Antioxidant activity

Despite the use of treatments known to induce oxidative stress and the production of compounds (flavonoids, betalains) with documented antioxidant activity, DPPH assessed antioxidant activity was unable to detect the difference between either control group and their respective treatment. Several explanations exist for this; firstly, the antioxidant system of plants is composed of a complex mix of enzymatic and low-molecular-weight compounds. These have been shown to react independently of each other depending upon the type, intensity, and duration of ROS-inducing event (Gill and Tuteja, 2010, Vanacker et al., 1998). As such, it is quite feasible that net antioxidant activity of the control and treatment groups had not changed, but that various sub-groups of antioxidants adjusted in a compensatory manner to maintain the same gross antioxidant status; we have found that an analogous process involving glutathione peroxidase occurs in mammalian systems (Jacobson et al., 2007). Adding further complexity to this scenario is the fact that the redox status of individual antioxidants changes depending on whether the plant is in the induction, compensation or acclimatised stage of stress response (Polkowska-Kowalczyk et al., 2007). In addition, there is considerable ongoing discussion as to the relevance and most physiologically appropriate way to measure ROS production in planta and in vivo (Halliwell, 2009, MacDonald-Wicks et al., 2006).

3.6

Conclusion

Our data show that the metabolite profile of CR stress response is stress specific, with the induction of betalain pigment compounds upon UVB exposure and flavonoid compounds upon exposure to low temperatures. The results indicate that it is the presence of a permanent positive charge and visible pigmentation which is most important in providing protection against UVB damage. In response to low-temperature conditions, differential enzyme activation plays a major role in determining the changes in CR flavonoid profile with the preferential accumulation of less-substituted flavonoids, rather than the more often reported production of flavonoids with a dihydroxyl-substituted B-ring being observed. The accumulation of less-substituted minor flavonoids is likely to be an important process in the stress response of other species and is worthy of further investigation.

3.6.1 Acknowledgements

The authors would like to thank Mr Philip Andrews for propagating the plant material.

Adam Pirie is the recipient of an Australian Postgraduate Award funded by the Australian Government. This project was supported by the ARC Discovery and Linkage projects to Sergey Shabala.

4

Pirieol A from Carpobrotus rossii, a novel spinacetin

glycoside containing apiose and HMG moieties.

4.1

Abstract

Carpobrotus rossii (CR) is a plant native to Australia where it is found growing along its southern coastal areas. The plant has a history of traditional use by both indigenous Australians and early European settlers. Despite this history of use and the recent reports of in vitro pharmacological activity, no detailed structural analysis of the compounds contained have been published. This paper reports the elucidation of three CR flavonoids, primarily the predominant flavonoid compound produced by CR, as well as two related minor flavonoid compounds. We have shown these flavonoids to be comprised of a common spinacetin aglycone, and common glucose substituents. The presence/absence of apiose and 3-hydroxy- 3-methylglutaric acid (HMG) moieties determines whether the flavonoid is the predominant flavonoid (all substituents present) or one of the minor flavonoids (apiose or HMG missing, respectively). The predominant flavonoid's IUPAC name was determined to be 5-[[4-[3,4- dihydroxy-4-(hydroxymethyl)tetrahydrofuran-2-yl]oxy-3,5-dihydroxy-6-[2-(4-hydroxy-3- methyl-phenyl)-5,6,7-trimethyl-4-oxo-chromen-3-yl]oxy-tetrahydropyran-2-yl]methoxy]-3- hydroxy-3-methyl-5-oxo-pentanoic acid.

NB: Chapter 4 has been written in a format common to many organic chemistry journals, for example J Nat. Prod. In such journals there is often a combined results and discussion section when the determination of new structures (but no bioactivity) has occurred.

4.2

Introduction

The succulent halophyte Carpobrotus rossii (CR) (Schwantes, 1928) is a member of the Aizoaceae family (Blake, 1969) and commonly found growing along the coastline of southern Australia (Australia). The stresses associated with surviving in these locations, namely exposure to salinity, high light, poor nutrition, extremes of temperature, and aridity (Garcia-Mora et al., 1999) induce the plant to produce high levels of polyphenolic compounds which function as antioxidants in the photosynthetic machinery of the leaves (Chapter 2). Both indigenous Australians and early settlers consumed CR leaf material as a food, and also used it to treat a variety of ailments including burns, stings, and gastrointestinal disorders (Plomley et al., 1966, Watson, 2007).

Despite this history of use, no reports detailing the structure of compounds produced by CR has been published. Recent pharmacological investigations have shown that the extract is flavonoid rich, has strong antioxidant activity, and inhibits the release of inflammatory cytokines (IL-10, TNF-α, MCP-1) in vitro (Geraghty et al., 2011). Additionally, preliminary data indicate that consumption of crude CR extract may have beneficial effects on cardiovascular disease risk by lowering the concentration of lipid levels in vivo (Geraghty, DP, personal communication).

UPLC-UV and -MS/MS analysis of CR leaf extracts from plant samples collected during a previous field survey (Chapter 2) showed that the leaves contained a suite of flavonoid compounds (Figure 4.1a, 4.1b). Tandem MS/MS analysis revealed that many of these compounds formed common daughter ions, indicating the presence of common underlying structures (Figure 4.1c, 4.1d).

The flavonoid profiles of surveyed plants showed considerable intra- and inter-site variation (data not presented). However, a dominant/co-dominant peak with a molecular weight (MW) of 784 was observed in all samples. This peak generated daughter ions common to many of the other flavonoids observed in the CR extract and so was selected as an appropriate candidate for detailed structural investigation.