Method development for extraction and HPLC analysis of kaempferol, chrysin and quercetin

Abstract

Flavonoids (different phytochemical compounds found in plants acting as phytoalexins), have been found to have potent antioxidant effects. This has led into studies conducted on its effect on the body in reducing oxidative stress in different diseases as they have the potential to scavenge free reactive oxidative species that causes harm to the body, causing degeneration of cells. In the search for medicines that can delay the progression of Alzheimer’s disease (AD), research into the use of flavonoids has become more popular based on the oxidative stress theory of AD. This has established its ability to reduce amyloid β plaque formation and tau protein formation in the different areas of the brain. This study aimed to develop a HPLC method for analysis of chrysin (flavone subgroup), kaempferol and quercetin (flavonol subgroup) after extraction from selected plant material (red and yellow onions and broccoli), identifying the flavonoid compounds, estimating their quantities and drawing comparisons between different processing and storage procedures. A rough screening method using TLC was also developed. A screening method was developed using TLC with two different mobile phases that proved adequate. This was a) toluene 80%: ethanol 20% and b) toluene 60%: ethyl acetate 30%: formic acid 10%. A HPLC method for analysis of chrysin, kaempferol and quercetin was developed and validated using the 10 mM phosphoric acid (H3PO4) 50%: methanol 25%: acetonitrile 25% as mobile phase with a flow rate set to 1 ml/min and the wavelength of detection set to 280 nm. A Kinetex® EVO C18 column (250 mm x 4.6 mm; 5 μm particle size and 100 Å pore size) was used. The plant material was processed to use whole, cut and blended plant material which were either fresh or frozen. Cooked plant material and grilled onions were also used for analysis of flavonoid content. Maceration, water bath extraction (WBE), ultrasonic extraction (USE) and microwave assisted extraction (MAE) were used to extract flavonoids from the prepared plant materials. Samples from these extractions were analysed on the HPLC. Comparisons were drawn between these different plant preparations, processing and extraction methods to establish the better method. In this study, best extraction results were achieved by MAE, followed by USE, maceration and lastly, WBE. A rough estimation of the chrysin, kaempferol and quercetin content led to the conclusion that insufficient concentrations of these flavonoids would be obtained from such small amounts of plant specimens used in this study. A more diverse diet with supplements of these compounds would be more beneficial to the AD patient, or any patient looking at natural treatment aimed at prevention.