CONCLUSION

This chapter presented and discussed the findings from several preliminary experiments and method optimisation studies that were required in the lead up to the primary experimental work conducted as part of this study. This included the optimisation of several parameters for HPLC- PDA/ESI-MSn _{analysis, such as the HPLC column to be used, the composition of the mobile phase} and the gradient program for each of the three bok choy extracts (i.e. hydroalcoholic, alkaline hydrolysate, and acid hydrolysate), as well as the optimum PDA detector wavelength for each extract, and the ESI-MSn _{ionisation conditions.}

The HPLC column chosen for this study was the Varian Polaris C18-A; 150 x 2.0 mm 3 µm as it gave the best resolution and peak shape of the major flavonol glycosides present in a hydroalcoholic onion extract. The best separation of the flavonol aglycones present in the acid hydrolysate bok choy extracts was achieved using a acetonitrile/water + 0.1% v/v formic acid mobile phase with the following gradient: 90/10 A/B – 10/90 A/B (0-30 minutes), 10/90 A/B (30- 35 minutes), 10/90 A/B – 90/10 A/B (35-36 minutes), 90/10 A/B (36-46 minutes). A superior separation of the flavonol glycosides and the flavonol glycoside-hydroxycinnamic acid derivatives found within the alkaline hydrolysate and hydroalcoholic extracts respectively, was

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 A bs or ba n ce ( at 570 n m ) Time (Hours)

HT-29 Growth Curve

100 cells/well 500 cells/well 1,000 cells/well 5,000 cells/well 10,000 cells/well 50,000 cells/well 100,000 cells/well

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achieved using a methanol/water + 0.1% v/v formic acid mobile phase with the following gradient: 95/5 A/B – 5/95 A/B (0-40 minutes), 5/95 A/B (40-45 minutes), 5/95 A/B – 95/5 A/B (45-46 minutes), 95/5 A/B (46-60 minutes). Therefore, the same conditions were used for both the alkaline hydrolysate and hydroalcoholic extracts, whereas the acid hydrolysate was analysed using a different mobile phase composition and gradient program.

The optimum PDA detector wavelength was determined to be 370 nm for flavonol aglycones in the acid hydrolysate extracts, 350 nm for flavonol glycosides in the alkaline hydrolysate extracts and 330 nm for the flavonol glycoside-hydroxycinnamic acid derivatives found in the hydroalcoholic extracts. It was observed that the broad absorbance maximum that arises from the absorption of the flavonol B-ring, referred to as band I, shifts approximately 20 nm lower with the addition of a glucose moiety to the flavonol aglycone, and a further 20 nm with the addition of a acyl moiety. This is known as a hypsochromic shift.

Analysis of the flavonol compounds present in the different bok choy extracts via ESI-MSn _was conducted in the negative ionisation mode using formic acid as the mobile phase additive. The MS was tuned using rutin, a similar flavonol compound to those analysed in the bok choy. The capillary temperature and capillary voltage were set to 350 ºC and -48.50 V respectively, which gave the optimal response for the tune compound.

The preparative HPLC conditions also required optimisation. The flow rate and injection volume required to be scaled up from the HPLC-PDA/ESI-MSn _{method, and the mobile phase gradient} program needed to be adjusted. The flow rate was scaled up from 0.2 mL/min to 10 mL/min for the preparative HPLC and the injection volume was scaled up from 5 µL to 1000 µL. The mobile phase composition was kept the same as the analytical HPLC method, however, the gradient was extended as this produced a better separation. The optimised gradient was as follows: 95/5 A/B – 5/95 A/B (0-60 minutes), 5/95 A/B (60-65 minutes), 5/95 A/B – 95/5 A/B (65-66 minutes), 95/5 A/B (66-76 minutes).

Other sample preparation/extraction parameters that were optimised included the hydrolysis time for the acid hydrolysis extraction method, as well as the syringe filter type used to filter each extract prior to chromatographic analysis. The optimum hydrolysis time was determined to be 4 hours for the acid hydrolysis method. Nylon filter discs were used for both the alkaline hydrolysate and hydroalcoholic bok choy extracts prior to analysis, however, PTFE filter discs were used for the flavonol aglycones in the acid hydrolysate extracts as it was observed that the flavonol aglycones adsorbed onto the nylon discs.

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The passage dilution factor for routine maintenance of the HT-29 cell culture model, and the cell density and time range to be used for the MTT proliferation assays were also optimised. A 1:10 dilution of HT-29 cells was the ideal dilution factor to reach 70% confluency of cells in three days and was therefore used to passage cells in between the MTT proliferation assays. A cell density of 10,000 cells/well was chosen for the assays as the exponential growth phase of the cells was within an ideal timeframe at this density. Initial MTT proliferation assays were conducted between 24-96 hours, after which all subsequent assays were conducted at the 72 hour time point. After completion of all preliminary and optimisation studies, the methods were employed for the primary experimental work, and all results from this work are presented and discussed in the following two chapters.

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CHAPTER V

5

IDENTIFICATION AND QUANTIFICATION OF

FLAVONOLS IN BOK CHOY CULTIVARS

5.1

INTRODUCTION

This chapter reports and discusses the results obtained from the HPLC-PDA/ESI-MSn _{study of} flavonol compounds present in three hardy varieties of bok choy (‘Sumo,’ ‘Karate,’ and ‘Miyako’) produced for the local market in Victoria, Australia. Information on these varieties, including growing conditions and preparation for analysis is given in 3.2 and 3.4.

The key flavonols of interest in this study include quercetin, isorhamnetin and kaempferol, their glycosylated derivatives, and other more complex conjugates that occur naturally within the bok choy plant, such as flavonol glycoside-hydroxycinnamic acid derivatives. This chapter is divided into the following sections:

1. Identification and quantification of the flavonol aglycones obtained after acid hydrolysis of freeze-dried, powdered bok choy (3.4.2), including verification of the method used. 2. Identification and structure elucidation of flavonol glycosides obtained after alkaline

hydrolysis of freeze-dried, powdered bok choy (3.4.3).

3. Identification and structure elucidation of flavonol glycoside-hydroxycinnamic acid conjugates and other naturally occurring flavonol compounds obtained by hydroalcoholic methanol/water extraction of freeze dried, powdered bok choy (3.4.4).