## 4 Discussion  
### XO Inhibitory Activity of C. morifolium Fractions  
Our study provides, for the first time, data showing that _C. morifolium_ sub-fractions are effective in XO inhibition and uric acid reduction. At the beginning, EtOAc fraction was shown to exhibit moderate (High: 71-100%; moderate: 41-70%; low: 0-40%) XO inhibition. This might be due to its higher polyphenic content as compared to the other fractions. _C. morifolium_ EtOAc fraction has been shown to contain significantly higher total phenolic and flavonoid content than other fractions in our preliminary study [14]. This is because natural phenols show solubility preference to solvents with intermediate polarity [15]--EtOAc in this study--rather than more polar or non-polar solvents. Thus, majority phenolic and flavonoid compounds of _C. morifolium_ flower in this study might have been drawn to the EtOAc fraction during solvent-solvent partitioning, which conferred its higher bioactivity.  
The results agree with [PERSON] et al. (2016) [16], according to whom _Chrysanthemum segetium_ EtOAc extract showed the highest polyphenol level and flavonoid contents. Although the inhibitory effect of a complex mixture could not be directly compared to a pure compound, the moderate XO inhibitory activity exhibited by the EtOAc fraction suggested that it might serve as a potential XO inhibitor. In other words, the EtOAc fraction might exhibit higher XO inhibition at higher purity or concentration. Therefore, it was subjected to bioassay-guided fractionation to obtain higher purity sub-fractions for further analysis.  
Kinetic studies indicated that the mode of XO inhibition for the RS fraction was competitive, whereas the crude extract, EtOAc, and PE fractions were shown to be non-competitive. Non-competitive inhibitor inactivates an enzyme by altering its active site or overall conformation [17]. This indicates that the inhibitor in the crude, EtOAc, and PE fractions does not compete for the XO active site with xanthine (substrate); but rather, inhibits the XO enzyme, possibly by causing conformational change to the XO active site. According to [PERSON] (2012) [18], pure polyphenic compounds, such as quercetin and kaempfer derivatives, possessed non-competitive XO inhibition. The RS fraction in this study inhibited XO enzyme via competitive inhibition. This indicated that the inhibitor present in the RS fraction might resemble the structure of xanthine, thereby competing for the same active site. Competitive inhibitor can only bind to the free enzyme but not enzyme-substrate complex, thus inhibition activity can be overcome by increasing the substrate concentration within the reaction [19]. According to [PERSON] et al. (2020) [20], polyphenols also inhibit XO competitively by binding to its active site, therefore the inhibitory activities of competitive inhibitors are directly proportional to their concentrations.  
The increased bioactivity of sub-fractions F7 to F11 might be due to the greater concentration of potent bioactive compounds present in the semi-purified sub-fractions as compared to the crude extract. Fractionation is a crucial step to increase the purity of bioactive compounds, and hence the bioactivity. In some cases, a mixture of compounds possesses higher bioactivity than a pure compound [21]. Hence, the anti-hyperuricemic effect of the semi-purified EtOAc sub-fractions was investigated in this study. Before that, cytotoxic assays were carried out to determine the range of non-cytotoxic concentration of the sub-fractions.