Abstract
Background
Consumption of vegetables and fruits has been associated with the prevention of various diseases caused by oxidative damage due to the abundant antioxidants these dietary components contain. Pūhā (<i>Sonchus oleraceus</i> L.) leaves have been reported to be rich in antioxidants and possess strong antioxidant activity; thus show potential to be formulated into nutritional supplements. However, comprehensive investigation of antioxidant activity of individual compounds in leaf extracts from the genus <i>Sonchus</i> has not been reported. In this thesis the key antioxidants in pūhā have been identified. The effects of sample preparation including leaf position, harvest time, handling and extraction conditions on the key antioxidants in pūhā leaf extracts have been investigated. Stability of the antioxidants in pūhā extracts under storage, which would affect the quality of the final product, was also studied. Finally, the cellular activity of pūhā extracts was determined in terms of cellular antioxidant activity and anti-aging effects.
Methods
Pūhā leaf extracts were prepared in 70% methanol in water (w/w). An online method using high pressure liquid chromatography (HPLC) to separate herbal extracts followed by post-column reaction with the 2, 2’-diphenylpicrylhydrazyl (DPPH) reagent, as a measure of antioxidant activity, was applied to screen individual peaks with antioxidant activity. These active peaks of interest were isolated through flash chromatography and semi-preparative HPLC; and subsequently, structurally identified using nuclear magnetic resonance (NMR) and mass spectrometry (MS). This online method was used for monitoring the changes of the key antioxidants in pūhā extracts during sample preparations and storage. Design of experiments was applied to investigate the stability of the key antioxidants in the pūhā (leaf or extract) stored at different temperatures (4, 25 and 50°C) and relative humidities (15, 43 and 75%) for 180 days. Antioxidant activity was assessed by both the DPPH free radical assay and the Cellular Antioxidant Activity (CAA) assay with HepG2 cells. Senescence-associated β-galactosidase (SA-β-Gal) activity, a biomarker for cellular aging, was used to determine the anti-aging responses of pūhā extracts to H<sub>2</sub>O<sub>2</sub> stress in WI-38 cells.
Results
Three key antioxidants were determined via the online method and identified as caftaric acid, chlorogenic acid and chicoric acid, with chicoric acid being the dominant compound, using MS and NMR. The antioxidant activity assessed by the online HPLC-DPPH assay was consistent with an offline DPPH assay. The highest concentration of chlorogenic acid was found in young leaves at the top of the plant; while middle leaves contained the highest total amount of antioxidant compounds. The three antioxidants degraded to unquantifiable levels after oven-drying. More than 90% of the antioxidants were retained by freeze-drying and air-drying. Pre-treatment with liquid N<sub>2</sub>, increasing the extraction temperature and time, and freeze-drying the crude extract did not enhance the yield of the key antioxidants. Both leaf and extract samples preserved > 90% of antioxidants, except those stored at 75% relative humidity. Leaf material had higher antioxidant concentrations and greater cellular antioxidant activity than that of corresponding extract samples. Pūhā exhibited high cellular antioxidant activity and significantly reduced the SA-β-Gal activity induced by H<sub>2</sub>O<sub>2</sub> stress.
Conclusion
The online HPLC-DPPH assay was validated as a useful screening tool for investigating individual compounds that contribute to antioxidant activity in leaf extracts. Optimised extraction conditions for the key antioxidant compounds were leaves from the middle of plant pre-treated with liquid N<sub>2</sub>, extraction at 25°C for 0.5 h and solvent removal by rotary evaporation. Freeze-drying was favourable drying technique to preserve more antioxidants in pūhā. To preserve antioxidant activity, it is preferable to store pūhā as dried leaf material rather than as a leaf extract. In cellular assays, pūhā leaf extracts exhibited high cellular antioxidant activity and effectively reduced stress-induced premature senescence. This thesis demonstrates the potential of pūhā leaves to be an excellent source for nutritional supplements.