via unstable flavanones, and the formation of dimers (reviewed by Joubert and De Beer, 2011) and coloured dibenzofurans (Heinrich et al., 2012). This susceptibility of aspalathin to oxidation results in a substantial reduction in its content in fermented rooibos (Joubert, 1996). Unfermented rooibos, on the other hand, is processed in such a manner as to minimise oxidation of its polyphenols and to retain its green colour; it is accordingly termed green rooibos (Schulz et al., 2003, Joubert and De Beer, 2011). Oxidation of the polyphenols in the course of fermentation and other processing methods leads to weaker antioxidative capabilities in red rooibos (Joubert et al., 2008). Aspalathin is not only the most abundant flavonoid in green rooibos, but it is one of its most potent radical scavengers (Von Gadow et al., 1997, Joubert et al., 2004, Krafczyk et al., 2009, Snijman et al., 2009). Japanese researchers were the first to report an anti-ageing effect on human skin (Joubert and De Beer, 2011). Juráni et al. (2008), using Japanese quails as ageing model, showed that rooibos, when fed to hens, hens contributed to the slowing of age-related decrease in egg production. Furthermore, other beneficial properties such as antimutagenic (Snijman et al., 2007), anti-inflammatory (Baba et al., 2009) and antidiabetic (Kawano et al., 2009) effects have been attributed to aspalathin and rooibos. Detailed summaries of in vitro and in vivo studies on the bioactivity of rooibos infusions and extracts, as well as its major flavonoids, were presented by Joubert et al. (2008) and Joubert and De Beer (2011).

Caenorhabditis elegans is a tiny free-living nematode, which is widely used as a model organism in different research fields. Due to its small size and short life span it has become an important model for ageing studies, which are facilitated by a large number of transgenic mutants expressing disease-related phenotypes (Hekimi and Guarente, 2003). For instance, daf-2 encoding an insulin receptor negatively regulates the fork head (FOXO) transcription factor DAF-16 in the insulin/IGF signalling pathway that is involved in metabolic diseases in humans (Kimura et al., 1997).

In this study, we investigated whether rooibos aqueous extract can protect C. elegans against oxidative stress caused by the prooxidant juglone or a high glucose environment. Our results suggest that aspalathin, as the main compound from green rooibos, can increase the life span of C. elegans under stress conditions through the insulin/IGF-1 signalling pathway and enhance oxidative stress resistance by up-regulating the expression of stress-response related genes.